Pickett, Haskel C.

SHAREOK repository

LIPASE ACTIVITY IN BOVINE MILK AND BLOOD By HASKEL C. PICKETT . 11 Bachelor of Science. Oklahoma State University Stillwater, Oklahoma 1950 ·'''.! Submitted to the faculty of the Graduate College of the Oklahoma State Univ,rsity ·fn partial fulfillment of the requirements for the degree•· of · · MASTER OF SCIENCE Julyt .. 2~h. 1967 LIPASE ACTIVITY IN BOVINE MILK AND BLOOD Thesis Approved: ~esisAdviser ~~uiltM Dean o~the Graduate College ii ACKNOWLEDGMENTS OKLAHOMA STATE UNIVERSITY LI SRA RY JAN 16 1968 ., ,. ~ ... -:, ,,._.,,. The author wishes· to·express his·appreciation to Dr. James B. Mickle, his·major adviser,·for his .supervision and constructive criti-cism during·the·experimental·work and .the.writing of this thesis. Appreciation also is· expressed to Dr. S. D •. Musgrave, Head of the Dairy Science Department·, who· was helpful in every way. The author wants to thank Mrs. Olive·Pryor, Mr.·MackTietz, and Mr. Don Haggerty, who helped with the experimental work. 659733 TABLE OF CONTENTS Chapter I,. INTRODUCTION AND LITERATURE REVIEW II. III. IV. METHODS RESULTS AND DISCUSSION. SUMMARY AND CONCLUSIONS REFERENCES iv . . Page 1 3 6 14 15 LIST OF TABLES Table I, Age, Calving, Estrous, and Health Information for Four Cows . ., . . . . . . . . . . . . . . . II. Lipase Activity in Milk and Blood of Individual Cows (Butyric Acid per Ml of Blood or Milk) •.•.. IIL Milk Production of Cows on Afternoons of Sampling IV. Analysis of Variance for Milk Lipase Activity Data LIST OF FIGURES Figure L Lipase Activity of.Milk and Blood. Cow. 561. 2. Lipase Activity of Milk and .Blood. Cow 477. . 3, Lipase Activity of Milk and Blood .. Cow .488. . 4. Lipase Activity of Milk and Blood. Cow 564. • V Page 5 9 11 13 Page 14 14 15 15 · CHAPTER I INTRODUCTION AND LITERATURE REVIEW Rancidity is a disagreeable flavor defect which sometimes occurs in raw milk. This actually is a combination of several flavors rather than a single one. It is caused by the enzYJile lipase which hydrolyzes milk fat producing free fatty acids that are responsible for the flavor defects. When it occurs, this flavor results inlost sales; thus it is of importance to the dairy industry in Oklahoma and.throughout the nation .. A great number of papers have been published about this flavor defect, most of which have no bearing on the present study; however, they have been reviewed by Johnson (6) and by Kolar (8). Several workers have concluded that the tendency for raw milk to develop rancidity is a function of the individual cow and that this ten~ dency varies · from day-to-day in . the · same· cow ( 2, .. 5) • Al though much has been accomplished recently towards understanding.the actions of lipase. in other systems, little progress has been made toward understanding the causes for the.differences inmilk lipaseactivity among cows. It is known that the lipase activity of the liver is higher in a laying. hen than it is in an unmature bird . .(4). In addition,. lipase activity inthe·mammary gland of guinea pigs has been found to increase markedly at parturition (9, 10), although Baldwin.has reported no such increase in the mammary tissue of cows (1) •. Although these.literature reports provide no clear-cut answers, it appears that there might be 1 2 some relation between the reproductive functions of,the animal body and. the lipase activity of certain.of its components. In.view of this, a study was made·at OSffof the relationship between estrous cycles of dairy cows and blood serum lipase activity.(3). In connection with this study, the relation between blood and milk lipase activity was measured. These variables and their.relation to rancid flavors are the subject of the present report. CHAPTER II METHODS The four cows used in this work were fed the same ration and were housed under similar conditions throughout the experiment (3), Informa-tion concerning ages, calving, estrus, and health for these animals is summarized in Table I. Sampling was started the first time a cow was observed in heat after she went on experiment. Blood and milk samples were obtained once a week during the middle of the estrus.cycle, and daily samples were taken starting two or three days before the day of observed heat and ending two or three days after the day of h~at. Weekly samples of blood and milk were taken after the cows were bred, at the end of the third or fourth estrous cycle. When taking samples, 200 ml aliquots of the afternoon milking were cabled to 0-SC in less than 5 minutes and held at that temperature until analyzed two days later. Aftermilking, 50 ml blood samples were taken from the jugular vein of each cow and.put into centrifuge tubes contain-ing 5 ml of a 3.2% sodium citrate solution. These mixtures were held at 0-SC until analyzed two days later •. Blood lipase activity was deter-mined by the procedure of Kern et al. (7), as modified by Haggerty (3). The procedure for determining milk lipase activity is summarized below. 3 1. Twenty--five ml of skim milk (obtained by centri-fuging the samples) were added to 25 ml of 0.2M "Tham" buffer at pH 9.61 and 2 ml of tributyrin substrate.2 2. Duplicate determinations were prepared fc;>r each milk sample. The lipase in one.duplicate, marked "blank," was.inactivated immediately by adding H Cl to reduce the pH. to 2 .• 0 or less. The other duplicate, marked "test," was incubated one hour at-37C in a shaking water bath, after which it was also inactivated with H Cl. 3. The fatty acids in both· determinations. (blank and test) were extracted with ether, then titrated with KOH to a tbymolphtholein end-point. The results were·corrected for the. blank, then expressed as grams.of butyric acid released per ml of skim milk. An effort was made to relate these milk lipase activity values to the acid degree value (ADV) milk and to its rancid taste. ADV values 4 were determined by·the procedure of Thomas et al. (11) and·tasted by two or three "trained" judges. 1A .buffer pH of· 8. 5 was used in prelii;ninary .. work which. caused highe1r:,:resul:~s ·l\'than'·;,were·:'.,ebtai-ned:::W!f.1tb;~.~he~pB·.:9\6~;ba,ff er.t·:;-- However, the · differenc:es~·obtained 'With·, the two buffers were·,_ consistent; thus, either buffer could be us·ed ···to· demonstrate• differences ,among. milk samples. 2This s:ubstrate'contained 15% tri.buty~i~-~~d,30%.bovine albumin (Fraction V from Sigma Chemical ·co~) .. : The mixture was warmed then homogenized·until a'stable·emuls±on'was.formed. 5 TABLE I AGE, CALVING, ESTROUS, AND,EEALTHa INFORMATION FOR FOUR COWS Identification Calving. Age b Date of Number Breed Date Years-Months 1st Heat 477 Jersey .. 9/21/66 5-2 10/26/66 488 Ayrshire 9/6/66 5-11 10/21/66 561 Ayrshire 9/19/66 4-1 10/11/66 564 Ayrshire 9/22/66 4-0 10/20/66 aHealth Records: 488 - Antibiotics 10-12 and 13 Glucose and B-12 11-7, treated for bloat 12-13. 561 - Antibiotics mastitis 9~20 and 2-2. 564 - Antibiotics mastitis 1-1 and 1-18. b Age on October 1, 1966. CHAPTER III RESULTS AND DISCUSSION The graphs (Figures I-,,IV) of the lipase dat~ (Table II) show maxi"." mum blood lipase values within.24 hours of.the day when the cow was observed to be in "heat." As. Haggerty has observed (3), the relation-ship between observed heat and .high blood lipase activity would probably have been more exact·had samples been.taken more often than once a day. These high lipase -values associated,.with heat .were preceded by low lipase values--the minimum values.occurring one or two days before estrus. In addition, the maximum v.alue of heat was followed by low lipase values with the minimum.occurring one or two days after estrus. Blood lipase activity values during the middle of the estrus cycle.were erractic; apparently the hormone actively associated with estrus was not the dominant factor duri~g this part.of the cycle. Nearly all of the changes _in .blood .lipase activity were. reflected by similar changes in milk lipase.activity. The relative lipase activ-ity of the milk was much greater .. than. that·.of · the blood,· however; so_ the changes were magnified in the.milk •.. The relationship between milk and blood lipase was·not exact in.terms.of.time, and.changes often occurred .in the blood the day before.they.occurred in the milk. The milk lipase data of the first.two heat periods were analyzed statistically with an analysis.of ,variance technique. For this; the maximum lipase values obtained within 24 hours of observed heat were 6 7 chosen together with the minimum values occurring one or two days before and after the day of heat. One milk,lipase value representing an.approx-imate average of the middle of the .. cycle also was chosen, These calcu-lations. (Table IV) indicated a significant difference between periods of the cycle (P<O. 01). Most. of this .difference could be accounted for by the variance between the low lipase.activities before and after heat, as compared to the high values obtained.during heat and during the middle of the cyc.J_e. There was a difference among cows (P<0.05), and the values obtained during the second.estrus cycle were higher than those obtained during the first cycle {P<0.05). The remainder of the data indicated that this gradual increase in lipase activity continued even after the cows had been bred •. When considering all the data, .Cow 477 appears to have a higher average milk lipase activity than.the other three cows, This animal also had the best health record (Table.I). She was the only Jersey on the experiment,\ the other three.animals being Aryshires. As such, her average milk production was somewhat.lower than the other three animals (Table III) and the average fat.content of her milk was higher. These data cannot demonstrate whether these,factors caused the observed dif-ferences in lipase activity .. However, their relationships might be interesting subjects for future study .. An effort was made to relate the.lipase values of this study (with tributyrin substrates)· to the taste.of the milk and to its "acid Degree Value" (ADV). This latter value is .a.measurement of lipase activity used in some of the earlier.research on.rancid flavors (6, 11). Although 31 different milk samples were.tested, none of these tasted rancid; so no positive correlation could be demonstrated. However, all the samples 8 had ADV values of less than. 3.0.and butyric acid values of less than 30. Thus, values higher than· these appear to· be necessary before the milk will taste ranGid. Date Oct, 11 17 20 21 22 23 24 26 27 28 29 30 31 Nov. 1 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23 26 27 28 29 30 TABLE II LIPASE ACTIVITY IN MILK AND BLOOD OF INDIVIDUAL COWS (BUTYRIC ACID PER,ML OF BLOOD OR MILK) COW NUMBER 477 488 561 564 Blood Milk Blood Milk Blood Milk Blood (g X 10,000) 5.1 18.8 5.1 20.3 5.1 21.5 6.8 6.4 19.4 8.6 5.5 25.7 5.5 24.7 5.9 21.5 6.7 9.3 28.1 8.2 25.7 5.4 26.7 5.7 23.1 7.5 28.3 6.7 19.7 8.0* 26.7 6.7 16.9 9.3 6.7 21. 7 10.0 10.3 30.6 5.1 23.2 5.9 27.4 8.7 4.6 25.2 5.7 5.9 27.3 8.0 7.7 28.0, 4.9 27.4 5.9 15.9 5.9 5.7 6.2 5.9* 21.5 8.5* 8.0 31.3 7.2 21.5 8.5 18.7 8.0 8.0 26.2 6.4 22.5 8.2 9.3 25.0 8.2 28.3 7.5 18.3 8.0 28.3 7.2 6.2 20.8 6.4 8.5 28.0 7.7* 24.8 8.7 24.6 6.2 ,22. 7 8.5 7.2 7.7 8.0 8.7* 27.9 6.4 27.4 8.0 25.5 7.2 24.3 10.0 8.2 27.0 8.0 24.1 6.4 8.0 9.0 29.0 5.9 10.3 5.4 25.5 12.3 6.7 21.9 10.7 9.2 27.4 8.5 26.2 8.0, 24.8 11.5 9,0 25.7 12.0 7.7 26.7 9 Milk 19.0 27.3 25.7 22.5 18.7 20.1 26.0 23.4 22.7 21. 7 20.4 19.0 18.7 23.9 23.1 24.1 21.1 24.3 22.4, 26.7 24.8 23.5 25.1 10 TABLE II (Continued) COW NUMBER 477 488 561 564 Date Blood Milk Blood Milk Blood Milk Blood 1'illk -·-(g X 10,000) Dec. 1 8.2 24.6 7.5 24.6 10.5 19.2 2 6.5 26.5 8.7 19.0 3 11.2 27.9 5 7.2 28.4 8.5 26.9 10.0 26.3 6 9.0 28.0 7 10.2 29.4 8 9.3* 32.6 7.2 8.4* 22.9 12.6 26.5 9 7.8 27.5 5.1 22.9 10 7.5 32.2 8.4 29.4 12 10.5 32.4 9.0 30.4 9.0 23.0 9.6 25.5 15 8.1 24.3 16 5.0 25.7 8.6 19.5 17 5.3 25.5 7.4 20.3 18 5.6 23.7 19 8.0 21. 7 5.0 23.7 20 7.7 26.7 5.0 23.5 6.8 20.3 8.0 25.9 21 5.7 26.0 8.0 22.1 22 24.9 9.5 17.1 28 8.6 29.0 7.4 23.3 Jan. 3 7.4 33.2 5.6 30.8 8.9 25.7 9.5 25.7 11 8.3 29.3 6.2 19.5 8.0 23.5 7.1 21.5 17 8.0 24.6 8.0 28.1 8.3 20.5 7.7 21.4 24 8.2 31.4 7.2 32.0 6.9 24.0 6.5 19.3 31 7.9 34.7 6.9 22.6 7.9 23.8 9.2 24.0 Feb. 8 13.7 29.0 8.4 26.9 8.1 20.5 10.6 21.0 14 6.2 26.0 5.9 28.6 6.2 27.7 11.5 27.1 21 9.3 27.1 7.2 27.7 8.4 23.7 10.3 22.2 28 7.8 29.0 5.9 25.6 10.6 22.7 10.6 25.0 Mar. 7 8.4 32.0 10.5 22.9 8.4 26.5 9.0 26.7 14 7.5 30.5 11.2 26.0 10.3 23.1 6.8 18.0 21 8.0 34.3 8.2 29.4 11.2 32.3 8.0 19.3 28 8.0 25.3 8.5 19.3 9.8 20.7 6.9 19.3 Apr. 4 9.8 32.3 8.0 21.8 9.8 24.8 9.2 23.1 11 TABLE III MILK PRODUCTION oF·cowS,ON;A.FTERNOONS