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Comparative methods of casein extraction

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Comparative methods of casein extraction

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Content COMPARATIVE METHODS OP CASEIN EXTRACTION A Thesis Presented to the Department of Chemistry University of Southern California In partial fulfillment of the Requirements for the Degree of Master of Science By Caroline Hayes Armstrong March 30, 1941 UM'l Number: EP41525 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI EP41525 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Dissertation Publishing Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 C , 4 \ This thesis, written by _B. _ _ CAROL!N E _ _ HA'YE S _ _ AM! ST _RO N G... under the direction of h.&£. Faculty Committee, and a p p ro v e d by a l l its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fu lfill m ent of the requirem ents f o r the degree of MASTER OF SCIENCE Dean Secretary D a te ..J .m § .s ... 1941. If** I I * * Faculty Committee CONTENTS SECTION page I. COMMERCIAL HISTORY OF CASEIN 1 Uses of Casein . 1 Chemistry of Casein 2 Sources of Casein 3 Comparison of Methods of Extraction 4 II. EXPERIMENTAL WORK 7 The Problem 7 Source and Composition of Soybeans Used 7 Procedure 7 Tables and Graphs Showing Results 12-13e III. ANALYSIS OF RESULTS 14 IV. SUMMARY 15 V. BIBLIOGRAPHY 16 COiMSRCIAL HISTORY OF CASEIN Uses of Casein The commercial development of casein started about 1890. The largest single user of casein is the paper coating industry. Prior to the introduction of casein • , animal glue was used as an adhesive or size to glue to the surface such pigments as clay , blanc-fixe , and 3atin white in making high grade book paper , lithograph paper , friction glaze and flint glaze papers. The second largest user of casein is the wood glue industry. The reason for the rapid rise of this glue was largely due to its water resisting qualities. Using casein as a base 9 glues may be compounded which are soluble in cold water , but are waterproof when hardened. Casein wood glues may be dissolved and applied cold without affecting the glue, or the resulting bond. The casein wood glues range in properties from slightly water-resistant types to glues that will pass the government Army and Navy specifications for airplanes. These must give a very high breaking strength even after the glued \>rood has been soaked for forty eight hours in water and tested immediately. In making kalsomine , animal glue dextrines were formerly used and are used to a greater or lesser extent today; however , they have no water resistance. Currently there are many dried powdered casein paints which require merely a mixture with water and are washable when dried. They are made in various qualities ranging from the silghtly washable kalsomine up to the' very insoluble ones which are suitable for exterior application. Much of this type of paint is made for inside factory painting where a surface* is desired that is more washable than a casein kalsomine, but less water- resistant than a casein outside paint. Practically all of the plastic paints which have been so popular in the past few years are pfepared with casein as the binding material. In recent years several manufacturers have produced satisfactory casein paints of a paste type using water as the carrying vehicle. Chemistry of Casein Casein is a protein having an approximate molecular weight of 32,000. It has a specific gravity of about 1.26 and behaves as a weak acid. It is insoluble in water, alcohol, and other neutral organic- solvents, however it is readily soluble in water containing hydroxides or carbonates of the alkali earth metals. Its isoelectric point is at a pH of 4.6 o at which point its solubility is 0.11 grams per liter at 25 C. Casein contains a total of 21 amino acids including the following which are listed together with their percentages. Glutamic acid- 22%, hydroxy glutamic acid- 11%, proline- 8%, valine- 8%, leucine-8^, lysine- 8% are the amino acids found in greatest abundance in casein. 