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Characterization of the edible bird’s nest the “Caviar of the East”
Food Research International 38 (2005) 1125–1134 www.elsevier.com/locate/foodres
Characterization of the edible bird’s nest the “Caviar of the East” Massimo F. Marcone
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Department of Food Science, Ontario Agricultural College, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 24 January 2005; accepted 18 February 2005
Abstract A few species of swiXets (genus Aerodramus) build edible nests that are consumed by humans worldwide, as a delicacy known as the “Caviar of the East” or as a medicinal food. This study reports on the compositional properties of two types of nest, the white nest and the red “blood” nest. The order of composition (from lowest to highest) was found to be identical for both types of nests, i.e., lipid (0.14–1.28%), ash (2.1%), carbohydrate (25.62–27.26%) and protein (62–63%). It was also found that both nests share a common 77 KDa protein that has properties similar to those of the ovotransferrin protein in eggs. This protein may be partially responsible for the severe allergic reactions that sometimes occur among young children who consume edible bird’s nest products. It was found that SDS–PAGE electrophoretic Wngerprinting might serve as a useful analytical technique for diVerentiating between white and red nests and for determining if the more expensive “blood” nest was adulterated with the less expensive white nest. Also evaluated were diVerent analytical methodologies for detecting adulterants. Three of the most common adulterants found in retail bird’s nests are karaya gum, red seaweed, and tremella fungus, and they are routinely incorporated during commercial processing prior to Wnal sale. Using crude protein determination, it was found that these adulterants (which typically accounted for 2–10% of the Wnished nest), reduce the overall crude protein content of the genuine white bird’s nest by as much as 1.1–6.2%. A modiWed xanthoproteic nitric acid test for proteins proved to be a rapid, and simple test to detect adulteration in both whole and Wnely ground nests, and would be suitable in the Weld where analytical facilities are not readily available. After simple nitric acid treatment, visual examination and comparison of whole nests adulterated with karaya gum, red seaweed, and Tremella fungus against the authentic white nest revealed that levels of adulteration as low as 1.7%, 1.8%, and 3.5%, respectively, could be identiWed visually. In the case of Wnely ground nests, the visual detection level was higher for all three adulterants: 1.1% for karaya gum, 1.2% for red seaweed, and 2.0% for Tremella fungus. The use of a reXectance colourmeter rendered this test even more sensitive, allowing detection at even lower levels. 2005 Elsevier Ltd. All rights reserved.
1. Introduction “Edible bird’s nest” refers to the nest produced by several diVerent swiftlet species. Human consumption of these nests has been a symbol of wealth, power, and prestige, as well as being used medicinally in traditional Chinese medicine dating as far back as the Tang (618– 907 AD) and Sung (960–1279 AD) dynasties (Koon & Cranbrook, 2002). More than 24 species of insectivorous, ecolocating swiXets are distributed around the world, but only a few ¤
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produce nests that are deemed ‘edible’ (Koon, 2000). The majority of edible bird’s nests traded worldwide come from two heavily exploited species, the White-nest swiXet (Aerodramus fuciphogus) and the Black-nest swiXet (Aerodramus maximus), whose habitats range from the Nicobar Islands in the Indian Ocean to seacaves in the costal regions of Thailand, Vietnam, Indonesia, Borneo and the Palawan Islands in the Philippines (Koon, 2000; Koon & Cranbrook, 2002). The nests are built almost exclusively by the 7–20 g male swiXet over a period of approximately 35 days. The building material is composed almost entirely of a glutinous material found in saliva secreted from the swiftlet’s
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two sublingual salivary glands (Goh et al., 2001). The half-bowl, self-supporting shaped nests, weigh 1–2 times the swiXet’s actual body weight and are usually attached to the vertical concave face walls of inland or seaside caves (Fig. 1A and B). Harvesting of the edible bird’s nest for human consumption is a painstaking and often times dangerous operation for local collectors. Most nests are built hundreds of feet up on cave walls and require the use of temporary scaVolding made of locally collected bamboo or ironwood. After collection, the tedious process of cleaning approximately 10 nests takes a person approximately 8 h (Koon & Cranbrook, 2002). The nests are cleaned by soaking them in water until the nest cement is softened and the tightly bound laminae partially loosen. Small feathers and Wne plumage are then manually removed with tweezers with the cleaned strands subsequently being re-arranged and molded into chips of various shapes, air-dried, and packaged for sale around the world (Koon & Cranbrook, 2002) (Fig. 1C and D). Edible bird’s nest is not the only commercially available food product highly esteemed/priced for human consumption processed through an animal but also includes argan oil made from the argan nut that has passed through the digestive track of a goat and Kopi Luwak [the most expensive and rarest beverage/coVee in
Fig. 1. (A) Edible red “blood” bird’s nest (unprocessed); (B) edible white bird’s nest (unprocessed); (C) edible red “blood” bird’s nest (processed); (D) edible white bird’s nest (processed).
the world which passes through the GI track of a civet (Marcone, 2004)]. Hong Kong is considered the world’s largest importer and consumer of the processed nests with North America being the second largest market (Goh et al., 2001). World trade Wgures conservatively estimates that 17–20 million nests are harvested each year with the total weight being estimated at approximately two (2) metric tonnes (Goh, Chew, Shek, & Lee, 1999; Goh et al., 2001). After personal observation of trade practices in both Malaysia (Borneo) and Indonesia, the above stated values appear to be under estimates of actual world wide production. Often referred to as the “Caviar of the East”, the nest retails for anywhere from $2000.00 (for white nests) to $10,000.00 (for “red blood” nests) Canadian per Kilogram (Koon & Cranbrook, 2002) depending upon their grade. They are usually prepared for consumption by cooking them in a double boiler with sugar producing a gastronomic delicacy often known as ‘bird’s nest soup’ which is highly esteemed as a food tonic believed to have medicinal properties (Koon, 2000; Koon & Cranbrook, 2002). The only current scientiWc knowledge about the medicinal properties of these nest extracts is that they have haemogglutination inhibiting action against the inXuenza virus (Howe, 1961; Howe, Lee, & Rose, 1960). A more recent discovery demonstrated that partially puriWed swiftlet nest extracts possess the Wrst known avian epidermal growth factor (EGF) (Kong et al., 1987; Ng, Chan, & Kong, 1986). Unfortunately, much is still unknown about the compositional properties of the edible bird’s nest. It is quite clear, however, that the salivary nest cement is the main ingredient of the edible bird’s nest and is undoubtedly one of the most expensive food ingredients in the world (Koon & Cranbrook, 2002). As a result, the number of documented and suspected cases of ‘white’ bird’s nest adulteration with less expensive materials has risen sharply over the past several years (Goh et al., 2001; Law & Melville, 1994). In an eVort to increase the net weight of the nest prior to sale, a few re-occurring materials have consistently been identiWed as common adulterants including karaya gum, red seaweed, and Tremella fungus. These materials are usually incorporated during the processing stages at levels approximating 10% and are extremely diYcult to detect due to their similar color, appearance, taste and texture to the actual salivary nest cement. One of the adulterants often times incorporated during processing is karaya gum which is a dried exudation of the stem and branches of Sterculia urens (a member of the cacao family) and is insoluble in water. Instead, it absorbs water forming viscous colloidal sols with adhesive type properties similar to the nest cement. The white jelly fungus (Tremella fuciformisis) is another often used adulterant which when introduced in the form of thin slivers
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looks very much like the laminae strands of the edible bird’s nest. Perhaps the most used of adulterants are the carrageenan-bearing red seaweeds like Kappaphycus alvarezii which is cut into slivers and boiled therefore rendering them very diYcult to detect in the Wnal product. Since little is known nor is published about the composition of these nests, an investigation was conducted to elucidate if there is any substantial chemical diVerence among ‘lower’ grade white nests and premium red ‘blood’ nests. In addition, due to the recent raise in the rate of adulteration during processing, relatively rapid, simple, and eVective analytical methods were explored that could be used in the Weld for detecting possible anomalies at much earlier stages in the distribution chain.
2. Methods 2.1. Materials Unprocessed and processed edible white and red “blood” birds’ nests were collected from various locations from both Malaysia (Kuala Lumpur and the island of Borneo) and from locations on the island of Sumatra in Indonesia. These nests comprised those collected from both inland and seaside caves as well as man made bird houses in both countries.