OF SAMPLING COW NVMBER ~ 477 488 561 564 lb Oct. 11 23.0 13 17 27.0 20 29.0 23.0 21 27.0 22 26 .o. 23 25.0 24 36.0 30.0 24.0 26 14.0 28.0 27 22.0 29.0 29.0 28 22.0 31.0 29 28.0 30 29.5 31 21.0 28.0 24.0 Nov~ 1 28.0 23.5 2 28.0 28.5 3 19.0 27.0 27.5 24.0 5 32.0 22.0 6 21.0 18.5 7 21.0 12.0 27.0 29 .o · 8 24.0 32.5 9 19.0 25.0 10 20.0 23.0 26.5 25.0 11 20.5 26.5 31.0 12 22.0 13 18.0 21.5 14 19.5 36.0 29.0 28.0 15 21.5 26.0 16 21.0 23.5 17 18 19.0 19 17.5 29.0 24.0 20.5 20 20.0 21 24.5 23 21.5 12.0 24.0 26 28.5 25.0 27 18.0 18.0 28 18.0 25.0 22.5 24.0 29 28.5 29.0 30 30.0 25.5 12 TABLE III (Continued) COW NUMBER Date· 477 ~ 561 564 lb Dec. 1 20.0 23.0 24.0 2 24.0 28.0 3 25.0 5 27.0 19.0 23.0 6 19.0 7 20.5 8 17.0 16.0 23.0 9 17.0 19.0 10 16.0 21.5 12 19.0 26.0 21.0 19.0 15 6.0 16 16.0 20.0 17 18.5 20.0 18 20.0 19.0 19 28.0 20.0 20 15.5 28.0 20.0 19.0 21 20.0 19.0 22 24.0 19.0 28 13.0 17.0 Jan. 3 12.5 21.0 18.0 18.0 11 11.5 19.0 12.0 16.0 17 14.1 19.0 16.5 18.0 24 14.0 20.5 15.0 17,5 31 14.0 16.0 14.0 15.0 Feb, 8 13.0 21.5 16.5 14.0 14 16.0 22.0 15.0 15.5 21 15.0 21.0 14.5 14.0 28 13.5 21.0 13.5 16.0 Mar. 7 12.0 18.0 13.0 12.0 14 12.0 19.5 13.0 12.5 21 12.0 19.0 13.0 6.5 28 13.0 14.0 11.5 11.0 TABLE·IV ANALYSIS OF VARIANCE FOR MILK LIPASE ACTIVITY DATA Degrees Sum of of Source Squares Freedom Total 311.50 31 Estrus Cycles 23.80 1 Cows 125.03 3 Periods .of Cycle 76.20 3 (Before and After heat VS, other two periods) (75.65) (1) Interactions Estrus x Cows 2.a1, 3 Estrus x Periods 2.06· · 3 Cows x Periods 52.25 9 Estrus x Cows ·x Periods (error) 29.35 9 aP<0.05 bP<0,01 13 Mean Squares F 23.80 7.30a 41~68 12.79 b 25.40 7.79b 75.65 23.21b 0.94 0.29 0.69 0.21 5.81 1. 78 3.26 Lipase Activity 40 30 20 10 0 --~ ' _."\ ,, n J~\ I,----- \ \. / \ , Iv ... , ,, _, \. I\ I ..._ \ . \ ,,-, fa ,.J ,I J Oct. h J, Nov, Dec. --- Milk Blood Jan~ Figure 1. Lipase Activity of Milk and Blood. Cow 561. Lipase Activity 40 --- Milk Blood 30 !I""" ,,-.... ,,. _,, -- -, ,,., . ', .,,, ' ... , ~ , r, ,.. ,.. ... ., , , ,,. '- 1 I \• ',' • .,, .._ ._ .,. ,, - ., ' ... ., 20 10 0 Oct. Nov. Dec. Jan. Figure 2. Lipase Activity of:Milkand Blood. Cow 477. h. Day of observed heat. 14 Lipase Activity 40' .. 30 · --- Milk Blood. ,. " . .,,. - -, "'"' ' "' " ,. ' \ 15 ' ~ \ _, - - ""\ I ..f . \ . ,, ,'' . .... . - - , ,........ ' . V . . ./ .... - - I , ... ---20 10 0 Oct. Nov. Dec. Jan. Figure 3. Lipase·Acf;ivi-tyof Milk and·Blood. Cow 488. Lipa~e·Activity 40 30 · 20 10 0 --- Milk Blood ,", ---\ , ... .. " -... ~ ~ -.... - - -, , I I \ I \ _, ' '· ',,: ol \J ,., \ I -..... --- ._,, ~.,,. " It Oct. Nov. Dec. ! . " Jan. Figure 4. Lipase Activity of Milk and Blood •. Cow 564. h. Day of.observed heat. CHAPTER IV SUMMARY AND CONCLUSIONS The purpose of this experiment.was to measure the relationship between milk and blood lipase.in individual cows. The-relationships of these two variables to the· estrus;cycles of the cows and the rancid flavors in their milk also were observed. Milk and blood samples were taken from four cows throughout .. their .lactation. Daily samples were obtained from each cow starting two .or three days before each day of "heat" and. continuing