3 Sources of Casein Milk, from which casein is readily separated, has heen the principal source of this protein. However, soybean experi mentation is rapidly developing a new source. Alpha protein from soybeans has been found to possess the same properties as casein. Soybean cake, which is the residue after the press extraction of oil, is a fine source of casein having a protein content of approximately 45.3^. There is a United States Patent'1 on a process of obtaining casein from soybean cake using dilute sodium hydroxide as an extracting agent. The soybean, Glycinie Soja, is a polypetalous Dicotyl edon of the Leguminos&a family of the sub-family Papi1ionacue. It is indigenous to Southeastern Asia, and is a paramount food plant of that region. The cultivation of it has heen known to mankind for over four thousand years. At the present time there have been catalogued over one thousand different varieties. Manchuria produces the largest amount of soybeans for commercial purposes. They are one of the largest export crops, either as seed3, oil, flour, sauce, or milk of the Asiatic countries. Soybeans are by far the most important legume of the far east. They are a constituent of the daily diet of the natives, being used as a supplement to the ever prevalent rice. The seed of the soybean is the richest natural vegetable food known. It has a greater number of uses than . . . . . . . . . . . 4 - - any other comparable substance,both in the Orient and in other countries. Soybean flour, because of its low carbohydrate and high protein content is an excellent food for diabetics. Soybean milk, extracted from the seed is used in cooking and is recommended for feeding infants and invalids. Soybean sprouts are also used as food. The oil from the soybean is a superior drying oil of major importance. It is extracted from the seeds by expression or by treating the ground seeds with an organic solvent. It is halfway between linseed and cottonseed oils in its prop erties and is therefore sometimes classified as a drying oil and sometimes as a semi-drying oil. The soybean can be grown under diversified soil and moisture conditions, preferably in a temperate climate because of susceptibility to frost. In the past few years, soybeans have become a crop of major importance in the United States. Over half of the domestic crop is raised in the State of Illinois. The variety habitual to the United States is that known as ’Common Yellow’, those found in the Orient being the green varieties.. Comparison of Methods of Extraction Before casein can be extracted from soybeans, the oil from the beans must be removed; this can be done by press extraction , which is not too efficient, or by solvent extraction. The latter method removes practically all of the oil. The press cake must have further treatment to remove the remainder of the oil. It is often cheaper to use the press cake as a source of casein as it is a "byproduct of the soy bean oil industry. When whole, flaked soybeans are used and the oil is solvent extracted, the oil can be recovered and hasaa ready market. In extracting the casein, the fat extracted beans are treated with dilute sodium hydroxide (0.2%) for varying lengths of time. The resulting solution is centrifuged.and the supernatant liquid is drained off. The liquid contains the alpha protein which is precipitated by adding hydrochloric acid until a pH of 4.6 , the isoelectric point of casein, is reached. This precipitates out the casein, giving a yield of about 80^ of the total available protein. The casein is separated by centrifuging and is then redissolved in dilute alkali, preferably ammonium hydroxide and is again centri fuged until clear. The pH of the alkali is less than 7.5 . The protein is reprecipitated by adding hydrochloric acid until a pH of 4.8 is reached. This is washed with very dilute hydrochloric acid of pH 4.8. The curd is freed from water by pressing and by crumbling into ethyl alcohol. After standing a while, the alcohol is drained off and the casein is completely extracted in a continuous extractor, first with hot alcohol, and lastly with ether, followed by drying at a low temperature. Another method of purification uses in order in the drying process alcohol, acetone, toluene, and finishes with ether and drying at a low temperature. Still another method uses 0.5^ sodium carbonate as the extraction method instead of sodium hydroxide. 