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of 15 kV using a Hitachi S-4500 Field-Emmission Scanning Electron Microscope equipped with an “Quartz One” energy dispersive X-ray spectrometer. X-ray system was calibrated to measure elements from Boron and higher. Analyses were performed essentially as described by Houston, Moore, Favrin, and HoV (2004). All data analysis was performed by software supplied by Hitachi. 2.5. SDS–PAGE SDS–PAGE electrophoresis was performed on raw nest material after normalization for diVerences in protein content. Approximately 50 mg of Wnely ground nest material was dissolved in 1 ml of SDS–PAGE standard derivitization and procedure run according to the method outlined by Marcone and Yada (1997). Standard molecular weight proteins included, -lactalbumin, 14,400 Da ovalbumin, 43,000 Da, bovine serum albumin 67,000 Da; phosphorylase b, 94,000 Da (Pharmacia, LKB, Montreal, Canada). 2.6. Phenol–sulfuric acid reaction for carbohydrates Total carbohydrate content was determined as described by Dubois, Gilles, Hamilton, Rebers, and Smith, 1956. Glucose was used in preparing the standard curve ranging from 2 to 10 g per sample. Samples were allowed to stand until no further color change could be detected.
2.2. Colorimetric determination 2.7. Amino acid analysis The color (L*, a*, and b* values) of the raw edible bird nests was determined using a Minolta Chroma Meter CR-200b (Minolta Camera Co., Ltd., Osaka, Japan). All analyses were performed on three indivdually collected nests of each nest type (white or ‘blood’ nests) and duplicate tests performed on each nest. Ten individual readings were taken by placing nests perpendicular to the optical sensor. All readings were averaged. 2.3. Proximate analysis of bird’s nest and elemental (P, K, MG, and Ca) analysis Proximate analysis was performed as prescribed in the oYcial standard methods of the American Association of Cereal Chemists, Inc. (AACC, 1983) after the nests were Wnely ground using a coVee grinder (Black & Decker, Toronto, Ont.) and screened through a 125 mm standard sieve. For elemental analysis, 0.250 g samples of oven-dried nest material was wet digested and subjected to atomic absorption analysis as described by Marcone and Yada (1997). 2.4. X-ray microanalysis Uncoated samples of edible bird’s nest were attached to carbon stubs and were scanned at a high-voltage setting
Amino acid analysis was performed on the white and red “blood” birds’ nests exactly as described by Marcone and Yada (1997). 2.8. Fatty acid analysis Fatty acid analysis was performed as described by Christie (1982) with minor changes, was used to prepare the fatty acid methyl-esters. The fatty acid derivatives were analyzed using a Shimadzu Gas Chromatograph (Model GC-14 A, Man-Tech. Associates Inc., Guelph, Ont.), equipped with a split mode injection system, Xame ionization detector (FID), and fused silica capillary column (SP-2330) with 30 £ 0.25 mm ID and 0.25 M Wlm thickness (Supelco, Oakville, Ont.). The initial oven temperature was 145 °C and temperature programmed at a rate of 6 °C/min to 235 °C. The intial time was 2.0 min, and the Wnal hold time was 10 min. The injection port temperature was 260 °C, and the detector port temperature was 260 °C. The hydrogen gas and air Xow rate were adjusted at 30 and 300 ml/min, respectively, and the carrier hydrogen gas rate was 1.0 ml/min. The sample volume was 0.1 l and the data were integrated with a Shimadzu Model C-R4A Chromatopac (Man-Tech Associates Inc., Guelph, Ont.). The fatty acid
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composition was determined using standard fatty acid methyl esters (Sigma–Aldrich, Oakville, Ont.). 2.9. Triacylglyceride analysis The AOCS oYcial method (Ce 5b-89, 1995) after slight modiWcation to the mobile phase ratio was used to determine the TAG composition of extracted lipid from bird’s nests. The separation was performed on two Econosil C18 columns (5 m, 4.6 £ 250 mm, Alltech, DeerWeld, IL) placed in series. The analysis was carried out isocratically with a mobile phase consisting of 60:40% (v/v) acetone:acetonitrile (v/v). Sampes (5%) were dissolved in HPLC grade acetone and 20 l aliquots were automatically injected onto the column (Waters 700 Satellite WISP, Millipore, Milford, MA) and eluted at a Xow rate of 1 ml/min. The column was equilibrated at 30 °C with a sensitivity scale at 64. The TAG were identiWed by comparing retention times to pure standards purchased from Sigma–Aldrich (Oakville, Ont.).
A DUO-Trio test diVerence analyses was conducted by presenting 24 panelists (12 male and 12 females) an unadulterated control and a pair of coded samples, one of which was identical to the control and one adulterated to a known level. The panelists were asked which sample was diVerent. Progressingly, lower levels of each of the three adulterants were tested to determine at which level 95% conWdence interval of the panelists could still pick out the adulterated sample. All samples were treated with nitric acid just prior to presentation to the panelist. Tests were performed in triplicate. 2.13. Statistical analysis All analyses were performed on three individual samples unless otherwise indicated. Statistical analysis was performed using a SAS Statistical Analysis System package. SigniWcant diVerences among samples were determined by Duncan’s multiple range test (p 6 0.05) (SAS, 1990). 2.14. Results and discussion
2.10. Scanning electron microscopy Nest fragments were Wxed at 23 °C with 5% gluteraldehyde in 0.05 M phosphate buVer pH 7.0 for 2 h. After 10 washes in phosphate buVer, they were post-Wxed in 1.0% osmium tetraoxide in the same buVer for 1 h at 23 °C. The Wxed fragments were rinsed with phosphate buVer and dehydration was achieved using an ethanol series. Samples were then critical point dried using carbondioxide. Nest fragments were then mounted on aluminum stubs and coated with gold/palladium (60/40) to a thickness of 25–30 nm using an Anatech Hummer VII sputter coater (Alexandria, VA). Following coating, the nest fragments were viewed under a Hitachi S-570 Scanning Electron Microscope with a high voltage setting of 10–20 kV. 2.11. Xanthoproteic acid test for proteins Whole and ground white edible birds’ nests were tested for protein by application of three drops (approximately 100 l) of concentrated nitric acid to their respective nest material and comparison of color development performed exactly 20 s after application. Concentrated nitric acid reacts with tyrosine, phenylalanine, and tryptophan yielding red-yellow color products. 2.12. Determination of visual sensitivity in adulteration detection In order to determine the level of visual sensitivity for the detection of adulterated nests using the modiWed xanthoproteic acid test for proteins, panelists were asked to evaluate adulterated nests in the range of 0.5–10%.
A structural examination of both raw and processed white and red “blood” birds’ nests indicated that they were composed of repeatedly interwoven strands of salivary laminae cement (Fig. 1A–D). Fig. 1A and B shows the appearance of unprocessed nests where as Fig. 1C and D shows nests that have been processed/cleaned and ready for commercial sale to the consumer. Overall, the laminae strands composing both white nests and red “blood” nests were highly variable ranging from 2 to 4 mm in diameter. The strands from each nest can be easily distinguished from one another by a visual observation of their overall color (Fig. 1). The lower grade “white” nests had an oV-white hue while the red ”blood” nests exhibited an overall reddish “terracotta” color. The red color associated with the latter is neither water-soluble nor lipid/solvent extractable. Unfortunately, it has been known that on occasion white nests have been treated with red pigments which are either partially or wholly water-soluble so as to give the false appearance of the nest being of higher grade (i.e., red “blood” nest), and hence command a higher price from unexpectant consumers. Various tests were conducted on both types of nests to determine their respective composition. The order of composition (from lowest to highest) was found to be identical for both types of nests, i.e., lipid, ash, carbohydrate, and protein (Table 1). Test results from both the white and red nests indicated that lipids constituted the smallest measured fraction but also that there were diVerences between the white and red nests. The white nest contained only minute trace amounts of lipid, i.e., approximately 0.14% whereas the red nest contained approximately 1.28% or 9
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Table 1 Composition analysis of edible birds’ nests and common adulterants White bird’s nest Proximate analysis (%) Moisture Ash Fat Protein Carbohydrate Elemental analysis (ppm) Sodium (Na) Potassium (K) Calcium (Ca) Magnesium (Mg) Phosphorus (P) Iron (Fe)
7.50a 2.10a 0.14a 62.0d 27.26c
Red “blood” bird’s nest 8.00b 2.10a 1.28b 63.0d 25.62b
650c 110a 1298c 330b 40b 30c
700a 165b 798b 500a 45b 60d
Fatty acid analysis (%) (P) Palmitric C16:0 (O) Steric C18:0 (L) Linoleic C18:1 (Ln) Linolenic C18:2
23a 29a 22a 26a
26a 26a 22a 26a
Triacylglycerol (%) PPO OOL PLnLn Monoglycerides Diglycerides
16a 13a 19a 31b 21a
14a 15a 18c 27a 26a
Karaya gum
17.52c 16.11b 1.20b 0.70b 74.47d 70a 6900b 1642d 3040c 10a 10a
Red seaweed
44.63d 33.94d 2.32c 0.40a 18.71a 50,350d 31,640e 1840d 6100d 90c 20b
Tremella fungus
4.50a 7.64c 2.22c 8.60c 77.04e 180b 26,440d 190a 520a 4060d 20b
Values in category row with the same letter are not signiWcantly diVerent (p 7 0.05).
times more than the white nest (Table 1). This preliminary result could possibly be another deWnitive test to diVerentiate between the two nest types. Further examination of this lipid component revealed that each nest type had similar mole fractions (i.e., percentages) especially those fatty acids of nutritional signiWcance, namely palmitic C16:0 (P), stearic C18:0 (O), linoleic C18:1 (L), and C18:2 (Ln). Interestingly, triacylglycerol analysis revealed the presence of over 52% mono- and diglcerides with equimolar ratios of PPO, OOL, PLn Ln composing the rest of the TAG present. The origin or function of such high content of mono- and diglycerides is not yet understood. It may be that the extremely high relative humidity of the caves (>80%) together with surface condensation may have lead to hydrolytic cleavage of the triacylglyerols. The action of enzymes found in the bird’s saliva needs also to be taken into consideration. Further investigation into the area will be required especially on freshly made nests before any deteriation can occur to them. The amount of inorganic ash in both nests was virtually identical at 2.1% (Table 1). Although the total amount of minerals from each nest were identical, atomic absorption analysis indicated signiWcant diVerences in the total amounts of various nutritionally important minerals, namely calcium, potassium, magnesium and iron. White nests were found to be much higher in calcium as were the red nests which were found
to be much higher in potassium, magnesium and iron. All red nests tested were found to have typically higher levels of iron. The results of the carbohydrate analysis of both white and red nests are presented in Table 1. The test results indicate that this was the second highest occurring component in all of the nests with some diVerences, notably that the lower-grade white nests had slightly more total carbohydrate content than the higher-grade red “blood” nests. By far the most abundant component in both nest types was crude protein with the red “blood” nests having a slightly higher but not signiWcantly higher (p 7 0.05) level (63%) compared to the white nests (62%). Amino acid analysis also revealed that both nest types had very similar amino acid proWles that were rich in certain, but not all essential amino acids (Table 2). To learn more about the types of proteins that constitute this particular dominant fraction within the respective nests, SDS–PAGE electrophoresis was performed. The results demonstrate that each nest type possesses its own unique protein Wngerprint, indicating that each is composed of diVerent protein species (Fig. 2), suggesting that not only could this analytical procedure potentially serve as a method to diVerentiate between these two diVerent nests, but also as a method of determining the identity and/or purity of a nest for possible regulatory purposes. The staining intensity of their constituent protein bands might also be useful in assessing if an
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Table 2 Amino acid determination of white and red “blood” edible birds’ nests Amino acidA Aspartic + asparagines Threonine Serine Glutamic + glutamine Glycine Alanine Valine MethionineB Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
White edible bird’s nest 9.5 4.4b 15.4a 7.0a 5.9a 4.0a 10.7a 0.8a 10.1a 3.0a 10.1a 6.8a 3.5a 3.3a 5.4a
Red “blood” edible bird’s nest 10.0a 5.3b 15.9a 7.0a 4.8b 4.7b 11.1a 0.0b 10.7a 3.4b 9.0b 6.7a 2.8b 2.6b 6.1b
Values in category row with the same letter are not signiWcantly diVerent (p 7 0.05). A Molar percent basis. B By oxidation.
adulteration of the premium red “blood” nest with the lower grade “white” nest has occurred. In addition, areas around the protein bands were observed to be surrounded by substantial streaking, which is commonly observed for highly polymerized protein complexes (Fig. 2). Furthermore, both nest types share one band with a molecular weight of approximately 77 kDa, the protein being glycosylated to approximately the same extent for each type (i.e., 2.7%). The molecular weight of the common protein in both nest types had a strong band at 77 KDa that is very similar in molecular weight to the highly allergenic ovotransferrin protein in eggs. It has been demonstrated that ovotransferrin is glycosylated (Williams, Elleman, Kingston, Wilkins, & Kuhn, 1982) and, interestingly, it is glycosylated to the same level as the 77 kDa protein band in the nests (2.8%). It is still unclear, however, if an ovotransferrin-like protein is synthesized elsewhere other than in eggs but these tentative results demonstrate that there is some semblance of similarity between the protein in eggs and that found in the birds’ nests. Further amino acid sequence studies will help to further elucidate how these proteins are inter-related. Interestingly, the red (terracotta) color of the “blood” nest is very similar to the color of puriWed ovotransferrin in its iron complexed state whereas the “white” colored nest is similar in color to ovotransferrin in its non-iron complexed form. This also supports the proposed theory that the protein in both nest types is at least ovotransferrinlike, very similar to ovotransferrin in eggs. Further supporting this theory is the diVerence in the iron contents of both nest types as indicated by atomic absorption analysis. Scanning electron microscopy combined with X-ray microanalysis performed at various locations on
Fig. 2. (I) SDS–PAGE (reducing 1 l of 1 mg¡1 protein solutions) applied to Homogenous 20 Phast gels (Pharmacia LKB). (lanes S) standard [-lactalbumin, 14,400 Da; soybean trypsin inhibitor, 20,100 Da; carbonic anhydrase, 30,000 Da; ovalbumin, 43,000 Da; bovine serum albumin, 67,000 Da; phosphorylase b, 94,0000 Da] (1), ovotransferrin; (2), total protein – red ‘blood’ bird’s nest; (3), total protein – white bird’s nest. (II) Densitometric scan of gel (all labels are the same as in (I).
the surface of the red nests indicate relatively higher levels of iron compared to that of the white nests. These analyses also indicated that the distribution of various elements in the nests are highly variable indicating possible compositional diVerences in the secreted saliva during the 35-day period when the nests are being built (Fig. 3). Since proteins constitute the major compositional fraction of both studied nest types, it is not uncommon that several medical studies have shown the occurrence of a severe allergic reaction among some young children who have consumed ‘bird’s nest soup’ (Goh et al., 2001). The symptoms of these allergic reactions were similar to those induced by egg-like proteins. Interestingly, these studies also noted that bird’s nests-induced allergies were the most common cause of anaphylaxis reported at the National Hospital in Singapore even surpassing other well-deWned food allergens such as cow’s milk or egg in younger children and peanut or crustacean seafood in older children (Goh, Chew, Chua, Chay, & Lee, 2000; Goh et al., 1999). In North America as well as the
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Fig. 3. Scanning electron microscopy combined with X-ray microanalysis of red “blood” edible bird’s nest. (A) X-ray microanalysis of “red” nest portion. (B) X-ray microanalysis of “white” nest portion.
rest of the world, however, there is no or very little information as to how many children have experienced bird’s nest-induced anaphylaxis. The medical community may not even be aware of its existence or properly trained to diagnose it. Practitioners of traditional Chinese medicine have consistently indicated that the consumption of birds’ nest soup is beneWcial for the treatment of a variety of health problems (Koon & Cranbrook, 2002). It is often administered to the elderly and very young alike who are recovering from various types of infections. Studies have indicated that ovotransferrin is both bacteriostatic and bacteriocidal in nature (Ibrahim, Sugimoto, & Aoki, 2000). If the protein identiWed in these nests is in fact an ovotransferrin-like protein, then it could be reasonably involved in or responsible for Wghting infection just like the ovotransferrin in eggs. This may be a possible reason as to why edible bird’s nest soup has been consistently consumed throughout the centuries as prescribed traditional Chinese medicine. The salivary nest cement is the most important ingredient in the edible bird’s nest and is undoubtedly one of the most expensive food ingredients in the world. Often times, however, it has been found adulterated with Tremella fungus, karaya gum or red seaweed (Figs. 4 and 5). These three materials are typically only incorporated to a maximum of 10% to increase the overall net weight of the Wnished nest. These compounds are ideal adulterants since their color, taste, and texture are similar to that of the genuine birds nests salivary cement and are therefore often diYcult to detect. Scanning electron microscopy did not prove to be an eVective technique for determining the existence nor the type of adulteration in edible bird’s nest except for those containing added red seaweed. Unlike these adulterated with tremella fungus or plant resin, particles of red sea
Fig. 4. (A) Red seaweed; (B) karaya gum (plant resin); (C) Tremella fungus.
weed were easily distinguishable from strands of birds’ nest cement. Cross-sections of seaweed strands contained very detailed striations on raised surfaces which were distinguishable from the high amophorous strands of bird’s cement (see inset of micrograph; Fig. 6). Compositional analysis of these common adulterants revealed that they contain minerals in diVering amounts and ratios rendering each with a unique and distinguishable mineral proWle (Table 1). Since iodine and salt (NaCl) are present in red seaweed, but are not typically found in genuine birds nest, presumptive tests for iodine (iodine spot test) and salt (silver nitrate spot test) could
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Fig. 6. Scanning electron micrograph of seaweed cross-section found in adulterate nest. Not striations in box.
Fig. 5. (A, C, and E) Edible white bird’s nest (processed); (B) edible red seaweed adulterated white bird’s nest (processed); (D) edible karaya gum (plant resin) adulterated white bird’s nest (processed); (F) edible tremella fungus adulterated white bird’s nest (processed).
quickly and easily be used to detect non-heat treated seaweed adulterants (results not shown). Unfortunately, most violators heat treat their seaweed prior to adulteration, decreasing both the iodine and NaCl (to the same extent) levels and thereby limiting detection test usage. Nests containing abnormally high potassium compared to pure birds nest material could indicate adulteration with red seaweed and/or Tremella fungus, which are both relatively high in this mineral. On the other hand, nests with abnormally high levels of phosphorous could indicate adulteration with Tremella fungus, whereas high amounts of sodium could indicate adulteration with red seaweed.
Closer compositional examination of the three adulterants – namely karaya gum, red seaweed and Tremella fungus – revealed that they were relatively low in overall crude protein, i.e., 0.7%, 0.4% and 8.6%, respectively, compared to the 62% of pure white birds nest material (Table 1). Intentional additions of as little as 2% to as high as 10% (with 10% being the most commonly found level of adulteration found) substantially decreased the overall total protein content of the nest ranging from 1.1% to 6.2%, as measured by total protein analysis. Therefore, comparison of the total protein contents of suspected adulterated birds nest versus the pure edible bird’s nest could potentially be used to determine nest authenticity. Amino acid analysis revealed that white nest protein was substantially high in two speciWc aromatic amino acids, namely phenylalanine and tyrosine (Table 2). Concentrated nitric acid reacts with aromatic amino acids in proteins to produce a yellow-red color in proportion to the amount of protein present. This xanthoproteic acid test for proteins could therefore potentially be used to detect adulterated nests for sale on the market. All whole adulterated nests produced a visibly less intense yellowred reaction when treated with nitric acid when compared to the same test performed on the genuine white nests. When pieces (1.0 £ 1.5 cm) of nests that were artiWcally adulternated with karaya gum, red seaweed and
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Fig. 7. (A) Whole edible red seaweed adulterated white bird’s nest xanthoproteic nitric acid test); (B) whole pure edible white bird’s nest (xanthoproteic nitric acid test).
Tremella fungus were presented to panelists, their detection down to 1.7%, 1.8% and 3.5%, respectively, could be made. Fig. 7 shows two such nest pieces (one pure and one adulterated with 5% red seaweed) treated directly with nitric acid. The unadulterated (pure nest) was much darker in color than the adulterated one due to the higher content of protein. In the Wnely ground nests, the use of this test permitted the vision detection of adulteration to levels of 1.1% (karaya) gum, 1.2% (red seaweed), 2.0% (Tremella fungus). Fig. 8 shows the color diVerence that could be expected with adulteration of 10% typically found in the market place. This may be due to the more uniform distribution of protein and therefore more uniform color development in the test material. The use of a reXectance colourmeter made this test even more sensitive down to 0.2% for karaya gum, 0.3% for red seaweed, and 0.5% for Tremella fungus. Table 3 shows typi-
Table 3 Minolta colorimetric reading of ground bird’s nest with variable amounts of adulterants treated with nitric acid (xanthoproteic acid test) Typical adulteration level 2%
Seaweed Karaya gum Tremella fungus Pure Nest
5%
10%
L*
a*
b*
L*
a*
b*
L*
a*
b*
56a 55a 50a 43
48c 50c 50c 58
32a 30a 25a 25
60b 62b 55b
40b 40b 45b
37 35 27‘
65c 65c 60c
37a 35a 40a
42c 40c 30c
Values in each row of the same descriptor, i.e., all L*, a* or b* (separately) with the same letter are not signiWcantly diVerent (p 7 0.05).
cal L*, a*, b* values for adulteration reading between 2% and 10% which are typical ranges found in the market place.
Fig. 8. (A) Crushed edible karaya gum adulterated white bird’s nest (10% adulteration); (B) crushed edible white bird’s nest (10% adulteration) (all treated with nitric acid – xanthoproteic nitric acid spot test).
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In conclusion, this study reports on the actual compositional properties of both white and red “blood” nests revealing that they both contain predominately proteinous material followed by carbohydrate, ash and lipid (in descending order). SDS–PAGE was found to be an adequate analytical method to diVerentiate between these two nest types whereas a modiWed xanthoproteic acid test was found to facilitate the diVerentiation detection of “whote” nests that were adulterated with commonly used materials. When pieces (1.0 £ 1.5 cm) of nests artiWcally adulterated with karaya gum, red seaweed and Tremella fungus were tested, panelist could detect their presence down to 1.7%, 1.8% and 3.5% inclusion, respectively. Fig. 7 shows two nest pieces directly treated with nitric acid and shows the diVerence in color between adulterated and pure nests.
Acknowledgements The author thank Dr. Anthony Clarke of the Department of Microbiology for performing amino acid analysis and Dr. Alexandra Smith of the Department of Food Science for her assistance in X-ray microanalysis.
References AACC (American Association of Cereal Chemists). (1983). Approved Methods of the American Association of Cereal Chemists. 8th Ed. St. Paul, MN: AACC. AOAC (Association of OYcial Analytical Chemists) (1995). Liquid chromatographic mehtod for determination of triglycerides in vegetable oils in terms of ther partition numbers; summary of collaborative study. Journal of AOAC International, 77C4, 954– 957. Christie, W. W. (1982). A simple procedure for rapid transmethylation of glycerolipids and cholesterylesters. Lipid Research, 23, 1072– 1075. Dubois, H., Gilles, K. A., Hamilton, J. R., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356.
Goh, D. L. M., Chew, F.-T., Chua, K.-Y., Chay, O.-M., & Lee, B.-W. (2000). Edible “Bird’s nest”-induced anaphylaxis: an under-recognized entity?. Journal of Pediatric, 137, 277–279. Goh, D. L. M., Chew, Y.-N., Shek, F.-T., & Lee, B.-W. (1999). Alergy net-pattern of food-induced anaphylaxis in children of an Asian community. Allergy, 54(1), 84–86. Goh, D. L. M., Chua, K.-Y., Chew, F.-T., Seow, T. K., Ou, K. L., Yi, F. C., & Lee, B. W. (2001). Immunochemical characterization of edible bird’s nest allergens. Journal of Allergy and Clinical Immunology, 107(6), 1082–1088. Houston, D. M., Moore, A. E. P., Favrin, M. G., & HoV, B. (2004). Canine urolithisis: a look at over 16,000 urolith submissions to the Canadian Veterinary Urolith Center from February 1998 to April 2003. Canadian Veterinary Journal, 45, 225–230. Howe, C., Lee, L. T., & Rose, H. M. (1961). Collocalia mucoid: a substrate for Myxovirus neuraminidase. Archives of Biochemistry and Biophysics, 95, 512–520. Howe, C., Lee, L. T., & Rose, H. M. (1960). InXuenza virus sialidase. Nature, 188, 251–252. Ibrahim, H. R., Sugimoto, Y., & Aoki, T. (2000). Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism. Biochemica et Biophysica Acta, 1523, 196–205. Kong, Y. C., Keung, W. M., Tip, T. T., Ko, K. K., Tsao, S. W., & Ng, M. H. (1987). Evidence that epidermal growth factor is present in SwiXet’s (Collocalia) nest. Comparative Biochemistry and Physiology, 87(2), 221–226. Koon, L. C. (2000). Features – Bird’s nest soup – Market demand for this expensive gastronomic delicacy threatens the aptly named edible-nest SwiXets with extinction in the east. Wildlife Conservation, 103(1), 30–35. Koon, L. C., & Cranbrook, Earl of (2002). Swiftlets of Borneo – Builders of edible nests (pp. 1–171). Sabah, Malaysia: Natural History Publication (Borneo) SDN., B.H.D. Law, A. S. M., & Melville, D. S. (1994). International trade in SwiXet nests with special reference to Hong Kong. Cambridge: TraYc International. Marcone, M. F. (2004). Composition and properties of Indonesian palm civet coVee (Kopi Luwak) and Ethiopian civet coVee. Food Research International, 37, 901–912. Marcone, M. F., & Yada, R. Y. (1997). Evidence for the phosphorylation and glycosylation of the amaranth 11S globulin (Amaranthin). Food Biochemistry, 21, 341–369. Ng, M. H., Chan, K. H., & Kong, Y. C. (1986). Potentiation of mitogenic response by extracts of the Swiftlet’s (Collocalia) nest. Biochemistry International, 13(3), 521–531. SAS Institute. (1990). SAS user’s guide: statistics; statistical analysis system: Cary, NC. Williams, J., Elleman, T. C., Kingston, I. B., Wilkins, A. G., & Kuhn, K. A. (1982). The primary structure of len ovotransferrin. European Journal of Biochemistry, 122, 297–303.
Characterization of the edible bird’s nest the “Caviar of the East” Massimo F. Marcone
¤
Department of Food Science, Ontario Agricultural College, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 24 January 2005; accepted 18 February 2005
Abstract A few species of swiXets (genus Aerodramus) build edible nests that are consumed by humans worldwide, as a delicacy known as the “Caviar of the East” or as a medicinal food. This study reports on the compositional properties of two types of nest, the white nest and the red “blood” nest. The order of composition (from lowest to highest) was found to be identical for both types of nests, i.e., lipid (0.14–1.28%), ash (2.1%), carbohydrate (25.62–27.26%) and protein (62–63%). It was also found that both nests share a common 77 KDa protein that has properties similar to those of the ovotransferrin protein in eggs. This protein may be partially responsible for the severe allergic reactions that sometimes occur among young children who consume edible bird’s nest products. It was found that SDS–PAGE electrophoretic Wngerprinting might serve as a useful analytical technique for diVerentiating between white and red nests and for determining if the more expensive “blood” nest was adulterated with the less expensive white nest. Also evaluated were diVerent analytical methodologies for detecting adulterants. Three of the most common adulterants found in retail bird’s nests are karaya gum, red seaweed, and tremella fungus, and they are routinely incorporated during commercial processing prior to Wnal sale. Using crude protein determination, it was found that these adulterants (which typically accounted for 2–10% of the Wnished nest), reduce the overall crude protein content of the genuine white bird’s nest by as much as 1.1–6.2%. A modiWed xanthoproteic nitric acid test for proteins proved to be a rapid, and simple test to detect adulteration in both whole and Wnely ground nests, and would be suitable in the Weld where analytical facilities are not readily available. After simple nitric acid treatment, visual examination and comparison of whole nests adulterated with karaya gum, red seaweed, and Tremella fungus against the authentic white nest revealed that levels of adulteration as low as 1.7%, 1.8%, and 3.5%, respectively, could be identiWed visually. In the case of Wnely ground nests, the visual detection level was higher for all three adulterants: 1.1% for karaya gum, 1.2% for red seaweed, and 2.0% for Tremella fungus. The use of a reXectance colourmeter rendered this test even more sensitive, allowing detection at even lower levels. 2005 Elsevier Ltd. All rights reserved.
1. Introduction “Edible bird’s nest” refers to the nest produced by several diVerent swiftlet species. Human consumption of these nests has been a symbol of wealth, power, and prestige, as well as being used medicinally in traditional Chinese medicine dating as far back as the Tang (618– 907 AD) and Sung (960–1279 AD) dynasties (Koon & Cranbrook, 2002). More than 24 species of insectivorous, ecolocating swiXets are distributed around the world, but only a few ¤
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produce nests that are deemed ‘edible’ (Koon, 2000). The majority of edible bird’s nests traded worldwide come from two heavily exploited species, the White-nest swiXet (Aerodramus fuciphogus) and the Black-nest swiXet (Aerodramus maximus), whose habitats range from the Nicobar Islands in the Indian Ocean to seacaves in the costal regions of Thailand, Vietnam, Indonesia, Borneo and the Palawan Islands in the Philippines (Koon, 2000; Koon & Cranbrook, 2002). The nests are built almost exclusively by the 7–20 g male swiXet over a period of approximately 35 days. The building material is composed almost entirely of a glutinous material found in saliva secreted from the swiftlet’s
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two sublingual salivary glands (Goh et al., 2001). The half-bowl, self-supporting shaped nests, weigh 1–2 times the swiXet’s actual body weight and are usually attached to the vertical concave face walls of inland or seaside caves (Fig. 1A and B). Harvesting of the edible bird’s nest for human consumption is a painstaking and often times dangerous operation for local collectors. Most nests are built hundreds of feet up on cave walls and require the use of temporary scaVolding made of locally collected bamboo or ironwood. After collection, the tedious process of cleaning approximately 10 nests takes a person approximately 8 h (Koon & Cranbrook, 2002). The nests are cleaned by soaking them in water until the nest cement is softened and the tightly bound laminae partially loosen. Small feathers and Wne plumage are then manually removed with tweezers with the cleaned strands subsequently being re-arranged and molded into chips of various shapes, air-dried, and packaged for sale around the world (Koon & Cranbrook, 2002) (Fig. 1C and D). Edible bird’s nest is not the only commercially available food product highly esteemed/priced for human consumption processed through an animal but also includes argan oil made from the argan nut that has passed through the digestive track of a goat and Kopi Luwak [the most expensive and rarest beverage/coVee in
Fig. 1. (A) Edible red “blood” bird’s nest (unprocessed); (B) edible white bird’s nest (unprocessed); (C) edible red “blood” bird’s nest (processed); (D) edible white bird’s nest (processed).
the world which passes through the GI track of a civet (Marcone, 2004)]. Hong Kong is considered the world’s largest importer and consumer of the processed nests with North America being the second largest market (Goh et al., 2001). World trade Wgures conservatively estimates that 17–20 million nests are harvested each year with the total weight being estimated at approximately two (2) metric tonnes (Goh, Chew, Shek, & Lee, 1999; Goh et al., 2001). After personal observation of trade practices in both Malaysia (Borneo) and Indonesia, the above stated values appear to be under estimates of actual world wide production. Often referred to as the “Caviar of the East”, the nest retails for anywhere from $2000.00 (for white nests) to $10,000.00 (for “red blood” nests) Canadian per Kilogram (Koon & Cranbrook, 2002) depending upon their grade. They are usually prepared for consumption by cooking them in a double boiler with sugar producing a gastronomic delicacy often known as ‘bird’s nest soup’ which is highly esteemed as a food tonic believed to have medicinal properties (Koon, 2000; Koon & Cranbrook, 2002). The only current scientiWc knowledge about the medicinal properties of these nest extracts is that they have haemogglutination inhibiting action against the inXuenza virus (Howe, 1961; Howe, Lee, & Rose, 1960). A more recent discovery demonstrated that partially puriWed swiftlet nest extracts possess the Wrst known avian epidermal growth factor (EGF) (Kong et al., 1987; Ng, Chan, & Kong, 1986). Unfortunately, much is still unknown about the compositional properties of the edible bird’s nest. It is quite clear, however, that the salivary nest cement is the main ingredient of the edible bird’s nest and is undoubtedly one of the most expensive food ingredients in the world (Koon & Cranbrook, 2002). As a result, the number of documented and suspected cases of ‘white’ bird’s nest adulteration with less expensive materials has risen sharply over the past several years (Goh et al., 2001; Law & Melville, 1994). In an eVort to increase the net weight of the nest prior to sale, a few re-occurring materials have consistently been identiWed as common adulterants including karaya gum, red seaweed, and Tremella fungus. These materials are usually incorporated during the processing stages at levels approximating 10% and are extremely diYcult to detect due to their similar color, appearance, taste and texture to the actual salivary nest cement. One of the adulterants often times incorporated during processing is karaya gum which is a dried exudation of the stem and branches of Sterculia urens (a member of the cacao family) and is insoluble in water. Instead, it absorbs water forming viscous colloidal sols with adhesive type properties similar to the nest cement. The white jelly fungus (Tremella fuciformisis) is another often used adulterant which when introduced in the form of thin slivers
M.F. Marcone / Food Research International 38 (2005) 1125–1134
looks very much like the laminae strands of the edible bird’s nest. Perhaps the most used of adulterants are the carrageenan-bearing red seaweeds like Kappaphycus alvarezii which is cut into slivers and boiled therefore rendering them very diYcult to detect in the Wnal product. Since little is known nor is published about the composition of these nests, an investigation was conducted to elucidate if there is any substantial chemical diVerence among ‘lower’ grade white nests and premium red ‘blood’ nests. In addition, due to the recent raise in the rate of adulteration during processing, relatively rapid, simple, and eVective analytical methods were explored that could be used in the Weld for detecting possible anomalies at much earlier stages in the distribution chain.
2. Methods 2.1. Materials Unprocessed and processed edible white and red “blood” birds’ nests were collected from various locations from both Malaysia (Kuala Lumpur and the island of Borneo) and from locations on the island of Sumatra in Indonesia. These nests comprised those collected from both inland and seaside caves as well as man made bird houses in both countries.
1127
of 15 kV using a Hitachi S-4500 Field-Emmission Scanning Electron Microscope equipped with an “Quartz One” energy dispersive X-ray spectrometer. X-ray system was calibrated to measure elements from Boron and higher. Analyses were performed essentially as described by Houston, Moore, Favrin, and HoV (2004). All data analysis was performed by software supplied by Hitachi. 2.5. SDS–PAGE SDS–PAGE electrophoresis was performed on raw nest material after normalization for diVerences in protein content. Approximately 50 mg of Wnely ground nest material was dissolved in 1 ml of SDS–PAGE standard derivitization and procedure run according to the method outlined by Marcone and Yada (1997). Standard molecular weight proteins included, -lactalbumin, 14,400 Da ovalbumin, 43,000 Da, bovine serum albumin 67,000 Da; phosphorylase b, 94,000 Da (Pharmacia, LKB, Montreal, Canada). 2.6. Phenol–sulfuric acid reaction for carbohydrates Total carbohydrate content was determined as described by Dubois, Gilles, Hamilton, Rebers, and Smith, 1956. Glucose was used in preparing the standard curve ranging from 2 to 10 g per sample. Samples were allowed to stand until no further color change could be detected.
2.2. Colorimetric determination 2.7. Amino acid analysis The color (L*, a*, and b* values) of the raw edible bird nests was determined using a Minolta Chroma Meter CR-200b (Minolta Camera Co., Ltd., Osaka, Japan). All analyses were performed on three indivdually collected nests of each nest type (white or ‘blood’ nests) and duplicate tests performed on each nest. Ten individual readings were taken by placing nests perpendicular to the optical sensor. All readings were averaged. 2.3. Proximate analysis of bird’s nest and elemental (P, K, MG, and Ca) analysis Proximate analysis was performed as prescribed in the oYcial standard methods of the American Association of Cereal Chemists, Inc. (AACC, 1983) after the nests were Wnely ground using a coVee grinder (Black & Decker, Toronto, Ont.) and screened through a 125 mm standard sieve. For elemental analysis, 0.250 g samples of oven-dried nest material was wet digested and subjected to atomic absorption analysis as described by Marcone and Yada (1997). 2.4. X-ray microanalysis Uncoated samples of edible bird’s nest were attached to carbon stubs and were scanned at a high-voltage setting
Amino acid analysis was performed on the white and red “blood” birds’ nests exactly as described by Marcone and Yada (1997). 2.8. Fatty acid analysis Fatty acid analysis was performed as described by Christie (1982) with minor changes, was used to prepare the fatty acid methyl-esters. The fatty acid derivatives were analyzed using a Shimadzu Gas Chromatograph (Model GC-14 A, Man-Tech. Associates Inc., Guelph, Ont.), equipped with a split mode injection system, Xame ionization detector (FID), and fused silica capillary column (SP-2330) with 30 £ 0.25 mm ID and 0.25 M Wlm thickness (Supelco, Oakville, Ont.). The initial oven temperature was 145 °C and temperature programmed at a rate of 6 °C/min to 235 °C. The intial time was 2.0 min, and the Wnal hold time was 10 min. The injection port temperature was 260 °C, and the detector port temperature was 260 °C. The hydrogen gas and air Xow rate were adjusted at 30 and 300 ml/min, respectively, and the carrier hydrogen gas rate was 1.0 ml/min. The sample volume was 0.1 l and the data were integrated with a Shimadzu Model C-R4A Chromatopac (Man-Tech Associates Inc., Guelph, Ont.). The fatty acid
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composition was determined using standard fatty acid methyl esters (Sigma–Aldrich, Oakville, Ont.). 2.9. Triacylglyceride analysis The AOCS oYcial method (Ce 5b-89, 1995) after slight modiWcation to the mobile phase ratio was used to determine the TAG composition of extracted lipid from bird’s nests. The separation was performed on two Econosil C18 columns (5 m, 4.6 £ 250 mm, Alltech, DeerWeld, IL) placed in series. The analysis was carried out isocratically with a mobile phase consisting of 60:40% (v/v) acetone:acetonitrile (v/v). Sampes (5%) were dissolved in HPLC grade acetone and 20 l aliquots were automatically injected onto the column (Waters 700 Satellite WISP, Millipore, Milford, MA) and eluted at a Xow rate of 1 ml/min. The column was equilibrated at 30 °C with a sensitivity scale at 64. The TAG were identiWed by comparing retention times to pure standards purchased from Sigma–Aldrich (Oakville, Ont.).
A DUO-Trio test diVerence analyses was conducted by presenting 24 panelists (12 male and 12 females) an unadulterated control and a pair of coded samples, one of which was identical to the control and one adulterated to a known level. The panelists were asked which sample was diVerent. Progressingly, lower levels of each of the three adulterants were tested to determine at which level 95% conWdence interval of the panelists could still pick out the adulterated sample. All samples were treated with nitric acid just prior to presentation to the panelist. Tests were performed in triplicate. 2.13. Statistical analysis All analyses were performed on three individual samples unless otherwise indicated. Statistical analysis was performed using a SAS Statistical Analysis System package. SigniWcant diVerences among samples were determined by Duncan’s multiple range test (p 6 0.05) (SAS, 1990). 2.14. Results and discussion
2.10. Scanning electron microscopy Nest fragments were Wxed at 23 °C with 5% gluteraldehyde in 0.05 M phosphate buVer pH 7.0 for 2 h. After 10 washes in phosphate buVer, they were post-Wxed in 1.0% osmium tetraoxide in the same buVer for 1 h at 23 °C. The Wxed fragments were rinsed with phosphate buVer and dehydration was achieved using an ethanol series. Samples were then critical point dried using carbondioxide. Nest fragments were then mounted on aluminum stubs and coated with gold/palladium (60/40) to a thickness of 25–30 nm using an Anatech Hummer VII sputter coater (Alexandria, VA). Following coating, the nest fragments were viewed under a Hitachi S-570 Scanning Electron Microscope with a high voltage setting of 10–20 kV. 2.11. Xanthoproteic acid test for proteins Whole and ground white edible birds’ nests were tested for protein by application of three drops (approximately 100 l) of concentrated nitric acid to their respective nest material and comparison of color development performed exactly 20 s after application. Concentrated nitric acid reacts with tyrosine, phenylalanine, and tryptophan yielding red-yellow color products. 2.12. Determination of visual sensitivity in adulteration detection In order to determine the level of visual sensitivity for the detection of adulterated nests using the modiWed xanthoproteic acid test for proteins, panelists were asked to evaluate adulterated nests in the range of 0.5–10%.
A structural examination of both raw and processed white and red “blood” birds’ nests indicated that they were composed of repeatedly interwoven strands of salivary laminae cement (Fig. 1A–D). Fig. 1A and B shows the appearance of unprocessed nests where as Fig. 1C and D shows nests that have been processed/cleaned and ready for commercial sale to the consumer. Overall, the laminae strands composing both white nests and red “blood” nests were highly variable ranging from 2 to 4 mm in diameter. The strands from each nest can be easily distinguished from one another by a visual observation of their overall color (Fig. 1). The lower grade “white” nests had an oV-white hue while the red ”blood” nests exhibited an overall reddish “terracotta” color. The red color associated with the latter is neither water-soluble nor lipid/solvent extractable. Unfortunately, it has been known that on occasion white nests have been treated with red pigments which are either partially or wholly water-soluble so as to give the false appearance of the nest being of higher grade (i.e., red “blood” nest), and hence command a higher price from unexpectant consumers. Various tests were conducted on both types of nests to determine their respective composition. The order of composition (from lowest to highest) was found to be identical for both types of nests, i.e., lipid, ash, carbohydrate, and protein (Table 1). Test results from both the white and red nests indicated that lipids constituted the smallest measured fraction but also that there were diVerences between the white and red nests. The white nest contained only minute trace amounts of lipid, i.e., approximately 0.14% whereas the red nest contained approximately 1.28% or 9
M.F. Marcone / Food Research International 38 (2005) 1125–1134
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Table 1 Composition analysis of edible birds’ nests and common adulterants White bird’s nest Proximate analysis (%) Moisture Ash Fat Protein Carbohydrate Elemental analysis (ppm) Sodium (Na) Potassium (K) Calcium (Ca) Magnesium (Mg) Phosphorus (P) Iron (Fe)
7.50a 2.10a 0.14a 62.0d 27.26c
Red “blood” bird’s nest 8.00b 2.10a 1.28b 63.0d 25.62b
650c 110a 1298c 330b 40b 30c
700a 165b 798b 500a 45b 60d
Fatty acid analysis (%) (P) Palmitric C16:0 (O) Steric C18:0 (L) Linoleic C18:1 (Ln) Linolenic C18:2
23a 29a 22a 26a
26a 26a 22a 26a
Triacylglycerol (%) PPO OOL PLnLn Monoglycerides Diglycerides
16a 13a 19a 31b 21a
14a 15a 18c 27a 26a
Karaya gum
17.52c 16.11b 1.20b 0.70b 74.47d 70a 6900b 1642d 3040c 10a 10a
Red seaweed
44.63d 33.94d 2.32c 0.40a 18.71a 50,350d 31,640e 1840d 6100d 90c 20b
Tremella fungus
4.50a 7.64c 2.22c 8.60c 77.04e 180b 26,440d 190a 520a 4060d 20b
Values in category row with the same letter are not signiWcantly diVerent (p 7 0.05).
times more than the white nest (Table 1). This preliminary result could possibly be another deWnitive test to diVerentiate between the two nest types. Further examination of this lipid component revealed that each nest type had similar mole fractions (i.e., percentages) especially those fatty acids of nutritional signiWcance, namely palmitic C16:0 (P), stearic C18:0 (O), linoleic C18:1 (L), and C18:2 (Ln). Interestingly, triacylglycerol analysis revealed the presence of over 52% mono- and diglcerides with equimolar ratios of PPO, OOL, PLn Ln composing the rest of the TAG present. The origin or function of such high content of mono- and diglycerides is not yet understood. It may be that the extremely high relative humidity of the caves (>80%) together with surface condensation may have lead to hydrolytic cleavage of the triacylglyerols. The action of enzymes found in the bird’s saliva needs also to be taken into consideration. Further investigation into the area will be required especially on freshly made nests before any deteriation can occur to them. The amount of inorganic ash in both nests was virtually identical at 2.1% (Table 1). Although the total amount of minerals from each nest were identical, atomic absorption analysis indicated signiWcant diVerences in the total amounts of various nutritionally important minerals, namely calcium, potassium, magnesium and iron. White nests were found to be much higher in calcium as were the red nests which were found
to be much higher in potassium, magnesium and iron. All red nests tested were found to have typically higher levels of iron. The results of the carbohydrate analysis of both white and red nests are presented in Table 1. The test results indicate that this was the second highest occurring component in all of the nests with some diVerences, notably that the lower-grade white nests had slightly more total carbohydrate content than the higher-grade red “blood” nests. By far the most abundant component in both nest types was crude protein with the red “blood” nests having a slightly higher but not signiWcantly higher (p 7 0.05) level (63%) compared to the white nests (62%). Amino acid analysis also revealed that both nest types had very similar amino acid proWles that were rich in certain, but not all essential amino acids (Table 2). To learn more about the types of proteins that constitute this particular dominant fraction within the respective nests, SDS–PAGE electrophoresis was performed. The results demonstrate that each nest type possesses its own unique protein Wngerprint, indicating that each is composed of diVerent protein species (Fig. 2), suggesting that not only could this analytical procedure potentially serve as a method to diVerentiate between these two diVerent nests, but also as a method of determining the identity and/or purity of a nest for possible regulatory purposes. The staining intensity of their constituent protein bands might also be useful in assessing if an
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Table 2 Amino acid determination of white and red “blood” edible birds’ nests Amino acidA Aspartic + asparagines Threonine Serine Glutamic + glutamine Glycine Alanine Valine MethionineB Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
White edible bird’s nest 9.5 4.4b 15.4a 7.0a 5.9a 4.0a 10.7a 0.8a 10.1a 3.0a 10.1a 6.8a 3.5a 3.3a 5.4a
Red “blood” edible bird’s nest 10.0a 5.3b 15.9a 7.0a 4.8b 4.7b 11.1a 0.0b 10.7a 3.4b 9.0b 6.7a 2.8b 2.6b 6.1b
Values in category row with the same letter are not signiWcantly diVerent (p 7 0.05). A Molar percent basis. B By oxidation.
adulteration of the premium red “blood” nest with the lower grade “white” nest has occurred. In addition, areas around the protein bands were observed to be surrounded by substantial streaking, which is commonly observed for highly polymerized protein complexes (Fig. 2). Furthermore, both nest types share one band with a molecular weight of approximately 77 kDa, the protein being glycosylated to approximately the same extent for each type (i.e., 2.7%). The molecular weight of the common protein in both nest types had a strong band at 77 KDa that is very similar in molecular weight to the highly allergenic ovotransferrin protein in eggs. It has been demonstrated that ovotransferrin is glycosylated (Williams, Elleman, Kingston, Wilkins, & Kuhn, 1982) and, interestingly, it is glycosylated to the same level as the 77 kDa protein band in the nests (2.8%). It is still unclear, however, if an ovotransferrin-like protein is synthesized elsewhere other than in eggs but these tentative results demonstrate that there is some semblance of similarity between the protein in eggs and that found in the birds’ nests. Further amino acid sequence studies will help to further elucidate how these proteins are inter-related. Interestingly, the red (terracotta) color of the “blood” nest is very similar to the color of puriWed ovotransferrin in its iron complexed state whereas the “white” colored nest is similar in color to ovotransferrin in its non-iron complexed form. This also supports the proposed theory that the protein in both nest types is at least ovotransferrinlike, very similar to ovotransferrin in eggs. Further supporting this theory is the diVerence in the iron contents of both nest types as indicated by atomic absorption analysis. Scanning electron microscopy combined with X-ray microanalysis performed at various locations on
Fig. 2. (I) SDS–PAGE (reducing 1 l of 1 mg¡1 protein solutions) applied to Homogenous 20 Phast gels (Pharmacia LKB). (lanes S) standard [-lactalbumin, 14,400 Da; soybean trypsin inhibitor, 20,100 Da; carbonic anhydrase, 30,000 Da; ovalbumin, 43,000 Da; bovine serum albumin, 67,000 Da; phosphorylase b, 94,0000 Da] (1), ovotransferrin; (2), total protein – red ‘blood’ bird’s nest; (3), total protein – white bird’s nest. (II) Densitometric scan of gel (all labels are the same as in (I).
the surface of the red nests indicate relatively higher levels of iron compared to that of the white nests. These analyses also indicated that the distribution of various elements in the nests are highly variable indicating possible compositional diVerences in the secreted saliva during the 35-day period when the nests are being built (Fig. 3). Since proteins constitute the major compositional fraction of both studied nest types, it is not uncommon that several medical studies have shown the occurrence of a severe allergic reaction among some young children who have consumed ‘bird’s nest soup’ (Goh et al., 2001). The symptoms of these allergic reactions were similar to those induced by egg-like proteins. Interestingly, these studies also noted that bird’s nests-induced allergies were the most common cause of anaphylaxis reported at the National Hospital in Singapore even surpassing other well-deWned food allergens such as cow’s milk or egg in younger children and peanut or crustacean seafood in older children (Goh, Chew, Chua, Chay, & Lee, 2000; Goh et al., 1999). In North America as well as the
M.F. Marcone / Food Research International 38 (2005) 1125–1134
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Fig. 3. Scanning electron microscopy combined with X-ray microanalysis of red “blood” edible bird’s nest. (A) X-ray microanalysis of “red” nest portion. (B) X-ray microanalysis of “white” nest portion.
rest of the world, however, there is no or very little information as to how many children have experienced bird’s nest-induced anaphylaxis. The medical community may not even be aware of its existence or properly trained to diagnose it. Practitioners of traditional Chinese medicine have consistently indicated that the consumption of birds’ nest soup is beneWcial for the treatment of a variety of health problems (Koon & Cranbrook, 2002). It is often administered to the elderly and very young alike who are recovering from various types of infections. Studies have indicated that ovotransferrin is both bacteriostatic and bacteriocidal in nature (Ibrahim, Sugimoto, & Aoki, 2000). If the protein identiWed in these nests is in fact an ovotransferrin-like protein, then it could be reasonably involved in or responsible for Wghting infection just like the ovotransferrin in eggs. This may be a possible reason as to why edible bird’s nest soup has been consistently consumed throughout the centuries as prescribed traditional Chinese medicine. The salivary nest cement is the most important ingredient in the edible bird’s nest and is undoubtedly one of the most expensive food ingredients in the world. Often times, however, it has been found adulterated with Tremella fungus, karaya gum or red seaweed (Figs. 4 and 5). These three materials are typically only incorporated to a maximum of 10% to increase the overall net weight of the Wnished nest. These compounds are ideal adulterants since their color, taste, and texture are similar to that of the genuine birds nests salivary cement and are therefore often diYcult to detect. Scanning electron microscopy did not prove to be an eVective technique for determining the existence nor the type of adulteration in edible bird’s nest except for those containing added red seaweed. Unlike these adulterated with tremella fungus or plant resin, particles of red sea
Fig. 4. (A) Red seaweed; (B) karaya gum (plant resin); (C) Tremella fungus.
weed were easily distinguishable from strands of birds’ nest cement. Cross-sections of seaweed strands contained very detailed striations on raised surfaces which were distinguishable from the high amophorous strands of bird’s cement (see inset of micrograph; Fig. 6). Compositional analysis of these common adulterants revealed that they contain minerals in diVering amounts and ratios rendering each with a unique and distinguishable mineral proWle (Table 1). Since iodine and salt (NaCl) are present in red seaweed, but are not typically found in genuine birds nest, presumptive tests for iodine (iodine spot test) and salt (silver nitrate spot test) could
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M.F. Marcone / Food Research International 38 (2005) 1125–1134
Fig. 6. Scanning electron micrograph of seaweed cross-section found in adulterate nest. Not striations in box.
Fig. 5. (A, C, and E) Edible white bird’s nest (processed); (B) edible red seaweed adulterated white bird’s nest (processed); (D) edible karaya gum (plant resin) adulterated white bird’s nest (processed); (F) edible tremella fungus adulterated white bird’s nest (processed).
quickly and easily be used to detect non-heat treated seaweed adulterants (results not shown). Unfortunately, most violators heat treat their seaweed prior to adulteration, decreasing both the iodine and NaCl (to the same extent) levels and thereby limiting detection test usage. Nests containing abnormally high potassium compared to pure birds nest material could indicate adulteration with red seaweed and/or Tremella fungus, which are both relatively high in this mineral. On the other hand, nests with abnormally high levels of phosphorous could indicate adulteration with Tremella fungus, whereas high amounts of sodium could indicate adulteration with red seaweed.
Closer compositional examination of the three adulterants – namely karaya gum, red seaweed and Tremella fungus – revealed that they were relatively low in overall crude protein, i.e., 0.7%, 0.4% and 8.6%, respectively, compared to the 62% of pure white birds nest material (Table 1). Intentional additions of as little as 2% to as high as 10% (with 10% being the most commonly found level of adulteration found) substantially decreased the overall total protein content of the nest ranging from 1.1% to 6.2%, as measured by total protein analysis. Therefore, comparison of the total protein contents of suspected adulterated birds nest versus the pure edible bird’s nest could potentially be used to determine nest authenticity. Amino acid analysis revealed that white nest protein was substantially high in two speciWc aromatic amino acids, namely phenylalanine and tyrosine (Table 2). Concentrated nitric acid reacts with aromatic amino acids in proteins to produce a yellow-red color in proportion to the amount of protein present. This xanthoproteic acid test for proteins could therefore potentially be used to detect adulterated nests for sale on the market. All whole adulterated nests produced a visibly less intense yellowred reaction when treated with nitric acid when compared to the same test performed on the genuine white nests. When pieces (1.0 £ 1.5 cm) of nests that were artiWcally adulternated with karaya gum, red seaweed and
M.F. Marcone / Food Research International 38 (2005) 1125–1134
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Fig. 7. (A) Whole edible red seaweed adulterated white bird’s nest xanthoproteic nitric acid test); (B) whole pure edible white bird’s nest (xanthoproteic nitric acid test).
Tremella fungus were presented to panelists, their detection down to 1.7%, 1.8% and 3.5%, respectively, could be made. Fig. 7 shows two such nest pieces (one pure and one adulterated with 5% red seaweed) treated directly with nitric acid. The unadulterated (pure nest) was much darker in color than the adulterated one due to the higher content of protein. In the Wnely ground nests, the use of this test permitted the vision detection of adulteration to levels of 1.1% (karaya) gum, 1.2% (red seaweed), 2.0% (Tremella fungus). Fig. 8 shows the color diVerence that could be expected with adulteration of 10% typically found in the market place. This may be due to the more uniform distribution of protein and therefore more uniform color development in the test material. The use of a reXectance colourmeter made this test even more sensitive down to 0.2% for karaya gum, 0.3% for red seaweed, and 0.5% for Tremella fungus. Table 3 shows typi-
Table 3 Minolta colorimetric reading of ground bird’s nest with variable amounts of adulterants treated with nitric acid (xanthoproteic acid test) Typical adulteration level 2%
Seaweed Karaya gum Tremella fungus Pure Nest
5%
10%
L*
a*
b*
L*
a*
b*
L*
a*
b*
56a 55a 50a 43
48c 50c 50c 58
32a 30a 25a 25
60b 62b 55b
40b 40b 45b
37 35 27‘
65c 65c 60c
37a 35a 40a
42c 40c 30c
Values in each row of the same descriptor, i.e., all L*, a* or b* (separately) with the same letter are not signiWcantly diVerent (p 7 0.05).
cal L*, a*, b* values for adulteration reading between 2% and 10% which are typical ranges found in the market place.
Fig. 8. (A) Crushed edible karaya gum adulterated white bird’s nest (10% adulteration); (B) crushed edible white bird’s nest (10% adulteration) (all treated with nitric acid – xanthoproteic nitric acid spot test).
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In conclusion, this study reports on the actual compositional properties of both white and red “blood” nests revealing that they both contain predominately proteinous material followed by carbohydrate, ash and lipid (in descending order). SDS–PAGE was found to be an adequate analytical method to diVerentiate between these two nest types whereas a modiWed xanthoproteic acid test was found to facilitate the diVerentiation detection of “whote” nests that were adulterated with commonly used materials. When pieces (1.0 £ 1.5 cm) of nests artiWcally adulterated with karaya gum, red seaweed and Tremella fungus were tested, panelist could detect their presence down to 1.7%, 1.8% and 3.5% inclusion, respectively. Fig. 7 shows two nest pieces directly treated with nitric acid and shows the diVerence in color between adulterated and pure nests.
Acknowledgements The author thank Dr. Anthony Clarke of the Department of Microbiology for performing amino acid analysis and Dr. Alexandra Smith of the Department of Food Science for her assistance in X-ray microanalysis.
References AACC (American Association of Cereal Chemists). (1983). Approved Methods of the American Association of Cereal Chemists. 8th Ed. St. Paul, MN: AACC. AOAC (Association of OYcial Analytical Chemists) (1995). Liquid chromatographic mehtod for determination of triglycerides in vegetable oils in terms of ther partition numbers; summary of collaborative study. Journal of AOAC International, 77C4, 954– 957. Christie, W. W. (1982). A simple procedure for rapid transmethylation of glycerolipids and cholesterylesters. Lipid Research, 23, 1072– 1075. Dubois, H., Gilles, K. A., Hamilton, J. R., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356.
Goh, D. L. M., Chew, F.-T., Chua, K.-Y., Chay, O.-M., & Lee, B.-W. (2000). Edible “Bird’s nest”-induced anaphylaxis: an under-recognized entity?. Journal of Pediatric, 137, 277–279. Goh, D. L. M., Chew, Y.-N., Shek, F.-T., & Lee, B.-W. (1999). Alergy net-pattern of food-induced anaphylaxis in children of an Asian community. Allergy, 54(1), 84–86. Goh, D. L. M., Chua, K.-Y., Chew, F.-T., Seow, T. K., Ou, K. L., Yi, F. C., & Lee, B. W. (2001). Immunochemical characterization of edible bird’s nest allergens. Journal of Allergy and Clinical Immunology, 107(6), 1082–1088. Houston, D. M., Moore, A. E. P., Favrin, M. G., & HoV, B. (2004). Canine urolithisis: a look at over 16,000 urolith submissions to the Canadian Veterinary Urolith Center from February 1998 to April 2003. Canadian Veterinary Journal, 45, 225–230. Howe, C., Lee, L. T., & Rose, H. M. (1961). Collocalia mucoid: a substrate for Myxovirus neuraminidase. Archives of Biochemistry and Biophysics, 95, 512–520. Howe, C., Lee, L. T., & Rose, H. M. (1960). InXuenza virus sialidase. Nature, 188, 251–252. Ibrahim, H. R., Sugimoto, Y., & Aoki, T. (2000). Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism. Biochemica et Biophysica Acta, 1523, 196–205. Kong, Y. C., Keung, W. M., Tip, T. T., Ko, K. K., Tsao, S. W., & Ng, M. H. (1987). Evidence that epidermal growth factor is present in SwiXet’s (Collocalia) nest. Comparative Biochemistry and Physiology, 87(2), 221–226. Koon, L. C. (2000). Features – Bird’s nest soup – Market demand for this expensive gastronomic delicacy threatens the aptly named edible-nest SwiXets with extinction in the east. Wildlife Conservation, 103(1), 30–35. Koon, L. C., & Cranbrook, Earl of (2002). Swiftlets of Borneo – Builders of edible nests (pp. 1–171). Sabah, Malaysia: Natural History Publication (Borneo) SDN., B.H.D. Law, A. S. M., & Melville, D. S. (1994). International trade in SwiXet nests with special reference to Hong Kong. Cambridge: TraYc International. Marcone, M. F. (2004). Composition and properties of Indonesian palm civet coVee (Kopi Luwak) and Ethiopian civet coVee. Food Research International, 37, 901–912. Marcone, M. F., & Yada, R. Y. (1997). Evidence for the phosphorylation and glycosylation of the amaranth 11S globulin (Amaranthin). Food Biochemistry, 21, 341–369. Ng, M. H., Chan, K. H., & Kong, Y. C. (1986). Potentiation of mitogenic response by extracts of the Swiftlet’s (Collocalia) nest. Biochemistry International, 13(3), 521–531. SAS Institute. (1990). SAS user’s guide: statistics; statistical analysis system: Cary, NC. Williams, J., Elleman, T. C., Kingston, I. B., Wilkins, A. G., & Kuhn, K. A. (1982). The primary structure of len ovotransferrin. European Journal of Biochemistry, 122, 297–303.