milk of northern fur seal: composition, especially carbohydrate and protein

Milk of Northern Fur Seal: Composition, Especially Carbohydrate and Protein

SHUN'ICHI DOSAKO, SHIN'ICHI TANEYA, TOSHIAK| KIMURA, TOSHIHIRO OHMOR|, HIROAKI DAIKOKU, NORIKO SUZUKI, JUN'ICHt SAWA, KAZUHIKO KANO,

and SUMIO KATAYAMA Technical Research Institute

Snow Brand Milk Products Co., Ltd. 1--1--2, Minamidai

Kawagoe, Saitama, 350 Japan

ABSTRACT

The milk of northern fur seal was analyzed with special interest in carbohydrate and protein. High solids (61%) and fat (45.6%) were characteristics of its gross composition. Fa t ty acid distribution showed that more than 22% of the fat ty acids had carbon chains longer than 20 and that approximately 70% contained one or more double bonds. Analysis of free sugars showed no lactose but 123 mg/100 ml of myo-inositol. In carbohydrates bound to casein, .99% of sialic acid and .2% of glucosamine were determined. The amino acid composit ion of casein showed higher t ryptophan, lysine, serine, and glycine than bovine casein, and lower arginine, proline, and leucine. In whey, arginine, threonine, and valine were higher, whereas lysine, aspartic acid, isoleucine, and leucine were lower than in bovine whey. Amino acid patterns in casein and whey were similar to those of cat milk. The appearance of casein micelles was similar to bovine casein micelles. The mean diameter, however, was larger (approximately 330 rim). Electrophoretic pattern of casein showed five major bands in addition to minor components. One of the minor components was a glycoprotein, probably K-casein-like protein. The major whey protein had a mobil i ty similar to that of bovine 13-1actoglobulin. No &-lactalbumin- like protein was observed.

I N T R O D U C T I O N

Comparative studies on the composit ion of

Received August 24, 1982.

milk from different species have been of evolutionary and genetic trends (11, 15, 33). Data have been accumulated on easily obtained milks, such as from domestic animals, pets, laboratory animals, and humans. However, little is known about milk composit ion of seals. Lack of detailed information makes rearing infant seals in zoos difficult.

Milks of seals, as well as of polar bears, have distinct characteristics: high solid and fat contents and low lactose (33). Milk of the California sea lion has neither lactose nor ~-lactalbumin (24, 14). The northern fur seal has little lactose in its milk (26); thus, the young of these species seem not to require lactose in their diets. Therefore, what is the major carbohydrate in milks of seals in rela- tion to their nutrit ional requirements?

Regarding protein, only few data have been available so far. The amino acid composition of whole protein from the northern fur seal (2) and of casein from the harp seal (18) are already known. The electrophoretic pat tern of casein from the California sea lion showed two major components corresponding to ~- and t3-caseins (24). The major casein from the northern fur seal, however, was/3-casein (2), although no electrophoretic pat tern was presented. Thus, more intensive studies on the milk of seals are required.

The authors had a chance to obtain milk from the northern fur seal in the development of infant formula for seals. This work was to obtain detailed information on milk of northern fur seal with special interest in carbohydrate and protein. In addition to gross composition, fa t ty acid distribution, and amino acid composition, the electrophoretic patterns of casein and whey, electron micrographs of casein micelles, and analyses of free and bound carbohydrates are presented.

1983 J Dairy Sci 66:2076-2083 2076

MILK OF NORTHERN FUR SEAL 2077

M A T E R I A L S A N D METHODS

Milk of the Northern Fur Seal

Females were captured by the Japanese Fishers Agency at the Northern Sea of Okhotsk in August, 1978. Immediately after they were shot, the breasts were partially cut away to collect the milk on compression. Milk was stored at - 20°C , and part of it was supplied to us. After we determined gross and mineral composition, milk was centrifuged at 15,000 × g for 30 min at 30°C to remove fat and small amounts of insolubles. Fat was subjected to fat ty acid determination. A part of the defatted milk was used for electron microscopic observation and amino acid analysis of the whole protein. The pH of the rest then was adjusted to 4.6 with 1 N HC1 to obtain casein (precipitate) and whey (supernatant) fractions. Casein further was washed twice and freeze-dried. Whey was neutralized with 1 N NaOH, followed by dialysis against distilled deionized water. Dialysate, after evaporation, was subjected to analysis for free sugars. Retentate was collected and freeze-dried.

Gross and Mineral Composition

Total solids were determined by drying at 105°C until weights were constant. Fat and protein contents were determined by the Roese-Gottlieb and Kjeldahl methods (N × 6.38), respectively. Ash was determined after burning at 530°C for 12 h in an oven. Carbo- hydrate content was estimated by subtracting fat, protein, and ash from total solids.

Sodium, potassium, calcium, magnesium, copper, zinc, and iron were determined by the atomic absorption method on a Shimadzu model AA--650 Atomic Absorption Spectro- photometer . Phosphate and chloride were analyzed by the methods of Allen (1) and Volhard (20).

Determination of Hemoglobin

For us to estimate the quanti ty of blood contaminated in the milk, hemoglobin in blood and milk was determined by the azide- methemoglobin method (29).

Fatty Acid Analysis

Lipids were extracted with a mixture of

chloroform and methanol (2:1). Extracts were washed with distilled deionized water and then evaporated according to the method of Folch et al. (6). Lipids were saponified with 20% KOH- ethanol. After we prepared methyl ester with HCl-methanol, specimens were subjected to gas-liquid chromatography on a Shimadzu Gas Chromatograph GC-5A. The 3 mm × 300 cm column was packed with 15% diethyl glycol succinate (DEGS). Nitrogen was carrier gas.

Determination of Free Sugars

Sialic acid was determined by the method of Warren (30). Neutral and amino sugars were analyzed with gas-liquid chromatography after tr imethylsi lylat ion according to Sweely et al. (27). The column was packed with 5% SE-30. A GC/MS (HITACHI 063 gas chromatograph-RMU 6M mass spectrometer) was employed for assignment of structure.

Determination of Carbohydrates Bound to Casein

Freeze-dried casein was hydrolyzed gently with .1N H2804 at 80°C for I h to release sialic acid. The hydrolysate was subjected to ion-exchange chromatography with Dowex-t (CH3COO-- type). Sialic acid released was determined by the method of Warren (30) with N-acetyl neuramic acid as a standard. Neutral and amino sugars were released by hydrolysis under conditions of 2 N H2SO4, 100°C, 6 h, and 4 N HC1, 100°C, 15 h, respectively. After trimethylsililation, neutral and amino sugars were determined with gas-liquid chromatography (27).

Amino Acid Analysis

Duplicate samples were hydrolyzed with 6N HCI at 110°C for 22 h prior to analysis on a Nippon Denshi JLC-6AH amino acid analyzer. Tryptophan was determined after hydrolyzing in the presence of 6N Ba(OH)2 at 110°C for 48 h (10). Cystine was determined as cysteic acid after performic acid oxidation (10).

Electron Microscopic Observation

Defatted milk was diluted with 50 mM cacodylate buffer (pH 6.1) and subsequently fixed with 2.45% glutalaldehyde. After staining positively with saturated uranyl acetate, the

Journal of Dairy Science Vol. 66, No. 10, 1983

2078 DOSAKO ET AL.

spec imen was shadowed with Pt-C. E lec t ron micrographs were taken th rough a HITACHI HU-12A e lec t ron microscope . The size of casein micelles was measured with a Shimadzu size f r equency analyzer SF-20.

Polyacrylamide Gel Electrophoresis (PAGE)

The SDS-PAGE and urea-disc-PAGE were wi th 10% (32) and 7.5% acrylamide gels con- taining 4.5M urea and m e r c a p t o e t h a n o l (21). Gels were s tained wi th Coomassie bril l iant blue (32) or Schiff 's reagent (7).

peak-1

'o 2 . . . . 2 0 60 80 100 120 Time (m,n)

Figure 1. Gas-liquid chromatograph of free sugar in milk of northern fur seal.