7 II EXPERIMENTAL WORK The Problem This investigation was undertaken for the purpose of determining the efficiency of the various methods of extract ing casein from soybeans. Because of the wide and steadily increasing scope of the uses of casein, the development of commercial sources other than milk is important. Little work has been done on this problem in America although considerable effort has been expended in the Orient and in Argentina. Source and Composition of Soybeans Used The soybeans used were of the common yellow variety from the northern part of Illinois. The following analyses were made according to the methods of the Association of Official Agricultural Chemists. Each figure given represents an average of at least two determinations which checked to within 0.1 % of the percentage. The Illini beans contained: 42.78 % prb&ein, 5.03 % moisture, 19.65 % fat, 4.26 f> starch, 2.45 % fiber, -and 3.07 % ash the remainder being undetermined. Procedure The standard methods of protein extractions vary but little from one another. The protein is extracted from oil- free meal by dilute alkali or alkaline salt solutions followed by precipitation with acid. There has been little investiga tion on the optimum conditions for this extraction. Smith and Circle^ did some work on this, but they considered only the hydroxides and chlorides of sodium and calcium and com binations of the chlorides with various acids. Some research has been undertaken in the Orient using sodium and potassium carbonates.2 The series of experiments undertaken in this research deals with the study of dilute solutions of sodium carbonate, sodium hydroxide and sodium chloride, used as extracting agents. The pH values were obtained with a glass electrode pH meter of the portable type. All pH values above 9.0 were checked against a set of standard buffer solutions. The soybeans were ground in a crude drug mill and were stored in glass stoppered bottles. The samples were not kept very long after grinding and extracting as there is a tendency for the protein to decompose if allowed to stand for a long period of time. Because of the high oil content of the beans, it is necessary first to extract the oil. This was done by the solvent method. 1 Smith, A. K., and Circle, S. J. Ind. and Eng. Chem. 30, pp. 1414-18 (1939) 2 Chang, H.Y. and Chang, T. H. Journ. of Chem. Engr. (China) 4, pp 177-9 (1937) R.H. McCarroll, chief chemist, Pord Motor Company, recommends normal hexane or a petroleum naphtha with a boiling range of 60°C. to 70°C. as the best solvents.'1 ' In this research, petroleum naphtha was employed as it was more easily obtained and much cheaper than normal hexane. The best results in the fat extraction process are obtained with two to three times as much solvent as meal. There is practically complete fat extraction if this amount of solvent is allowed to percolate through the meal for ten minutes,meal is then heated to 100°C. The Pord Motor Company chemists have developed a method of fat extraction which is unique. Solvent is added at the upper end of an inclined tube,ten inches in diameter, while the meal is carried up through the tube by a screww. conveyor. The rate of rotation of the conveyor is adjusted so that ten minutes are required for the meal to reach the top where it is transferred to a steam jacketed tube. The latter tube volatilizes any adhering solvent. Two and one half gram samples of the fat extracted beans were agitated by means of an air stream with one hundred milliliters of solutions of sodium hydroxide and sodium carbonate of varying concentrations. After time inter vals of one half hour, one hour and two hours, ten iiilli- liters were removed and an analysis of the nitrogen content made. The nitrogen determinations were run in duplicate by 1 McCarroll,R.H. "Agricultural Developments" (A paper presented at a meeting of Agricultural Chemists 1938} 10 the Gunning, Arnold modification of the Kjeldahl method. The average precision of these determinations was better than one half of one percent. Before removal of the samples, the whole was centrifuged for ten minutes. The temperature of the extracting solutions varied from 18° to 22°C. The non protein ;nitrogen was not determined, but has been reported « • * , , " - - . . . by Hamilton as 5.55^ of the total nitrogen.