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California State University, Northridge CALIFORNIA STATE UNIVERSITY, NORTHRIDGE FOLACIN STABILITY IN SNOW PEAS: Effects of Cooking Methods and Extraction Procedures A thesis submitted in partial satisfaction of the requirements of the degree of Master of Science in Home Economics by Carolyn Tepper August, 1985 The Thesis of Carolyn Tepper is Christine H. Smith, Ph. D. Tung-Shan Chen, Ph.D., Chairman Califor·nia. State Univer·:.it>', Nor·thridge ACKNOWLEDGEMENT I would 1 ike to thank the members of mY graduate committee tor their guidance and encouragement: Dr. E. Audrey Clark for her Kind words and knowledge. Christine for her continual support and confidence. Dr. Tung-Shan Chen, who as my major advisor, showed exceedinq patience and kindness. Because of his vast knowledge in this area of research, I was able to master a small part of would also 1 ike to thank my parents, Robert and Renee, who taught me understanding and courage to excel. am extremely grateful to my fiance, Mark J. Sirof. Without his help (in lab, computers, and much more), and continual encouragement, I would have never of made it this far. I would especially 1 ike to thank the nutritionists and directors at my office; Deborah E. Beavers-Ross, Martha Gautschi, and Jane Rascoe, to name a few. thank them for their understanding, kind words of encouragement, and giving me the ualuab~ time needed to complete my thesis. iii 1='::.,-,!:r : ·--.·~-- APPROVAL PAGE . ! j I!! LIST OF TABLES '.} j i J T r·T --.l ·=·! OF FIGUREE ABSTRACT Cha.p ter- 1. INTRODUCTI GJ···l . Introduction C' Definitions.. ~· 2 ~ REt..) I EIAI OF LITERATURE History of Folacin 6 Chemistry and Structure of Folacin 7 Folacin Requirements in Man 10 F~lacin DeficiencY 10 Microbiological Assay . Effect of Ascorbic Acid Added During E;.~tr-acion II • , , - • 14 Effect of pH and Buffers on Folacin . Folacin Content in Chinese Vegetables . 18 CooKing Method and Folacin Retention 20 Microwave Cooking and Folacin Retention 22 Stir-fr:ting Steaming 25 3. ~1ATER I ALS At~D t1ETHODS 27 f'1ater· i al 27 ChB.pter- •")7 Microorganism for Assay Chemical Reagents and Culture Media 27 Equipment . t··1a i n ten an c e He thods . • 29 Pr·epar.::d ion of 1,)egeh.ble-::. 30 Prel iminar·y Studies . It It 30 Effect of Cooking on Folacin Content of Snow Peas Microbiological AssaY 36 Data An a 1::.·sis . 4. RESULTS AND DISCUSSION . 46 Pr·el iminar::-' Studies •• 46 Evaluation of Buffers and pH for Folacin Edr·act ion 46 Use of Ascorbic Acid During Extraction Procedure .. 51 Relation Between the Size of the Snow Pea and its Folacin Content •. Folacin Content in Various Pea Sizes 56 Stab i 1 i ty of Fc•l2,c in in the Fr·ozen Ex tr3.ct·:; 58 Compar· i son of Fc•ur Cooking t1e thods for Fo 1 ac in Retention . .;50 5. CONCLUS! ONS AND RECOt1r1ENDAT IONS ..., .... REFERENCES •· i: Chapter Page n" APPENDICES • • I I s 1: ~ I .. • • s • • t s I • • ~ • • • Cl4 l 7 ,-...,.. ;,_ l ·:0 l OF TA8LEE Ta.bl e 1. Recommended Dietary Allowances of Folic Acid 2. Variations in Ascorbic Acid Concentration Used in Extraction of Foodstuffs and Variations in Source of Conjugase Used Prior to Folacin Assay 17 3. pH Values: Types of Buffers Used During Preliminary Extraction Studies 4. Protocol of Standard Curve Tube Preparation 41 5. Effect of pH and Type of Buffer on Folacin in 49 6. Effect of Ascorbic Acid Concentration in the Extraction Buffer on the Folacin Activity of 53 7. Folacin Content of Various Sizes of Snow Peas (Whole) and Pea Portion Alone 8. Stabi 1 i ty of Fol d.C in in Sno1A1 Pea Extracts Stor·ed Frozen . 59 9. Snow Peas: Cooking Methods, Cooking Time, CooKed !..·..It • , l..<lt •. Loss. and Broth 1-.Jo 1 urne • . 62 10. Effect of Cooking on Folacin Content of Snow Peas 64 LIST OF FIGURES ,-, ill Chernica1 Str-uctur·e of Fr:tl ic Acid 7 2. Folacin Activity in Raw Snow Peas: Effect of pH and Buffer Dufing Extraction ..... 48 3. Folacin Activity in Raw Snow Peas: Effect of Percent Ascorbic Acid Added to Extraction Buffer .• 55 Effects of Cooking Methods and Extraction Procedures c.::..r·ol Yn Tepper· Master of Science in Home Economics The folacin activity of snow peas <Pisum sativum) was determined by Lactobacillus casei assay. Several variables were examined to determine the optimal extraction conditions for assaying the folacin activity in snow peas. Folacin in fresh snow peas were found to be affected by the type and pH of the extractinQ buffer and the level of ascorbic acid added to the extraction buffer. The optimum buffer for folacin retention during extraction was found to be sodium phosphate buffer at pH 6.0, and the optimum level of ascorbic acid in the buffer during extraction was found to be 0.45%. The size of the r·a,,J snot,J pea a.lso a.ffected the folacin content. Larger snow peas have a higher folacin content ( 170 pg/100g) than I the sma.ll er· snm'J peas ( 91 pg.. /1 OOg). the auer·.age size sno:/J peas t;,ter·t? us.ed throughout this stud;.', These~ sn-m.