RESULTS AND DISCUSSION

Gross and Mineral Compositions

Table 1 shows gross and mineral composi - t ions. High solids and fat con ten t s were as already known. Results, excep t for phosphorus , agreed wi th A shwor th et al. (2). Carbohydra te con t en t may be inaccurate because errors encoun te r ed in the de t e rmina t ion of each cons t i tu te are accumula ted when calculated by difference.

the 10 ml or 29.7 ml f rom 100 ml of milk) con ta ined .73 -+ .02 g of hemoglob in per 100 ml. Blood con ta ined 15.3 + .3 g of hemoglob in per 100 ml. Therefore , if the hemoglob in was conf ined to the serum, the a m o u n t of b lood in milk was (.73 x 29 .7 /15 .3) or 1.4 ml per 100 ml. If, however , the hemoglob in was evenly d is t r ibuted in the water in the milk, then the

Contamination with Blood

Ten milliliters o f whole milk was de fa t t ed and centr i fuged at 135,000 × g for 1 h at 10°C to remove casein micelles. Serum (2.97 ml f rom

TABLE 2. Fatty acid distribution of milk from northern fur seal.

Fatty acid (wt%) (wt%) a

TABLE 1. Gross and mineral composition of milk from northern fur seal.

(%) (%)a (rag%) (mg%) a

Total solid Fat Protein Carbohydrate Ash

Na K Ca

61.0 65.6 45.6 54.2 12.4 9.2

2.4 .14 .6 .56

52.1 83.8 56.7

Mg 14.1 C1 119.1 P 119.3 Cu .37 Zn .90 Fe .35

80

124 53

10:0 .4 12:0 .3 .2 14:0 6.6 6.5 14:1 .3 1.1 15:0 .4 0 15:1 .4 . . . . br 16:1 .2 . . . . 16:0 20.0 16.0 16:1 10.3 9.4 17:0 .2 1.3 17:1 .9 . . . . 18:0 1.9 2.4 18:1 34.5 26.7 18:2 1.9 3.8 20:0 or 18:3 .2 17.4 20:1 6.8 4.1 20:2 1.0 . . . . 20:4 or 22:0 2.4 0 22:1 1.0 5.4 22:2 5.2 20:5 4.8 . . . . 24:0 .7 . . . . 22:5 1.5 . . . . 22:6 3.7 . . . .

aData from Ashworth et al. (2). aData from Ashworth et al. (2).

Journal of Dairy Science Vol. 66, No, 10, 1983


myoi nositol TMSOCH=C-CHOTMS 309 I

TMSOH__O+TMS OTMS 191

217

I,. I

2OO

265

I,, 1] 10~ I~ ~'7

, , , _ L . !

100 300 L00

M-15-TMSOH ~0"7 M.~

Ii ,M-TMSOH61~-I5 I .I llm

I I

500 600 m/e

northern fur seal peak- 1

I i I I I

100 200 300 400 500 d I

600 m/e Figure 2. Mass spectrograph of peak 1 appeared in gas-liquM chromatograph and authentic rnyo-inositol.

blood content of the milk was (.73 x 39/15.3) or 1.9 ml per 100 ml,

Fatty Acid Distribution

Fatty acid distribution in milk fat from the northern fur seal is given in Table 2. Fatty acid composition agreed with Ashworth et al. (2) except for a lower content of 18:3 acid. Only .3% of fatty acids had carbon chains shorter than 14 whereas the fatty acid content of chain length above 20 carbons was 22.1%. Approxi- mately 70% of total fatty acids had one or more double bonds. Characteristics are similar for whales, grey seal (5), cat, and dog (16).