* On the addition of the dispersing solution, the sol uble sugars go into solution. Galactans which compose the material of the cell walls, will swell up, absorbing water and will mechanically remove into solution as a bulky sus- pensoid. Legumelin and proteose go into solution. Gummy substances which are partially soluble also go into the ex tract in the form of a colloidal solution or mucilaginous suspensoid. These mechanically suspended impurities cannot be separated from the solution by simple filtration as they immediately choke the filter paper making it water proof and preventing the passage of any more filtrate. One method of avoiding this is to wash the ground beans with a 0.1^ solu tion of acetic acid as it prevents the dissolution of the globulins. However, since this procedure extracts nearly one tenth of the total proteins, it was not adopted. The proteingextraction solution was centrifuged to separate 1 Hamilton, T. S., Uyei, H., Baker, J.B. and Grindley,. H. S. Journ. Am. Chem. Soc. 45 p 815 (1923) such insoluble material from the soluble matter and the supernatant liquid was removed "by decantation. The protein was removed by carefully adding dilute hydrochloric acid until the isoelectric point was reached, at which time a white, flocculent precipitate appeared. "When this precipit ate was filtered and allowed to (ffry in air, a brownish, gummy, glue-like mass was formed.* In order to prevent the formation of such a mass, the precipitate was washed with an hydrochloric acid solution of a pH of 4.6, followed by washing with alcohol and finally was crumbled into 95^ alcohol. After standing for some time, the alcohol was drained off and the precipitate*after being washed with a mixture of xylene and acetone, was allowed to dry at a low temperature. The product of this procedure was a granu lar white material. The substance did not absorb mib&sture from the air and revert to the gummy mass. For further purification, a double precipitation was performed using very dilute ammonium hydroxide as-,a solvent. After crumbling the final precipitate into 95^ alcohol, it was extracted very thoroughly in a Soxhlet extractor first with alcohol and finally with ether, after which process it was dried at a low temperature. Tables and Graphs Showing Results The following table of results represents the average values of duplicate determinations whose precision was one 12 half of one percent or "better. Dilute sodium hydroxide was the extractive agent. The first column indicates the number of millequivalents of "base per gram of meal, while the per centage columns indicate the effect of increasing the length of time of contact of the meal with the extraction solution. Table I Meq. HaOH per gm. meal pH % of Total nitrogen Dispersed - § ■ hr. contact 1 hr. 2hrs 5.12 — 94.9 95.3 95.3 4.50 — 95.4 95.6 95.7 3.12 — 96.3 96.3 96.3 2.75 12*1 95.7 95.9 95.9 .57 10.8 94.4 94.8 95.0 .34 9.3 93.2 93.4 93.4 .12 7.9 89.6 90.1 90.3 Table II Meq. HagCO*; per gm.meal pH % of Total Fitrogen Dispersed llhr. contact 1 hr. 2 hrs 4.102 — 95.3 95.5 95.5 3.093 - - 96.3 96.5 96.5 2.024 11.1 97.4 97.5 97.7 1.32 10.8 95.1 95.4 95.6 .960 9.2 94.1 94.3 94.3 .805 8.8 92.8 93.1 93.2 The following table was obtained as were the two prev ious tables, the extractive agent in this instance being a tenth normal solution of sodium chloride to which were added the indicated amounts of sodium hydroxide. Meq. Table III Meq. FaOH per gm. meal pH ■ % of Total nitrogen Dispersed -|hr. contact 1 hr. 2 hr s. 95.1 95.3 95.4 12.1 94.9 95.1 95.2 11.6 94.5 94.6 98.6 11.1 94.6 94.6 94.7 3.10 2.75 1.24 .8 9 13a GRAPH I Showing relationship between / of available protein extracted and milli-equiv: lentc of sodium hydroxide/ gram ' 1 i i ! I 9 4 ’ tVt i - + 4 4tvt RTt I: *0. 640E . ES$E R 5 / . o MILLER. lOi AN»il 13 b GRAPH II Showing relationship between % of available protein extracted and millequivalents of sodium carbonate used per SI 30 83. - o v ra rr i I n j ? r tg Q v-1h4 si n i 1 T r Lt M f - .. . 11 ■ .... --- ... 1 1 1 i ■ | l t ■ I - • I i. .... • • } "A ,i_: I b 1 11 i , ' t _ i i i I 1 i i i ■I i i 4 ■ J Z . _ 4-- t .4. _ _ __ !; 1 1. -t— 4- 4... i 1: ! i i - 1 . \ r ; t j i t 1 l.t h “f t t / i t 4t i t “ ' 'i' ... .... -fr i - j , i ++ U ! i il | 1 \ j f i —c i i s i ! ~ - 4- 4 ^ -• i 11 i i i : 1 1 1 j; 1 1 L.. 1 I 4 4 s ; — - - .... - F I 1i i 1 | 1 t I 1 • . . . . . __ ■ ■ tr -f-+ - 1 i t : + 4- i *- 4 H + 1 | x ■ ‘ i 1 ■ | j 1 1 [ ■ i ' 11 ... imi \ t i 4 _ \ i * i M I u C t r F p r - 4—4 - . 1 [ 4-t 1 - ... T TtT 44 -4-- 1 .:.r 4.. f 1 <L> + 4 X u i •4 J . • i i " t i i { ■ ■ I * • i - • rV i ; : I 1 • , i ■t 1 1 I 1 4 - 4 - ! t i . I ) ! 4 -- 1 • * ■ ... 