Milk Science Vol. 52, No. 1 2003 Original Reports
Yak butter lipid composition and vitamins in comparison with cow butter lipids
Dhanapati Neupaney1), Shigefumi Sasaki2),Jin-boKim1), Makoto Ishioroshi1), and Kunihiko Samejima1) 1)Faculty of Dairy Science, Department of Food Science, Rakuno Gakuen University, Ebetsu-shi, Hokkaido, Japan 0698501 2)Hokkaido Food Processing Research Center, Ebetsu-shi, Hokkaido 0690836, Japan
Abstract: Fatty acid compositions, vitamin A, and tocopherols in the butter lipid of the purebred of yak animal were determined by GLC and HPLC. The cholesterol content of yak butter and lipid oxidation kinetics were investigated by GLC and gravimetric methods. The butter produced from yak milk exhibited a strong ‰avor. This was caused due to the presence of a high concentration of short-chain saturated fatty acids in this butter. The major fatty acids in yak lipids were 160, 181, 140, 180, 40and80 in descending order. Retinol content in yak butter was 402.3 mg/100 gm fat. Alpha (a) tocopherol in yak butter was higher (2.6 mg/100 gm fat) than in cow butter (1.9 mg/100 gm fat), although g tocopherol was signiˆcant in cow butter (440 mg/100 gm fat). Tocopherols ( band dforms) were found in insigniˆcant amounts in yak and cow butter. Cholesterol content in yak butter was pronounced (325 mg/100 gm fat) than in cow but- ter (223 mg/100 gm fat). The cow milk lipid oxidation rate was faster and higher compared to the yak milk lipid. Polar lipid content in yak butter lipid was higher than in cow butter lipid.
reported to be rich in fat (6～10),protein(5.5 Introduction: ),andminerals2). Yak milk fat comprises a major portion of the Himalayan people's food in- The yak is a large animal that looks like a walk- take. These people have been consuming yak but- ing shag rug and has been used for its milk since ter from ancient times as food, medicine to heal ancient times. People in the highlands of the wounds and they also use it to worship the gods Himalaya use yak milk for the production of due to its purity. In Himalayan areas there is no cheese, churpi, ghee, butter, buttermilk, and dahi regular supply of plant-originated oils, so the only (yogurt). oil utilized is yak butter and ghee in their daily diet. Yaks are found in the highlands of the Nepalese Yak butter is only deˆned by organoleptic Himalayas, Tibet, Indian Kasmir, Mongolia, and parameters such as taste, odor and color. Present- Bhutan up to the altitude of 5200 m above sea lev- ly, yak butter is being used in the Himalayas as a el. They can survive in temperatures as low as －40 primary ingredient to produce a variety of food °C with little atmospheric oxygen. The estimated items and diŠerent dairy products including butter yak population (Bos grunniens)1) in the world is tea. Thus, more emphasis must be given to the about 12 million. Their maximum life span is 25 study of yak milk fat and fatty acid composition for years. Yaks usually graze on grasses, herbs, and its wide range of use. lichens in meadows during summer season and Lipids serve as precursors for beneˆcial biologi- consume straw and dry leaves in the winter season. cally active compounds such as prostaglandins, Yaks have di‹culty ˆnding su‹cient dry grasses steroid hormones, and bile acids1). Although there during winter, so their milk production decreases. is controversy as to the role of saturated and trans The female yak gestation period is 9 months, and fatty acids in cardiovascular disease, the science she gives birth to a single calf. The female yak behind the eŠects of dietary fat on human health is yields a maximum of 1～2Lmilk/day. Its milk is so complex that there are no simple and compre- 第巻 hensive answers to the question in the ˆeld at the present time. Yak milk fat has a unique taste and Preparation of fatty acid methyl esters (FAME). speciˆc ‰avor with a high nutritive value as claimed by the Himalayan people. Yak cheese and Lipids were saponiˆed with 1 N alcoholic potas- butter are the major and most expensive milk sium hydroxide by heating at 90°C for 50 min. products sold in Nepalese market. Thousands of Saponiˆed and unsaponiˆed matters formed during Nepalese farmers are being engaged in yak milk heating were separated by diethyl ether and water. production due to the great market demand for yak Then the fatty acids were extracted from the mix- milk products. ture with diethyl ether. Fatty acid methyl ester The objective of this study is to investigate the (FAME) of yak or cow lipid was prepared by the fatty acid compositions and fat-soluble vitamins in addition of BF3 (7,v/v) in anhydrous methanol yak butter as well as yak lipid oxidation kinetics by heating at 100°Cfor15min.ThentheFAME and then compare with those of fresh cow butter was extracted, clariˆed, dried, and stored at －30 lipids. °Cbeforeuse.
Materials and Methods GLC of FAME
The yak butter was produced in the Himalayas Fatty acid methyl ester preparations were inject- of Nepal and the cow butter was prepared at the ed (1 ml ) using the split mode. The carrier gas was experimental farm of Rakuno Gakuen University, helium, and the split ratio was 1001 at 250°C. Japan. Selected purebred of yaks at mid-lactation Oven temperature was programmed for 70°Cfor4 period were chosen for milking. Yaks were grazed min, then increased from 70 to 175°Cat13°C/min, on mature meadows when milked for butter held at 175°C for 27 min, raised to 215°Cat4°C/min production. No commercial feed or medical treat- and ˆnally held at this temperature for 31 min. ment had been used for these yaks. Yak and cow Chromatograms were documented with a comput- butter was produced by traditional technology, ing integrator (Turbochrom Work Station).Stan- churning the cream in a wooden butter churn. dard fatty acid methyl ester mixtures were used to HPLC and GLC-grade n-hexane, acetic acid, calibrate the gas chromatograph system using methanol, sodium chloride, pyrogallol acid, ethy- reference standards (GLCReference standard, lene acetate were purchased from Kanto Chemi- fatty acid methyl esters: GLC90). cals Industries, Ltd., Japan. Identiˆcation of fatty acids was made by com- paring the relative retention times of fatty acid Extraction of yak and cow butter lipids: methyl ester and peak areas from samples with those of the standards. The FAME standard con-
Lipids were extracted from yak and cow butter tained C4 to C24 fatty acid methyl esters, which by a modiˆcation of the method of Bligh and were purchased from Nu-Chek Prep, Inc., USA. Dyer3). Yak or cow butter was dissolved in chlo- roform and methanol (21, v/v).Thechloroform HPLC method for tocopherols and vitamin A: was removed by rotary vacuum evaporator to ex- tract lipids. In the subsequent step the residual A measured amount of yak or cow lipid (1.5 gm) mass was mixed with chloroform, methanol and was mixed with 0.5 ml of (1,w/v) NaCl, 10 ml water (843, v/v). The composite mass was of (3,v/v) pyrogallol acid and 1 ml of (60,w/ sedimented and the lower phase containing lipids v) KOH. The mixture was heated at 70°Cina was collected. The chloroform portion was re- water bath for 30 min. Then the mix was cooled to moved from the lipid mass by a vacuum evaporator room temperature and combined with 22.5 ml of (1 and the extracted lipids were stored at －45°Cbe- ,w/v) NaCl and 15 ml of (19, v/v) ethylene fore use. acetate: n-hexane. The composite mix was cen- trifuged at 3000 rpm for 5 min and the upper layer 第号 was collected, evaporated, and concentrated. Fi- nally, the concentrated sample was dissolved with Polar and neutral lipid extraction n-hexane and used in the subsequent experiments. and identiˆcation The standard solutions of the vitamins for analyzing butter lipid samples were prepared to a The column chromatography technique was ap- concentration of 1 mg/ml. Normal-phase HPLC plied to separate the polar and neutral lipids in yak with UV detector (Hitachi, Co., Ltd),andcolumn and cow butter lipids. The TLC technique was ap- from Shimadzu, Co., Ltd, were used to analyze the plied to distinguish the quality of polar and neutral samples. The HPLC was set at 30°Candthe‰ow lipids. TLC plates were at ˆrst dried in hot air oven rate was ˆxed 1 ml/min using a mixture of at 110°C for 2 hrs. After heating, the plates were methanol and water (91, v/v) as an eluent. The cooled and 10 ml of each lipid was introduced onto eŒuent was monitored at 325 nm. Vitamin A, and the TLC plates and eluted with hexane, diethyl all trans-retinol (95) were purchased from Sigma ether, and acetic acid (80301, v/v). In the sub- Chemical Co., USA. Vitamin E which contained a sequent step, the plates were sprayed with 50 bgand dtocopherols was used as the standard. H2SO4 and heated indirectly at 180°Cfor30min. Tocopherols and retinol were quantiˆed with an The polar and neutral lipid bands in the TLC plate external standard method in which quantiˆcation wereidentiˆedbycomparisonwithstandard.Polar was based on peak areas. and nonpolar fatty acid standards were used from the stock of our laboratory chemical store. GLC procedure for yak and cow lipid cholesterol Fatty acid extraction and methylation of polar and neutral lipids were proceeded as mentioned el- Yakorcowlipidwassaponiˆedwith1Nalcohol- sewhere. ic KOH by heating at 100°C for 1 hr. In subsequent step, the mixture was cooled to room temperature Results and Discussion and combined with petroleum ether and water (1 1, v/v). The cholesterol containing lipid mass was Table 1 shows the fatty acid composition of yak extracted and dissolved with 1 ml n-hexane for and cow butter lipid. Major fatty acids in yak lipid GLC analysis. This fresh lipid sample was analyzed were 160, 181, 140, 180, 40and80. by GLC (Perkin Elmer Auto system―XL, USA). Among the fatty acids, palmitic acid in yak butter Helium gas was used as carrier with a 251split represented a maximum of 25.1 and it was more mode, and a ‰ame ionization detector was used. The column temperature was maintained at 200°C. The injector and detector were held at 250°C. The Table 1 Fatty acid composition of yak and cow butter lipid. peak area was integrated and compared with the Fatty acids Fatty acid in yak lipid Fatty acid in cow lipid values from the working standards of cholesterol to () () calculate the concentration of cholesterol in the 405.1 3.0 603.6 1.3 unknown sample. 804.8 1.8 1004.1 2.3 Estimation of auto oxidation of lipid 1204.5 3.1 140 9.3 13.5 The weight-gain method was applied to observe 1411.5 1.2 160 25.1 32.9 the changes in quality of yak and cow butter lipids. 1612.4 2.5 Yak and cow lipid samples (0.46±0.01 gm) were 1808.5 9.1 placed in small petri dishes and kept in an unlight- 181 18.3 19.7 ed condition at 40°C. The oxidation induction 2001.9 2.0 period in lipids was gravimetrically estimated by 2100.1 0.1 2200.1 0.1 the weight-gain method and deˆned, as the time re- Other 10.7 7.4 quired to increase the weight of lipid by 0.3. 第巻 abundant at 32.9 in cow butter. Yak and cow but- The alpha (a) tocopherol level in yak butter ter contained small amounts of 210 (0.1) and was higher (2.6 mg/100 gm fat) than in cow butter 220 (0.1). Cow butter contained low amount (1.9 mg/100 gm fat) although gtocopherol was of short chain saturated fatty acids (11.5) com- signiˆcant in cow butter (440 mg/100 gm fat). paredwithyakbutter(22.1). Yak milk lipid con- Tocopherols ( b and dforms) were not sig- tained 22.1, 38.3 and 28.9, short chain, medium, niˆcantly detected in yak and cow butter. The and long chain fatty acids, respectively. Cow milk tocopherol content in yak and cow butter is shown lipid contained 11.5, 50.1, and 31, short chain, in Table 2. Tocopherols act as antioxidants de- medium chain and long chain fatty acids, respec- pending upon the structure of fats and oils10,11). tively. This result was comparable with Engle et They are considered to be primary or chain break- al.4) and Ramaswamy et al.5) who reported the con- ing antioxidants in free radical chain reactions, centration of medium chain fatty acids 51.38 and which can convert lipid radicals to more stable 50.95, respectively. Milk fat has a relatively high products, thus extending the shelf life of edible fats content of short chain fatty acids, which are lack- and oils12). Among the eight naturally occurring ing in other natural food fat. The easy digestion of tocopherols, atocopherol has the greatest biologi- milk fat is due to the speciˆc geometrical arrange- cal activity in humans13,14,15). Tocopherols play a ment of short chain saturated fatty acids6),which role in the prevention of some chronic diseases can be easily digested compare with other fats7,8) such as heart disease and some cancers16). and immediately can be converted into energy in Tocopherols also play an important role in immune the liver and utilize in the body. response and reduce the propensity of platelets to Our HPLC results showed that yak and cow but- adhere to the blood vessel wall17,18). ter lipid contained 402.3 and 691.5 mgretinol/100 Cholesterol content in cow and yak butter is gm of butter, respectively (Table 2). Average vita- shown in Table 2. The GLC result showed that the min A content in cow milk is 126 IU/100 gm cholesterol content in yak butter was signiˆcant milk1,7). Retinoic acid is required for the transport (325 mg/100 gm fat) than cow butter (223 mg/ of mannose units in the synthesis of certain 100 gm fat). The GLC results also resolved peaks glycoproteins and it functions to regulate gene ex- of other sterols in less signiˆcant amounts. pression in antiviral, antitumor, and im- Cholesterol is a structural element of cell mem- munomodulatory processes9). Milk contains vita- branes and acts as a precursor of bile acids and min A in preformed condition, i. e., retinol and steroid hormones6,19). In plasma, cholesterol and retinyl esters, on the other hand, carotenoids are lipids occur as complexes with protein20).Improv- pro-vitaminAwhicharederivedfromplant ing the intake of essential fatty acids can control resources1). Humans need vitamin A for growth, excess cholesterol deposition in the body. Dairy reproduction, resistance to infection, maintenance foods are not high in either fat or cholesterol. In- and diŠerentiation of epithelial tissues, stability take of fermented dairy products in the diet also and integrity of membrane structure and the helps to reduce the level of cholesterol in the process of vision. body21). For normal functioning of the human body 1 gm of cholesterol is needed daily. The results of the present study show that the Table 2 Retinol, tocopherol and cholesterol content in yak yak lipid oxidation rate was slower compared to and cow butter lipids (mg/100 g lipid). cow lipid at 40°C in unlighted conditions. The Fat soluble components yak butter cow butter average oxidation rate in yak and cow butter lipid Tocopherol (T) at 40°C was 0.21 and 0.26 mg, respectively. The aT 2600 1900 bT ≦1 ≦1 results are shown in Fig. 1. The slow rate of oxida- gT 230 440 tion of yak butter lipids may be due to the presence dT ≦1 ≦1 of high concentrations of short chain saturated fat- Retinol 402.3 691.5 ty acids. The high concentration of tocopherols Cholesterol 325000 223000 may also contribute to prolong the shelf-life of yak 第号
Table 3 Polar and neutral lipids in yak and cow butter (wt. ).