Free Sugars

Figure 1 shows gas-liquid chromatography of free sugars in the whey. Although Schmidt et al. (26) suggested small amounts of lactose in the milk of the northern fur seal, no lactose was in our specimen. Little lactose has been reported in the milk of sea otter (13). Because the only peak appeared to be myo-inositoI based on retention time, GC/MS was performed to assign the peak to authentic myo-inositol. As in Figure 2, the peak coincided completely with authentic myo-inositol; its content was 123 mg/100 ml. Myo-inositol content of bear (12), rabbit, hamster, Guinea pig, and pig milks were


2080 DOSAKO ET AL.

33 to 56 mg/100 ml; contents in milks of rat, Polynesian rat, and cotton rat ranged from 61 to 92 mg/100 ml (4). Bovine and human milks, however, contain less myo-inositol: 4 to 11 and 14 to 33 mg/100 ml, respectively (19, 23). Although the content of myo-inositol varies widely, not only with species but with their diets and stage of lactation (4), the milk of the northern fur seal contains the highest amount among the animais reported.

No or low lactose in the milk of the northern fur seal is of interest with respect to nutrition of infants. Because myo-inositol promotes the growth of infants in mice, rats, or domestic fowl (17), remarkably high contents of m y o - inositol might be responsible for the growth of infants of the northern fur seal.

Carbohydrates Bound to Casein

Component sugars of casein from various sources already have been reported by Baker and Lauer (3). Carbohydrates bound to casein from the northern fur seal, however, have not been determined. Results are summarized in Table 3 along with results concerning free sugars. In our specimen, .99% of sialic acid and .2% of gtucosamine were determined, although no hexose was detected. Glucosamine seemed to be almost the same as in other species. Sialic acid, however, was high, next to that of polar bear or pig (3).

Amino Acid Composition

Table 4 shows amino acid composition of whole protein, casein, and whey isolated from the milk of northern fur seal. The amino acid composition of whole protein agreed with Ashworth et al. (2). Compared to the amino acid composition of casein from harp seal (18), casein from the northern fur seal had high amounts phenylalanine and proline and little serine and tryptophan. Tryptophan, lysine, serine, and glycine were lower than in bovine casein, whereas arginine, proline, and leucine were higher. A similarity of amino acid pattern is in caseins from cat and dog milk (22, 33) with the exception of somewhat less methionine in casein from the northern fur seal. The amino acid composition of whey, however showed higher arginine, threonine, and valine, and lower lysine, aspartic acid, isoleucine, and leucine than bovine whey. The amino acid

TABLE 3. Free and component carbohydrates in milk of northern fur seal.

Free s u g a r Component sugar Carbohydrate (mg/lO0 ml) (g/lO0 g casein)

Sialic acid nd a .99 Lactose nd . . . . Amino sugar nd . . . . Neutral sugar . . . . nd Galactosamine . . . . . 2 Myo-inositol 123 . . . .

and, not detected.

composition of whey was similar to that of cat whey (22), although glycine slightly low in whey from cat milk.

Casein Micelles

Figure 3 shows an electron micrograph of casein micelles from the northern fur seal. Spherical micelles were similar to those of bovine micelles. The micelles appeared to be composed of submicelles although detailed fine structure was not clear. Volume frequency distributions of micelles are in Figure 4. The mean diameter was approximately 330 nm. The

TABLE 4. Amino acid compositions of whole protein, casein, and whey from northern fur seal (g/100 g protein).

Amino acid Whole protein Casein Whey

Trp 1.75 .55 1.50 Lys 6.61 4.85 6.10 His 3.04 2.87 2.35 Arg 5.32 3.66 5.03 Asp 8.74 6.11 8.71 Thr 5.02 3.06 5.93 Ser 3.95 3.37 3.50 Glu 16.49 18.79 13.02 Pro 8.66 10.43 4.97 Gly 1.52 .38 2.37 Ala 3.95 2.73 4.67 Cys 2.05 .55 2.52 Val 6.46 4.54 6.18 Met 2.81 1.13 2.06 lie 4.33 3.91 3.61 Leu 10.11 9.94 8.06 Try 4.71 4.81 3.96 Phe 4.48 4.46 3.48



Figure 3. Casein micelles in milk of northern fur seal.

diameter of bovine casein micelles is approximately 100 to 150 nm (31) whereas micelles of the northern fur seal milk are two or three times larger.