until two .or .. three days after the day of heat. Weekly samples were taken during .the .middle of each estrous cycle, and this weekly sampling continued after the cows were bred at the end of the third or fourth estrous cycle. High blood lipase values occurred within 24 hours of-observed "heato" These maximum··values were bracketed by lower ·values with mini-mums -occurring one. or· two days.before .and one or two days after the day of "heat," Changes in blood lipase.activity were reflected, and magni-fied, ip. the milk, although these .. changes· often occurred a day after they were observed in the·blood. Most of the milk samples tested in this study were not rancid; however, milk· lipase activities higher than 30 (units of butyric acid) might indicate potential rancidity. 16 (1) Baldwin, R, L. 1966. Several Species." REFERENCES "Enzymatic Activities in Mammary Glands of .d_. Dairy Sci., 49: 1533. (2) Cannon,· R. Y., and Rollins, G .. H. 1964. "Effect of Source of Nutrients and Nutritional Level-of the Cow on Development of Hydrolytic Rancidity in Milk." J. Dairy Sci., 47: 41. (3) Haggerty, D, M. 1967.· "Influence.of Estrus Cycle on Level of Blood Plasma Lipase Activity." M.S. Thesis, Okla. State Univ. (4) Hawkins, R. A., and Heald, P .. J., 1966. "Lipid Metabolism and the Laying Hen IV Synthesis of.· Triglycerides by Slices of Ovian Liver· in Vitro." Biochim Biophys Acta., 116: 41. (5) Jensen, R. G, 1959. "Spontaneous Lipolysis in Milk Throughout the Stages of Lactation of Individual Cows." .d_. Dairy Sci., 42: 1619. (6) Johnson, P. E., and Von Gunten, R. L. 1962. "A Study of Factors. Involved in the Development of Rancid.Flavor in Milk." Okla. Ag.· Expt. Sta. Bul., B-, 593. (7) Kern, F.; Jr., Steinmann, Laura, and Sanders, B. B. 1961. "Mea-surement of Lipoprotein Lipase Activity in Post Heparin Plasma: Description of Technique." ·l_. Lipid Res., 2: 51. (8) Kolar, C. W., Jr, 1960. "Relationships Between Certain Short-Chain Fatty·Acids, Total Fat Acidity and Rancid Flavors in Milk." M.S. Thesis, Okla. State Univ, (9) McBride, 0. W., and Korn, E. D. 1963. "The Lipoprotein Lipase of Mammary Gland and the Correlation· of its Activity to Lacta-. tion~" J. Lipid- Res., 4: 17. (10) Robinson,·D, S. 1963. "Changes in the Lipolytic Activity of the Guinea Pig Mammary Gland at Parturition." .d,, Lipid Res., 4: 21. (11) Thomas, E. L., Nielson, A. J,, and Olson, J, C., Jr, 1955. "Hydrolytic·Rancidity· in Milk." Amer.~ Rev., 17:1:50. 17 VITA Haskel C+ Pickett Candidate for the Degree of Master of Science Thesis: LIPASE ACTIVITY IN BOVINE MILK AND BLOOD Major Field: Dairy Science Biographical: Personal Data·: Born February .14, .1921, at Atlee, Oklahoma, Education: Finished grade school at Cyril, Oklahoma; high school at Lavaca, Arkansas; B .. s. degree at Oklahoma State University; M.S. at Oklahoma University; and a candidate for Master of Science degree from Oklahoma State University on July 28, 1967; 

Lipase Activity in Bovine Milk and Blood

https://shareok.org/bitstream/11244/26450/1/Thesis-1967-P597l.pdf

Lipase Activity in Bovine Milk and Blood

Abstract

Similar works

Full text

Available Versions

SHAREOK repository