4 - - A i- t r r" d r ’ 1 1 1 • i | 1 ■ 1 ’ j". — - -.1 1 • f i r 1 . 1 i • i -f- ' -H- — i- v - i • v t 1 1 - — 4 H- ++ 4 - c s ’I t r 4 i > w ~ it t. • 4-7 f \ m - 1 ! -W- -4- ■ 4 / i 4 -t- * 7 ‘ ,++ r - f +, n i ; 1 1 .... 4“ : y „ !_ — — • • f 11 ■ + 4 + 44 +Jl V ‘ i t 1 r ! ■ 1 1 i ■ ! 1 ■ 1 1 1 ++ ] I "■f 1 ” ‘ £ ; 1 1 4 -4 - 4 -+--4 - --4 - 4.. -4- * 4 - 1 | - U h j V h---t- t t r + 1 4 -- I i-H- -1 - - . . . h I ; 44 - ' I 1 I'M 4 4 1 1 ' i i 1 • » l ■ - I I ■ I ■ ! 4 _ l_ —p jT C I,. 1 u ! ' t ' ' 1 . 1 1 !; - ; ■ V r - 1 ■ " 1" >, '1! 1 I i ■ t 444 - ■ f ■ 1 i r ; r ! ' i 4 - 4 4 _. 4 -i- " 1 r n -b4 - . . . -i- 1 -~ t- rtp 4 -4 - i t r t n 4 * -H -h [ k -1 F' t - t ; . t . . . 1 *h- T - t t - 4 -4- 4 I 1 4 | n r - T f -4 + t 4 4 e .cicM m t ).'3 3 f z a K l-- V : t t t r , ! I K ^4 1 ■ NO. 6405. MILLER. LOS«*»_cLES 13 c GRAPH III , Showing relationship between % of available protein extracted and millequivalents of sodium hydroxide used in a 0,1 JT sodium chloride solution per gram of soybean mea! t~- 30 2-0 2.5 3-0 3 . 5 i. o 14 III ANALYSIS OF RESULTS This research shows that an optimum jrield of nitrogen ous material can he obtained from soybean meal by the use . of a 0,3$ solution of sodium carbonate when the ratio of soybean meal is 5 to 200. This maximum yield is 97.4$ of the • * .... . total available nitrogen. A similar maximum yield is obtained by the use of a 0.25$ solution of sodium hydroxide, other factors remaining the same. The latter method is less efficient, producing only 96.. $ of the total available nitrogen. The presence of sodium chloride decreases the extractive effect of the sodium hydroxide by one percent. All three of these methods produce better results than does the use of hydrochloric.acid solutions. Yields of 80 $ are unusually high for the latter method. A single extraction one half hour in duration is adequate to remove the greater part of the protein. If the material is extracted for a longer time, the amount of pro tein obtained above that extracted in the first half hour is practically negligible and not worth the time involved. A second extraction yields less than one percent! whereas, three percent additional protein should have been available. The extremely low cost and availablility of sodium carbonate are the concluding arguments in favor of this material over the use of sodium hydroxide. 15 IV SUMMARY The many uses of casein have been reviewed in this paper* The chemical properties and constituents were pointed out inasmuch as these factors vitally affect the methods used^for extraction. It was shown that there is a far greater proportion of casein in mfcal obtained from soybeans than in milk. The ease and methods of extraction of casein from soy beans were compared* A review of work done in the laboratory, on various methods of extraction, was made and the results of this investigation were carefully tabulated and graphed for easy comprehension. BIBLIOGRAPHY Journals Hung Yuan Chang and Te Hui Chang Journal of Chemical Engineering (China) vol. 4 p. 177-9 (1937) Hui Hsieh, Tung Pao Jen and Ping Kuon Chang Chiao Tung Univ. Research Institute Annual Report vol. 3 p. 63-70 (1936) Michaelis,L. and Pechstein, H. Biochem. Zeitschrift p. 260-268 (1912) Smith, A.K., and Circle, S.J. Industrial and Engineer ing Chemistry vol. 30 pp 1414-1418 (1939) Hamilton, T.S., Uyei,H.,Baker, J.B. and Grindley, H.S. Journal American Chemical Society vol.45 p815 (1923) Books and Papers Sutermeister, E. and Brotm, P.L. Casein and its Indust rial Applications. Am. Chem. Soe. Holograph 2 nd Edition. Rheinhold. Publishing Company (1939) McCarroll, R.H. "Agricultural Developments,H(A paper presented at a meeting of Agricultural Chemists 1938)

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Creator Armstrong, B. C. H (author)

Core Title Comparative methods of casein extraction

Degree Master of Science

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, organic,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), Brinton, Paul H.M.P. (committee member), Johnstone, G.R. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-788715

Unique identifier UC11348028

Identifier EP41525.pdf (filename),usctheses-c17-788715 (legacy record id)

Legacy Identifier EP41525.pdf

Dmrecord 788715

Document Type Thesis

Rights Armstrong, B. C. H.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA