Composition and oligosaccharides of a milk sample of the giant panda, Ailuropoda melanoleuca

Comparative Biochemistry and Physiology Part B 135 (2003) 439–448

Composition and oligosaccharides of a milk sample of the giant panda, Ailuropoda melanoleuca Tadashi Nakamuraa,*, Tadasu Urashimaa, Taiji Mizukamia, Michihiro Fukushimaa, Ikichi Araia, Tatsudo Senshub, Koji Imazuc, Tatsuko Nakaoc, Tadao Saitod, Zhiyong Yee, Hong Zuoe, Kongju Wuf a

Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan b Department of Animal Feeding and Management, School of Agriculture and Veterinary Medicine, Kitasato University, Higashi 23-35-1, Towada, Aomori 034-8628, Japan c Adventure World, Shirahama-cho, Nishimurome-gun, Wakayama 649-2201, Japan d Department of Bio Production, Graduate School of Agriculture Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai, Miyagi 981-8555, Japan e Chengdu Research Base of Giant Panda Breeding, Fu Tou Shan, Northern Suburb, Chengdu, Sichuan 610081, PR China f Chengdu Zoological Garden, Huan Xian, Northern Suburb, Chengdu, Sichuan 610081, PR China Received 18 June 2002; received in revised form 27 March 2003; accepted 31 March 2003

Abstract A milk sample from a captive giant panda (Ailuropoda melanoleuca), obtained at 13 days postpartum, contained 7.1% protein, 1.6% carbohydrate, 10.4% lipid and 0.9% ash. The ratio of casein to whey proteins was 5.0:2.1. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of the whey protein fraction showed the presence of at least two major proteins other than a-lactalbumin and b-lactoglobulin. SDS-PAGE and urea-gel electrophoresis showed that as-casein is not a major component. The proportions of triacylglycerol, cholesterol, cholesterol esters and phospholipid were 90.5, 5.3, 0.96 and 3.1%, of the total lipid, respectively. The dominant saccharide in the panda milk was Gal(a1-3)Gal(b1-4)Glc (isoglobotriose). The milk contained, in addition, lesser amounts of lactose, Gal(a1-3)Gal(b1-4)wFuc(a1-3)xGlc (fucosyl isoglobotriose), Neu5Ac(a2-3)Gal(b1-4)Glc (39-N-acetylneuraminyl-lactose), Neu5Ac(a2-6)Gal(b1-4)Glc (69-N-acetylneuraminyl-lactose) and Neu5Ac(a2-3)Gal(b1-4)wFuc(a1-3)xGlc. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Giant panda; Milk; Oligosaccharides; Structure; Casein; Whey proteins; Lipids; Fatty acid

1. Introduction The milk of all mammalian species can be assumed to contain all the nutrients required for the growth and development of the neonate. The components in milk are specific proteins and fats which are easily digested within the intestine of *Corresponding author. Tel.: q81-155-49-5567; fax: q81155-49-5577. E-mail address: [email protected] (T. Nakamura).

the young, as well as carbohydrates, mostly lactose and minerals and other biofunctional components. The proportions of these components in milk differ among mammalian species and are also different at different stages of lactation even within one species. Data on the composition of the milk of various species are of interest since it provides critical information on the nutritional requirements of the young and is essential for the design of milk formulas for infant animals that need to be

1096-4959/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1096-4959Ž03.00093-9

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hand-reared (Jenness and Sloan, 1970; Jenness, 1974). The giant panda is a rare and endangered mammal inhabiting bamboo forests of China. The adult animal has a body length of 120–150 cm and weighs 75–160 kg but the newborn infant is extremely altricial, its body length and weight being approximately 15 cm and 90–130 g, respectively (Nowak, 1999). At present there are fewer than 1000 surviving giant pandas, which should be protected both in the wild and in zoos. There are only a few previous reports which describe the gross composition of milk from the giant panda (Lyster, 1976; Hudson et al., 1984; Liu et al., 1997). There are very few opportunities for the collection of the milk of a species as rare as the giant panda and lactating pandas are difficult to milk, but further studies are desirable to establish accurate values for the various constituents and to permit comparisons between the milk of different individual animals. This paper provides information on the composition of a sample of milk from a captive giant panda and includes, for the first time, a detailed description of the chemical structures of its most prominent carbohydrates. 2. Materials and methods 2.1. Materials The milk sample (3 ml) was obtained in September, 2000, from a giant panda ‘Mei-Mei’ (6 years old) bred with another giant panda (‘EiMei’, 8 years old) in Adventure World, Wakayama Pref., Japan. Numbers of suckling were confirmed 13 times during 13 days postpartum. From 42 min after suckling at 13 days postpartum, the milk was collected by hand with no prior administration of a hormone such as oxytocin. In sampling, symptoms such as the mastitis could not be recognized in the mammary gland of a giant panda. 39- and 69-N-Acetylneuramyllactose were purchased from Sigma Chemical Co., St. Louis. 39-NAcetylneuraminyl-3-fucosyllactose was obtained from Seikagaku Co., Japan. Isoglobotriose was isolated from ovine, bovine and caprine colostrum (Urashima et al., 1989, 1991, 1994). Gal(a13)Gal(b1-4)wFuc(a1-3)xGlc was isolated from a sample of polar bear milk (Urashima et al., 2000). 2.2. Milk composition Milk protein was calculated from its nitrogen content on the whole milk, which was determined

by a micro Kjeldahl procedure. A factor of 6.38 was used to estimate the total protein (Cunniff, 1980). For determination of the lipid, an aliquot of the milk (250 ml) was extracted with chloroformy methanol according to the method of Bligh and Dyer (1959). The carbohydrate content was determined by the phenol sulfuric acid method (Dubois et al., 1956). Lactose was used as the standard (Marier and Boulet, 1959). To determine the ash content, 100 ml of the milk were incinerated in a muffle furnace at 550 8C for 6 h; the resulting ash was then weighed. The concentrations of calcium (Ca), potassium (K), magnesium (Mg) and sodium (Na) were determined by subjecting the ash to atomic absorption spectrophotometry. 2.3. Characterization of the milk protein Skim milk was obtained from the whole milk by removal of the upper layer after centrifugation at 3000=g for 30 min. The casein and whey proteins were prepared from skim milk by isoelectric precipitation. The casein and whey proteins were subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) with a 4.5% stacking gel and 15% running gel (Laemmli, 1970). Urea-PAGE (7 M urea) was performed according to the modified method of Melachouris (Melachouris, 1969; Ullrey et al., 1984). Gel concentrations were 4.5% for the stacking gel and 10% for the running gel. Casein was dissolved in the stacking gel buffer (0.062 M Tris–HCl buffer, pH 6.7) containing 7 M urea. The electrophoresis was done using bromophenol blue as a marker at 4 8C at a constant voltage of 100 V in the stacking gel and 200 V in the running gel. The gel was stained with Coomassie Brilliant Blue G-250. 2.4. Fatty acid composition Two-step single-dimensional thin-layer chromatography (TLC) of the total lipid was performed on a Silica Gel 60 plate (Merck); the solvents were chloroformymethanol (one-step; 95:5, vyv) and chloroformyacetoneymethanolyacetic acidy water (two-step; 50:20:15:10:5, by volume). Detection was done by spraying with 5% sulfuric acid in methanol and heating at 150 8C for 10 min. The fatty acids of each lipid class, separated

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Fig. 1. (a) SDS-PAGE comparison of whey protein and casein fractions of the giant panda milk with those of bovine milk. Lane 1: low molecular weight kits (Pharmacia), Lane 2: whey protein fraction of the giant panda milk, Lane 3: casein fraction of the giant panda milk, Lane 4: whey protein fraction of bovine milk, Lane 5: casein fraction of bovine milk. (b) Electrophoretic comparison of the casein of the giant panda milk with that of bovine milk using polyacrylamide gels containing 7 M urea (Urea-PAGE). The protein concentration of each sample was 1 mgyml. Lane 1: casein of bovine milk (10 ml), Lane 2: casein of bovine milk (5 ml), Lane 3: casein of giant panda milk (5 ml), Lane 4: casein of giant panda milk (10 ml). The gel was stained with Coomassie Brilliant Blue G250.

by TLC, were esterified in HCl–methanol (5 mly l) for 2 h at 125 8C (Nakano and Fischer, 1977) and analyzed using gas–liquid chromatography (Shimadzu 14A, Kyoto, Japan) with a 10% DEGS packed column (support: Shimalite W, 2 mm=2.6 m; Shimadzu) with nitrogen as the carrier gas (Fukushima et al., 1997). Identification and quantification of the fatty acids was performed by comparison of the retention time and area of each peak with those of standard fatty acid samples. 2.5. Analysis of oligosaccharides An aliquot of the milk (2 ml) was extracted with four volumes of chloroformymethanol (2y1, vyv). The emulsion was centrifuged at 4000=g for 30 min, and the lower chloroform layer and the denatured protein were discarded. The methanol was removed from the upper layer by rotary evaporation, and the residue was lyophilized as ‘carbohydrate fraction’. The carbohydrate fraction was dissolved in 2 ml of water and the solution was passed through a Bio Gel P-2 (-45 mm) column (2.6=100 cm). Elution was done with distilled water at a flow rate of 15 mlyh and 5 ml fractions were collected.

Aliquots (0.5 ml) of each fraction were analyzed for hexose with the phenol–sulfuric acid method and for sialic acid with the periodate-resorcinol method. Peak fractions were pooled and lyophilized. Fractions PND3, PND4 and PND5 (Fig. 2) were subjected to preparative TLC with acetoney2propanoly0.1 M lactic acid (2y2y1, vyv) as developing solvent. Their saccharides were detected by spraying with 5% sulfuric acid in ethanol and heating above a Bunsen burner. The components in these fractions with the following RLac values were purified by passage through a Bio Gel P-2 column under the same conditions as described above; PND3 with RLacs0.76, PND4 with RLacs 0.86 and PND5 with RLacs1.00. Each saccharide was subjected to 1H-NMR spectroscopy. The components in PND1 or PND2 were dissolved in 2 ml of 50 mM Tris–hydroxyaminomethane–HCl buffer (pH 8.7) and subjected to anion exchange chromatography using a DEAESephadex A-50 column (1.5=35 cm) equilibrated with the same buffer. The unadsorbed components were eluted with 300 ml of the buffer and the adsorbed components were then eluted with a linear gradient of 0.0 to 0.3 M NaCl in the buffer.

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2.6. 1H-NMR spectroscopy 1

H-NMR spectra were recorded in D2O (100.00 atom %D, Aldrich, Milwaukee, WI) at 600 MHz with a Varian INOVA 600 spectrometer operated at 293.1 K. Chemical shifts were expressed in parts per million down-field from internal 3-(trimethylsilyl)-1-propane sulfonic acid, sodium salt (TPS), but were actually measured by reference to internal acetone. 3. Results 3.1. Composition of the giant panda milk

Fig. 2. Gel chromatogram of the carbohydrate fraction from the giant panda milk on Bio Gel P-2 column (2.6=100 cm). Elution was done with distilled water at a flow rate of 15 mlyh and fractions of 5.0 ml were collected. Each fraction was monitored for total carbohydrate by the phenol–H2SO4 method (⽧) and for sialic acid by the periodate-resorcinol method (n).

Elution was done at a flow rate of 15 mlyh and fractions of 5 ml were collected. Aliquots (0.5 ml) of each fraction were analyzed for hexose. Peak fractions were pooled and lyophilized. The product was dissolved in 2 ml of water and passed through a Bio Gel P-2 column under the same conditions as described above. Peak fractions giving a positive result with the phenol–H2SO4 method were pooled and lyophilized. The components in PND-2, which eluted later than the void volume (20 ml) during anion exchange chromatography (Fig. 3), were further separated by high performance liquid chromatography (HPLC). The HPLC was performed using a Tosoh CCPM-II Intelligent pump with a TSKgel ˚ Amido-80 column (4.6=250 mm, pore size 80 A, particle size 5 mm, Tosoh Co., Tokyo, Japan). The mobile phase was 50 and 80% (vyv) acetonitrile (CH3CN) in 15 mM potassium phosphate buffer (pH 5.2). Elution was done using a linear gradient of acetonitrile from 80 to 50% at 40 8C at a flow rate of 1 mlymin. Eluted materials were detected by measuring the absorbance at 195 nm. The peak fractions of oligosaccharides were pooled, concentrated by rotary evaporation and dialyzed with distilled water using the MDS-8 microdialysis system (National Labnet Company, Inc., NJ, USA) followed by lyophilization.

The composition of the giant panda milk with respect to protein, lipid, carbohydrate and ash content is summarized in Table 1. Of the protein, the casein content was 5.02 gy 100 ml while the whey protein content was 2.08 gy100 ml. Fig. 1a compares the results of SDSPAGE of the whey protein and casein fractions of giant panda milk with those from bovine milk. They show that the giant panda milk is markedly different from bovine milk with respect to both the whey protein and the casein. The whey protein pattern from giant panda milk (Lane 2) showed a number of lightly stained zones whereas the bovine whey protein showed only two distinct bands of a-lactalbumin and b-lactoglobulin (Lane 4). The

Fig. 3. Anion exchange chromatogram of fraction PND2. A DEAE-Sephadex A-50 column (1.5=35 cm) equilibrated with 50 mM Tris–HCl buffer (pH 8.7) was used. Elution was done first with 300 ml of the same buffer and then with a linear gradient of the same buffer containing NaCl from 0 to 0.3 M. The flow rate was 15 mlyh and fractions of 5 ml were collected. The fractions were monitored by the phenol–sulfuric acid method.

T. Nakamura et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 439–448 Table 1 Milk composition in giant panda

Table 3 Assignment of 1H chemical shifts (ppm) of the neutral oligosaccharides PND3, PND4, PND5 from giant panda milk

Giant panda milka

Bovine milkb

78.37 21.63

87.0–87.7 12.3–13.0

Total protein Casein Whey protein

7.10 5.02 2.08

3.1–3.6 2.3–2.8 0.55–0.72

Carbohydrate

1.61

4.5–4.8

Lipids TG Sterol Sterol ester Phospholipids

10.38 9.39 0.55 0.10 0.32

Water Total solid

Ash Na K Ca Mg a b

3.1–4.0 3.06–3.95 0.01 Trace 0.02–0.03

0.94 0.084 0.167 0.132 0.016

0.7–0.8 0.035–0.050 0.140–0.155 0.115–0.125 0.011–0.014

Values are expressed as gy100 ml. Adapted from Walstra et al. (1984).

casein of the giant panda milk showed a prominent band, which corresponded to the b-casein of the bovine milk. The urea-gel electrophoretogram (Fig. 1b) shows more clearly the differences between the patterns of the caseins of the two species. as-Casein, which is a prominent component of bovine casein, was only a minor component of the panda casein fraction. Additionally, the mobility of the dominant component of panda casein, which was similar to that of bovine bTable 2 Fatty acid composition of giant panda milk Fatty acid

TG

CE

PC

PE

n-14:0 n-16:0 n-16:1(n-7) n-18:0 n-18:1(n-9) n-18:2(n-6) n-18:3(n-3) n-20:0 n-20:4(n-6) n-20:5(n-3) n-22:6(n-3) n-24:0

Trace 25.98 6.24 2.43 38.72 21.64 2.96 0.00 1.40 0.25 0.37 0.01

Trace 25.62 4.29 15.52 34.61 17.93 0.00 0.00 0.00 0.24 0.59 1.20

Trace 19.23 0.91 35.07 17.59 19.88 0.56 0.15 5.51 0.21 0.80 0.10

Trace 10.79 0.57 26.94 28.34 13.41 2.85 0.18 11.69 0.59 4.60 0.04

Total

100.00

100.00

100.00

100.00

Values are expressed as mol%.

443

Reporter Residue group

Chemical shifts (coupling constants, J) PND3 5.179 4.655 4.504 5.143 5.446 5.407

PND4 (3.9) (8.1) (7.6) (3.9) (4.2) (3.9)

5.225 4.669 4.524 5.146 –

PND5 (3.6) (8.0) (7.7) (3.8)

5.222 (3.9) 4.655 (8.1) 4.451 (7.8) – –

H-1

Glca Glcb Galb1-4 Gala1-3 Fuca1-3

H-4 H-5 H-6

Galb1-4 4.161 (2.9)a 4.184 (2.7)a Gala1-3 4.194 4.196 – Fuca1-3 1.190 (6.8)b

a J4,3, bJ6,5 PND3; Gal(a1-3)Gal(b1-4)Glc ± Fuc(a1-3) PND4; Gal(a1-3)Gal(b1-4)Glc PND5; Gal(b1-4)Glc

casein on SDS-PAGE, was lower than that of bovine b-casein on Urea-PAGE. The lipid content of our sample was found to be 10.4 gy100 ml (Table 1). Densitometry of a thin layer chromatogram of the lipids showed that triacylglycerols represented over 90% of the total lipids and that significant amounts of cholesterol, cholesterol esters and phospholipids were also present. The fatty acid composition of each of the four lipid classes is summarized in Table 2. The most prominent fatty acid of the triacylglycerols was oleic acid, followed by palmitic and linoleic acids. The carbohydrate content of our milk sample, as determined by the non-specific phenol–sulfuric acid method, was found to be 1.61 gy100 ml, which is much lower than that of bovine milk (Table 1). The oligosaccharides contained within the carbohydrate fraction are described in detail below. The ash content of our sample was 0.94 gy100 ml (Table 1). The milk concentrations of the major cations are also listed in Table 1. 3.2. Giant panda milk oligosaccharides The carbohydrate fraction from the giant panda milk separated into at least five peaks, named PND1 to PND5, during Bio Gel P-2 chromatography (Fig. 2). Sialic acid was contained in only two peaks, PND1 and PND2.

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The saccharides in fractions PND3, PND4 and PND5 were purified by TLC (Section 2.1) and characterized by 1H-NMR spectroscopy. The relevant chemical shifts are shown in Table 3. The 1 H-NMR spectrum of PND5 had the anomeric resonances of reducing a-Glc and b-Glc, and b(14) linked Gal at d 5.222, 4.655 and 4.451, respectively. Since the pattern was essentially similar to that of lactose, PND-5 was characterized to be Galb(1-4)Glc. The 1H-NMR spectrum of PND4 had the anomeric resonances of reducing a-Glc and b-Glc, and b(1-4) linked Gal and a(1-3) linked Gal at d 5.225, 4.669, 4.524 and 5.146, respectively. The spectrum had the characteristic resonance of H-5 of a(1-3) linked Gal at d 4.196 and H-4 of b(14) linked Gal, substituted at OH-3, at d 4.184. As the pattern was essentially similar to that of a39galactosyllactose, PND4 was characterized to be Gala(1-3)Galb(1-4)Glc (isoglobotriose). As the chemical shifts (Table 3) of the characteristic resonances of PND3 were essentially the same as those of Gala(1-3)Galb(1-4)wFuca(13)xGlc of polar bear milk, this oligosaccharide was characterized to be Gala(1-3)Galb(1-4)wFuca(13)xGlc. Its spectrum had the anomeric resonances of reducing a-Glc and b-Glc, and a(1-3) linked Gal, b(1-4) linked Gal and a and b of a(1-3)Fuc linked to reducing Glc at d 5.179, 4.655, 5.143, 4.504 and d 5.446 and 5.407, respectively, and H6 of a(1-3) linked Fuc at d 1.190. The shifts at d 4.194 and 4.161 were assigned to H-5 and H-4 of a(1-3) linked Gal and b(1-4) linked Gal, respectively. Fraction PND2 was subjected to anion exchange chromatography on DEAE-Sephadex A-50 (chromatogram in Fig. 3). The components which eluted at 65 ml were assumed to be negatively charged because they emerged in a retarded position in relation to the void volume. They were further separated by HPLC (Fig. 4). Six chromatographic peaks, named PND2-1 to PND2-6, were collected and characterized by 1H-NMR spectroscopy. As the chemical shifts (Table 4) in the 1H-NMR of PND2-2 were essentially similar to those of 39N-acetylneuraminyllactose, it was characterized to be Neu5Aca(2-3)Galb(1-4)Glc. The spectrum had the anomeric shifts of reducing a-Glc and bGlc, and b(1-4) linked Gal at d 5.221, 4.663, 4.532, respectively. The characteristic shifts at d 2.757 and 1.800 were assigned to H-3ax and H3eq of Neu5Aca(2-3), respectively, and the shift

Fig. 4. HPLC of fraction PND2. HPLC was performed using a Tosoh CCPM-intelligent pump on a TSKgel Amido-80 col˚ particle size 5 mm, Tosoh, umn (4.6=250 mm, pore size 80 A, Tokyo, Japan). The mobile phase was 50 and 80% acetonitrile in 15 mM potassium phosphate buffer (pH 5.2). Elution was performed using a linear gradient of acetonitrile from 80 to 50% at 40 8C at a flow rate of 1 mlymin. Peaks were detected by measuring the absorbance at 195 nm.

at d 4.114 was assigned to H-3 of b(1-4) linked Gal substituted by Neu5Aca(2-3) residue. The chemical shifts (Table 4) of PND2-4 were essentially similar to those of 69-N-acetylneuraminyllactose. It was therefore characterized to be Neu5Aca(2-6)Galb(1-4)Glc. The spectrum had the anomeric shifts of reducing a-Glc and b-Glc, and b(1-4) linked Gal at d 5.224, 4.668, 4.428, respectively. The characteristic shifts at d 2.711 and 1.745 were assigned to H-3ax and H-3eq of Neu5Aca(2-6), respectively. As the chemical shifts (Table 4) of PND2-5 were similar to those of 39-N-acetylneuraminyl-3fucosyllactose, it was characterized to be Neu5Aca(2-3)Galb(1-4)wFuca(1-3)xGlc. The spectrum had the anomeric shifts of reducing aGlc and b-Glc, b(1-4) linked Gal and a and b of a(1-3)Fuc linked to reducing Glc at d 5.178, 4.653, 4.501, 5.435 and 5.378, respectively. The shifts at d 1.763 and 1.796 were assigned to H3ax and H-3eq of a(2-3) linked Neu5Ac, respectively, and the shift at d 4.084 and 1.167 were assigned to H-3 of b(1-4) linked Gal substituted

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Table 4 Assignment of 1H chemical shifts (ppm) of the acidic oligosaccharides PND2-2, PND2-4, PND2-5 from giant panda milk Reporter group

Residue

H-1

Chemical shifts (coupling constants, J) PND2-2

PND2-4

PND2-5

Glca Glcb Galb1-4 Fuca1-3

5.221 (3.6) 4.663 (8.0) 4.532 (7.7) –

5.224 (3.8) 4.668 (8.0) 4.428 (8.0)

5.178 4.653 4.501 5.435 5.378

H-3ax

Neu5Aca2-3 Neu5Aca2-6

2.757 (4.7)a –

– 2.711 (4.7)a

2.763 (4.9)a –

H-3eq

Neu5Aca2-3 Neu5Aca2-6

1.800 (12.4b, y12.1c) –

1.745 (11.8b, y12.1c)

1.796 (13.7b, y11.3c) –

H-3

Neu5Aca2-3

4.114 (3.0)d

H-6

Fuca1-3

–

–

1.167 (6.3)e

NAc

Neu5Aca2-3 Neu5Aca2-6

2.030 –

– 2.028

2.029 –

(3.8) (8.0) (7.7) (4.4) (3.8)

4.084

PND2-2; Neu5Ac(a2-3)Gal(b1-4)Glc PND2-4; Neu5Ac(a2-6)Gal(b1-4)Glc PND2-5; Neu5Ac(a2-3)Gal(b1-4)Glc ± Fuc(a1-3) a J4,3. bJ3ax,4. cJ3ax,3eq. dJ3,4. eJ6,5.

by Neu5Aca(2-3) residue and H-6 of a(1-3)Fuc, respectively. Fractions PND2-1, PND2-3 and PND2-6 were not characterized by 1H-NMR spectroscopy since their quantities were too small. Similarly, the free oligosaccharides contained in fraction PND-1 could not be studied. 4. Discussion The protein content of our sample of giant panda milk was similar to a value of 7.05 gy100 ml reported by Hudson et al. (1984) for a sample collected at 8–9 months postpartum. Lyster (1976), however, obtained a value of 10.3 gy100 ml for a milky fluid collected from the mammary glands during a post mortem while Liu et al. (1997) reported that the protein content of giant panda milk was 6.34 gy100 ml at 1 day postpartum and approximately 5 gy100 ml from 5 to 15 days postpartum. The ratio of casein to whey protein in our sample (;2.4) was significantly different from that for bovine (4.0) milk, but similar to that for milk of the grizzly bear (2.5) and yezo brown bear (1.9) (Jenness, 1974; Ando et al., 1979; Walstra et al., 1984), which are phylogenetically

close to the giant panda (see below). Both SDSPAGE and Urea-PAGE indicated that the major casein component of the giant panda milk was bcasein, unlike bovine milk in which it is as-casein. The major component of human casein, also reported to be b-casein, has a lower mobility than bovine b-casein on Urea-PAGE because of a lower degree of phosphorylation (Groves and Gordon, 1970). The giant panda b-casein may similarly be phosphorylated less than bovine b-casein. The behavior of the whey protein of our panda milk on SDS-PAGE was similar to that reported by Lyster (1976) on starch gel electrophoresis. Evidently, the panda milk contains at least two major whey proteins other than a-lactalbumin and b-lactoglobulin. The slower mobilities of these proteins compared with those from bovine milk may be due to higher glycosylation. Although it was not possible at this stage to carry out accurate identification of each casein and whey protein band, our data illustrate that the proteins of giant panda milk are very different from those of bovine milk. This aspect will require further study. Previously published data for the lipid content of giant panda milk varied from 2.50 to 16.3 gy 100 ml (Lyster, 1976; Hudson et al., 1984; Liu et al., 1997). Liu et al. (1997) reported that the lipid

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concentration of giant panda milk decreased immediately postpartum, but then gradually increased after 5 days. Our value of 10.4 gy100 ml at 13 day postpartum therefore appears to be consistent with the observations of Liu et al. (1997). The phospholipid and cholesterol concentrations in our giant panda milk were much higher than those reported for bovine and human milk (Walstra et al., 1984), suggesting that these lipids may have special biological significance for infant pandas. The fatty acid composition of our milk sample was similar to that observed by Lyster (1976) with respect to the prominence of oleic and palmitic acids and the high proportion of unsaturated acids, but our sample contained more linoleic acid than that of Lyster (1976). The most prominent fatty acids in the milk of ruminants (cow, goat and sheep) are palmitic, stearic and oleic acids, but not linoleic acid. The staple food of the giant panda is bamboo, even though this species belongs to the Carnivora. It is of interest, therefore, that the fatty acid composition of giant panda milk is similar to that of a non-ruminant herbivore such as the horse, whose milks contain much palmitic, oleic and linoleic acid (Jenness, 1974). Our results confirm previous studies showing that the carbohydrate content of panda milk is low compared with that of most other species. Even so, the carbohydrate content of our milk sample, 1.61 gy100 ml, was considerably greater than the values of 0.16 and 0.298 gy100 ml reported by Lyster (1976) and Hudson et al. (1984), respectively, for the lactose content of giant panda milk, but these workers obtained their values using methods that were specific for lactose. Although lactose is the dominant saccharide in the milk of most mammals, there are many exceptions. For example, the milk of bears contains significant quantities of free oligosaccharides such as Gal(a13)Gal(b1-4)Glc (isoglobotriose) and Fuc(a12)Gal(b-4)Glc, in addition to lactose (Urashima et al., 1997, 1999, 2000). We used a non-specific method to determine the total content of the carbohydrate and the difference of over 1.3 gy100 ml between our value and the other two is probably due to the fact that the latter did not include saccharides other than lactose. This study, for the first time, provides detailed information on the saccharide components of giant panda milk. The major finding was that isoglobotriose was the dominant saccharide in our milk sample instead of lactose. Isoglobotriose has been

shown to be a dominant saccharide also in milk of the Ezo brown bear, the Japanese black bear and the polar bear. In this respect, therefore, the giant panda milk resembles that of bears. We were, however, unable to detect oligosaccharides containing A-(GalNAc(a1-3)wFuc(a1-2)xGal), B-(Gal(a1-3)wFuc(a1-2)xGal) or H-(Fuc(a12)Gal) antigens, or oligosaccharides containing lacto-N-neotetraose or lacto-N-neohexaose as core units, which are found in the milk of bears (Urashima et al., 1997, 1999, 2000). The giant panda (Subfamily Ailurinae) is phylogenetically close to bears insofar as it has been placed within the same family of Ursidae (Wilson and Reeder, 1993; Nowak, 1999); the saccharide patterns in bears and the giant panda thus seem to be roughly consistent with their phylogenetic relationship. Our panda milk contained more 39-N-acetylneuraminyllactose than 69-N-acetylneuraminyllactose. It is similar to the contents of sialyloligosaccharides in bovine milk but not in goat milk and human milk. 39-N-Acetylneuraminyl-3-fucosyllactose, which is found in human milk, was also observed in panda milk. On the other hand, oligosaccharides containing N-glycolylneuraminic acid, such as 39-N-glycolylneuraminyllactose which was major acidic oligosaccharides in ovine colostrum, were not found in the panda milk. It has recently been suggested that human milk sialyloligosaccharides serve not only as nutrients but also as anti-infection factors (Idota et al., 1995; Simon et al., 1997; Kawakami, 1997). The sialyloligosaccharides of giant panda milk may also fulfill this important function. The present results, together with previously published data, should aid in the design of milk replacers for infant pandas. Milk replacers are generally formulated from bovine milk fractions but it is clear that the composition of giant panda milk is very different from that of cow’s milk (Walstra et al., 1984). Peng et al. (2001) have recently reported that giant panda cubs fed on cow’s milk often fell ill and died, in contrast to cubs that were suckled on their mother’s milk. A possible reason for this is that bovine milk contains much more carbohydrate, i.e. lactose, than panda milk. It is known that infant kangaroos and other marsupials have a low tolerance for lactose insofar as they suffer from diarrhoea, dehydration and failure to thrive, and often die, when they are bottle-fed on cows milk or milk replacers of similar composition (Messer et al., 1989). These animals

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therefore have to be reared on special milk formulas that contain little or no lactose. In view of the low lactose content of giant panda milk, it is possible that panda cubs, like infant marsupials, have a low tolerance for lactose. It should be noted also that newborn giant pandas resemble newborn marsupials in being very altricial. Giant panda cubs that require hand-rearing might therefore benefit from being bottle-fed on low-lactose milk formulas similar to those being used for the handrearing of Australian marsupials. In view of the present results such formulas could be made even more suitable for giant panda cubs by supplementing them with small quantities of isoglobotriose and bovine sialyloligosaccharides. Acknowledgments We thank Dr M. Messer of the University of Sydney, Australia, for his invaluable suggestions and comments. This study was supported by a grants-in-aid for science research (B) from Japan Society for the Promotion Science. This study was partially supported by a grant from The 21st Century COE program (A-1), Ministry of Education, Culture, Sports, Science and Technology, Japan. References Ando, K., Mitsuomi, M., Isao, K., Kogo, Y., Katsumi, G., 1979. General composition and chemical properties of the main components of yeso brown bear (Ursus arctos yesoensis) milks. J. Coll. Dairying 8, 9–21. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cunniff, P. (Ed.), 1980. Official Methods of Analysis of AOAC International. sixteenth ed. The Association of Official Analytical Chemists, Washington, DC, pp. 10–33. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Fukushima, M., Matsuda, T., Yamagishi, K., Nakano, M., 1997. Comparative hypocholesterolemic effects of six dietary oils in cholesterol-fed rats after long-term feeding. Lipids 32, 1069–1074. Groves, M.L., Gordon, W.G., 1970. The major component of human casein: a protein phosphorylated at different levels. Archs. Biochem. Biophys. 140, 47–51. Hudson, G.J., Bailey, P.A., John, P.M.V., et al., 1984. Composition of milk from Ailuropoda melanoleuca, the giant panda. Vet. Rec. 115, 252. Idota, T., Kawakami, H., Murakami, Y., Sugawara, M., 1995. Inhibition of cholera toxin by human milk fractions and sialyllactose. Biosci. Biotechnol. Biochem. 59, 417–419.

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