Fatty Yak Cow acid Neutral lipids Polar lipids Neutral lipids Polar lipids
40 5.1 0.0 3.0 0.0 60 3.6 0.0 1.3 0.0 80 4.8 0.0 1.84 0.0 100 4.1 0.1 2.3 0.17 1100.04 ――― 120 4.5 14.3 3.1 20.88 140 9.3 11.5 13.5 11.97 141 1.01 0.01 1.2 0.1 Fig. 1 Oxidation kinetics of yak and cow butter lipids. 150 1.22 11.05 1.62 0.23 Yak and cow butter lipid samples were kept un- 160 25.0 25.36 32.9 26.25 der unlighted condition at 40°C, and the alterra- tion in weight was observed to analyze the lipid 161 2.5 3.3 2.9 0.18 oxidation pattern during time intervals. 170 0.52 0.45 1.03 0.23 180 8.5 15.33 9.0 12.05 181 19.0 5.04 19.0 17.01 182 2.84 0.49 2.02 1.9 lipids. Sensory and weight gain methods are most 183 1.17 0.13 0.15 ― accurate in predicting the stability of fats and 200 0.96 0.41 ―― lipids11). 201 0.008 0.004 ―― Neutral lipid content in cow butter lipid was 220 0.22 0.038 ―― higher (95) than in yak butter lipids (94).The 240 0.015 0.003 ―― polar lipid content of yak butter lipid was sig- ―not detected niˆcantly higher (3.89) in comparison to cow butter lipid (2.1). The results are presented in Table 3. Yak and cow butter exhibited less polar the yak is used for milking, and its butter is utilized lipid with short and long chain fatty acids. Sig- for food and medical purposes. The medical eŠects niˆcant amounts of polar lipids were detected be- of yak butter are still unknown. The Himalayan tween 120to181. people recommend drinking salted yak butter tea Yak butter was distinctly diŠerent in taste, to prevent high-altitude sickness. ‰avor and appearance in comparison to cow butter. At ordinary room temperature, yak lipid formed High cholesterol content and odor of yak butter a granulated structure and solidiˆed quickly. seems a remarkable obstacle to producing new Several researchers have reported that fatty acid foods using yak butter. The smell of yak butter is compositions in milk may changes due to animal quickly diŠused to the immediate surroundings feed, breed and incorporated minerals5,22,23).Frank when heated above melting point (40°C) and co- et al.24) reported that elephant and rabbit milk fat hered smell is detected for 810 hrs. The smell of contained 100 and 65, medium-chain fatty yak butter is thought to be awful by many foreign acids respectively. In yak milk medium chain fatty travelers in the Himalayas who and doubt on the acids were 38.3. We assumed that yak blood sanitation of local people. The results of this study must be adapted to high elevations for survival and concluded that the strong ‰avor of yak butter is grow under low atmospheric oxygen conditions. due to the high concentration of short chain satu- We also thought that the yak blood cells are small rated fatty acids. The results conˆrmed that the in size and numerous which may eŠect metabolic yak butter oxidation process was slower and less activities and also in‰uence the synthesis of biolog- pronounced than cow butter. ical compounds such as fatty acids. Dairy products are essential nutrients. The The ˆndings of this study may be helpful in major milk producing animals in diŠerent parts of designing a diet using yak milk for patients in the the world are the cow, buŠalo, camel, goat, sheep, Himalayas, as well as for the preparation of infant and mare. In the high altitudes of the Himalayas, and baby foods. We also hope that our results will 第巻