\1 pe.:~.s. ''Jer·e found to cont.ain appr·o~<imately 100l.lg./100g fctiacin and a three and c•ne-half ounce serving would provide 25.0% of the RDA for folacin. Cooking of the snow peas by micrcwaving, stir-frying, and steaming did not result in a significant loss of folacin. However, Cooking of the snow peas by microwaving, stir-frYing, and steaming did not result in a significant loss of folacin. However, boiling the :.nm·J peas caused a decrease in the fol.:1cin content. Most of the folacin lost was due to leaching into the cooking water. Therefore, attempts should be made to cooK with the least amount of water as possible. Chapter INTRODUCTION The Food and Nutrition Board of the National Academy of ~c1ences established Recommended DietarY Allowances (RDA) for folacin in 1968 (Food and Nutrition Board, 1980). The RDA for folacin are 400 ~9 for adults, with increased values for pregnant and lactating women <Food and Nutrition Board, 1980). Several studies have indicated that the average dietary intake of folacin is less than that of the RDA (Peterkin et al., 1981; Thenen, i982). The a.dequacy of folic acid inta~;e i:. uncer·tain <Nik-Daud and Bendar, 1983). It has been reported that much of the folic acid in foods is unstable during processing and cooKing, and may even be lost during storage <Briggs and Calloway, 1979). However, there is little reliable information avail.:>b1e on the f•Jlate contents. of foods and reports of instability may be due to errors in methodology of assay <Nik-Daud and Bender, 1983). It is necessary to establish the folacin content •Jf •.Jarious foods in order to determine if ther·e~ is adequate in ta!(e of folacin ba.s.ed on the RDA. At the present time there is 1 imited information on the amount c•f available folacin which is available in food consumed by the American pe•Jple (Heppner et al., 1972; Paul and So1.,1thgate, 11'78; Perloff and Buttrum, 1977). There are several methods of analyzing for folacin in foods. HovJe\Jer, the microbiologica.l assay is recommended <nein et al., 2 1'? :31 : Sh.:;., n ~ e t .; 1 . , 1 9:3 0 ) . The p r· e fer r· e d mi c roo r g an i -:.m i -; L.:..ctobe<.ci11u·~ cas~i <h ca-:.ei) since it r·esponds to a r,,Jider· ra.nqe of folate for·ms. (f'lalin, 1975). h casei •:.:..n utilize fc,J.:..te forms which haue a glutamic acid residue length of three or less <Tamura et a.l., 1972). The use c•f con._iugase deri•.Jed frc•m chicken p.:..ncrea.s results in folate forms with two glutamic acid residues (Leichter e t a 1 • , 1 978) • Phosphate-ascorbate buffer ha.·:. b~come .:;., s tandar·d edr·ac t i ng agent for folacin since 1961 <Herbert, 1961). The role of ascorbic acid in the folacin a·~say has been knmvn as a sta.bil izer· that Keeps the folates in the reduced forms, especially during autoclauing (Herbert and Bertino, 1967). Folic acid is gen~rally more stable in a neutral or alkaline pH than acidic pH <Blakely, 1969; Stokstad and Thenen, 1972). At pH of 4.0 or belOJ.JJ, folic acid is easily destroyed at 100°c. But at the same temperature folic acid is more stable above pH ~.0 (Dick et al ., 1948; Paine-Wilson and Chen, 1979). Recent studies have shown that the stability of pure folacin compounds can be increased in the presence of ascorbic acid <Chen and Cooper, 1979; o···sroin et al., 1975). Studies haiJe al·~o indicated that ascorbic acid afforded better protection than many other antioxidants, and at lorJJ concentrations <O·'Broin et al., 1975; Wilson and Horne, 1983). Lactobacillus easel was found to respond differ~ntly to changes in pH by the turbidimetric assay. Therefore, for the folacin analysis of foods, food hcmogenates should be adjust~d to a constant pH, and the same concentration of phosphate should be used throughout the assay procedure (Chen e t a 1 • , 1983). Th~ d i Her en t 3 growth responses of L. casei at various pH's have also been obser1Jed by Phillip·;; .:..nd !..Jright (1983). Vegetables are the maJor contributors of folacin in the diet <Leichter et al., 1978; Nik-Daud and Bender·, 1983).
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