Elect rophoret ic Patterns of Casein and W h e y

Figure 5 shows urea-disc PAGE patterns of caseins from bovine and northern fur seal. Casein from the northern fur seal had five major components (C], C2, C3, C4, and C5) in addition to minor components. Mobility of C1 was slightly faster than bovine ~sa-casein and

t ) ¢- ~D

O - 0~

E

Z.0

30

2o -1

I

o-L 0 332 664 996

D i a m e t e r ( r im) Figure 4. Volume frequency distribution of casein

micelles from northern fur seal.

~ - c a s e i n - - 4

l]-casein --~

as~-casein -~

-~. - ~ ~.Origin C5

c3

.,-,-~ ~ '" $2 : ...... C2

c St ~- ' Ct

A B C

Figure 5. Urea-disc polyacrylamide gel electrophoresis of caseins from bovine and northern fur seal. A: Bovine casein, B: northern fur seal casein stained with Coomassie brilliant blue, C: northern fur seal casein stained with Schiff's reagent.

that of C2 was between bovine &sa- and /3- caseins. There existed several minor components between C1 and C2. Mobilities of C3 and C4 were slower than bovine (]-casein and similar to mobility of human (]-casein (9, 28). Because mobility of (]-casein depends on not only charges but also on phosphate content (9, 28), C3 and C4 appeared to be /3-casein-like proteins with different amounts of phosphate. The C5 showed slow mobility, and no correspondence to any type of bovine casein. One of the most important caseins is K-casein, a glycoprotein that plays a major role in the stabilization of casein micelles in bovine (31) and human milks (35). The stabilization of caseins associated with formation of micelles in the milk of northern fur seal (Figure 3) suggested a K-casein- like protein. To detect glycoproteins, urea-disc PAGE was stained with Schiff's reagent, in Figure 5. Two bands ($1 and $2) were observed. Because staining with Schiff's reagent is sublet, a densitometric pattern is also given in Figure 6. Peak height for $2 was so small that it was doubtful to assign the $2 component a glycoprotein. The $1 component, however, seemed to be a glycoprotein, probably a K-casein-like protein. Most of the component carbohydrates (Table 3) might be bound to the S1 component. The mobility of the S1 component corresponded


2082 DOSAKO ET AL.

T T T $1 $2 Origin

Figure 6. Densitometric pattern of the northern fur seal casein stained with Schiff's reagent.

e- Origin

..4":

13'Lg

i e o- La

S C. Figure 7. SDS-polyactylamide gel electrophoresis

of c~-lactalbumin (A), bovine whey (B), and the northern fur seal whey (C).

to the minor c o m p o n e n t immedia te ly behind the C1 componen t , much faster than that o f bovine K-casein, probably due to the high con ten t of sialic acid (Table 3). Al though quant i f ica t ion was no t made, the con ten t of the $1 c o m p o n e n t appeared to be ex t remely low on the basis of the PAGE pat tern. The low K-casein-like prote in may be responsible for the larger mean diameter of casein micelles in the milk of nor thern fur seal (Figure 4) as the size of bovine casein micelles is larger wi th a decrease in K-casein (25).

Figure 7 shows SDS-PAGE pat terns of whey proteins f rom bovine and nor thern fur seal milks. The major i ty of whey prote in of the nor thern fur seal had a mobi l i ty similar to that of bovine ~-lactoglobulin. No band of mobi l i ty similar to that of bovine a- lac ta lbumin was observed, which agreed with the result that no lactose was de tec ted in our specimen (Table 3) because a- lacta lbumin is one of the subunits o f lactose snythetase (8).

ACKNOWLEDGMENT

The authors thank T. Sone o f our inst i tute for his encouragement . Technical advice and comment s f rom M. Shimizu, The T o k y o University, were greatly appreciated. The authors express appreciat ion to D. Hsieh, Harvard Medical School and Boston Children's Hospital, for his careful proofreading and comments .

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milk of northern fur seal: composition, especially carbohydrate and protein

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