be useful in optimizing yak cheese production tech- York. 229 (1994). nology including cheese ripening, taste, ‰avor and 10) A. K. Muhamad and F. D. Shahidi: J. Food Chem., 74, texture controls. Food technologists thinking of 431 (2001). utilizing yak dairy products, as a primary in- 11) U. N. Wanasundara and F. Shahidi: J. lipids. 4,51 gredient for the production of new food products (1997). will also beneˆt from our ˆndings. 12) M. Yoshida, M. Tatsumi and G. Kajimoto: J. Am. Oil Chem. Soc. 68,566(1991). Acknowledgements 13) P. Salo-Vaananen, V. Olilainen, P. Matila, K. Lehikoi- nen, E. Salmela-Molsa and V. Piironen: Food Chemis- This research work was completed under the try. 71,535(2000). JSPS support by the Ministry of Education, Cul- 14) L. J. Machlin: Vitamin E. A Comprehensive Treatise. ture, Sports, Science and Technology (Monbusho) Marcel Dekker, New York. (1980). of Japan. 15) M. L. Scoot: Advances in Our Understanding of Vita- min E. Fed. Proc 39, 2736 (1980). References 16) H. Ihara, I. Hirro, Y. Shino, N. Hashizume, M. Takase, J. Nagao and Y. Sumiyama: Nutr.Sci.Vitam., 1) N. P. Wong: Fundamentals of Dairy Chemistry.3rd ed, 46,257(2000). VanNostrandReinholdCompany,NewYork.(1988). 17) D. B. McCormick and H. L. Greene: Vitamins. In: 2) D.Neupaney,J.Kim,M.IshioroshiandK.Samejima: Tiez Textbook of Clinical Chemistry.2nded.BurtisCA, Milk Sci., 46, No. 2, 95 (1997). Ashwood ER ed. Philadelphia. (1994). 3) E. G. Bligh and W. J. Dyer: J. Can. Biochem. Physiol., 18) P.Weber,A.BendichandL.J.Machlin:Nutrition. 37, 911 (1959). 13,450(1997). 4) T. E. Engle, V. Fellner and J. W. Spears: J. Dairy Sci., 19) J. R. Sabine. Cholesterol, Marcel Dekker: New York. 84, 2308 (2001). (1977). 5) N.Ramaswamy,R.J.Baer,D.J.Schingoethe,A.R. 20) H. G. Kirschenbauer: Fats and Oils. Reinhold Publish- Hippen: J. Dairy Sci., 84: 2144 (2001). ing Corporation. New York. (1960). 6) E. Renner: Ann. Intern. Med., 103, 944 (1983). 21) I. Satomi and K. Samejima: Milk Sci., Vol. 50. No. 3 7) L. M. Lampert: Modern Dairy Products.3rd ed, Chemi- 113 (2001). cal Publication Co. New York. (1975). 22) W. Seas: J. Dairy Sci., 71,3179(1988). 8) J. W. C. Porter: Milk and Dairy Foods.OxfordUniver- 23) J. D. Sutto: J. Dairy Sci., 72, 2801 (1989). sity Press. London. (1975). 24) D. Frank, J. Gunstone, L. Harrod and F. B. Padlex: 2nd 9) W. S. Blaner and J. A. Olson: In: The Retinoids; Biolo- ed, The Lipid Handbook. Chapman and Hall. (1994). gy, Chemistry and Medicine.2nd ed, Raven Press. New 第号
ヤクおよび乳牛バター脂質の構成成分とビタミン類の比較
ネウパネ ダナパテイ1)・佐々木茂文2)・金 辰保1)・石下真人1)・鮫島邦彦1) 1)酪農学園大学酪農学部食品科学科，2)北海道食品加工研究センター
海抜5200 m 付近の高地に棲息している純系のヤクのミルクから作ったバターの脂肪酸組成とビタミン A およびトコ フェロール含量を GLC（気液ガスクロマトグラフィー）と HPLC（高速液体クロマトグラフィー）で測定し，ウシバ ターのそれらと比較した。ヤクバターのコレステロール含量と脂質酸化速度は，それぞれ GLC と重量測定法によって 判定した。ヤクバターは，短鎖の飽和脂肪酸を多く含んでいるので強い匂いを有している。脂質中の構成脂肪酸は多い 順に160, 181, 140, 180, 40, 80 であった。また，レチノール量は脂肪100 g 中に402.3 mg であった。ウシ バターの gトコフェロール量は，ヤクバターよりも多く，脂肪100 g 中に440 mg であるが，aトコフェロール量は， ウシバター（1.9 mg/100 g 脂肪）よりもヤクバター（2.6 mg/100 g 脂肪）の方が多かった。b と dトコフェロールは ヤクおよびウシバター中にほとんど検出できなかった。ヤクバター中のコレステロール量（325 mg/100 g 脂肪）は， ウシのそれ（223 mg/100 g 脂肪）よりも多かった。ウシバター脂質の酸化は，ヤクのそれに対して速くしかもその度 合いは高い。極性脂質含量はウシに比べてヤクバターの方が多かった。