- Email: [email protected]
Discriminating authentic Nostoc flagelliforme from its counterfeits by applying alternative ED-XRF and FTIR techniques
Food Chemistry 129 (2011) 528–532
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Discriminating authentic Nostoc flagelliforme from its counterfeits by applying alternative ED-XRF and FTIR techniques Angela Li ⇑, Yun Wei Yat, Wee Kim Yap, Chee Wei Lim, Sheot Harn Chan Food Safety Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, Singapore 169078, Singapore
a r t i c l e
i n f o
Article history: Received 20 April 2010 Received in revised form 7 January 2011 Accepted 16 January 2011 Available online 21 January 2011 Keywords: Nostoc flagelliforme Counterfeits Microscopy X-ray fluorescence Infrared spectroscopy
a b s t r a c t Nostoc flagelliforme is an edible blue-green algae belonging to the Nostocaceae family. It is recognised as a Chinese delicacy in south-eastern Asia and is widely consumed. Due to its high economic value and diminishing supply, as a result of overharvesting, counterfeits have often been found in the retail markets. Methods involving microscopy and histochemistry were conventionally applied to differentiate the authentic N. flagelliforme from its counterfeits. In this paper, we report an alternative analytical approach, using a combination of non-destructive energy dispersive X-ray fluorescence (ED-XRF) and Fourier-transform infrared (FTIR) spectroscopy, to achieve the objective of authentic N. flagelliforme verification. In view of the scarcity of this Chinese delicacy, such a non-destructive methodology would be ideal to preserve the integrity of the sample and yet provide a means to discriminate between authentic and counterfeit samples. Ó 2011 Published by Elsevier Ltd.
1. Introduction Nostoc flagelliforme is a terrestrial blue-green alga, grown in the arid or semi-arid geographic regions, including Algeria, China, Czechoslovakia, France, Mexico, Mongolia, Morocco, Russia, Somalia and USA. The alga adapts well to extreme environmental conditions, such as dramatic daily and yearly temperature variations and frequent wind, thereby demonstrating its ecological drought-adaptation and physiological heat-resistant capabilities (Gao, 1998). In China, it has been reported to be found in the northern and north-western regions: Qinghai, Xinjiang, Ningxia, Gansu, Shanxi and Shaanxi, Inner Mongolia and Hebei (Diao, 1996). N. flagelliforme is known to the southern Chinese population as ‘‘Facai’’, due to its black, hair-like appearance. This alga is widely used in Chinese cuisine especially during the festive seasons as its name ‘‘Facai’’ is a homonym with prosperity. The use of N. flagelliforme as a delicacy can be dated back to the Jin dynasty (A.D. 265– 316). In addition, it has also been recognised to possess herbal properties, as documented in the Compendium of Materia Medica, recorded more than 400 years ago (Gao, 1998). Rising demand for N. flagelliforme during the last century has led to extensive damage to the vegetation residing in northern and north-western China, thereby giving rise to desertification. As a result, the State Council of the People’s Republic of China imposed a
⇑ Corresponding author. Tel.: +65 6213 0735; fax: +65 6213 0749. E-mail address: [email protected] (A. Li). 0308-8146/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.foodchem.2011.01.054
ban on the collection and trading of N. flagelliforme in 2000, in an effort to begin the ecological conservation of northern China. This conservation strategy created a domino effect on the supply and demand food chain for ‘‘Facai’’. Specifically, it triggered a sharp reduction of supplies in countries outside China, causing a resultant price hike. Being a premium foodstuff, it has been transformed into a prime target for adulteration by profit-driven producers. Conventional methods used to differentiate the authentic alga from its counterfeits include microscopy, iodine staining colour test and elemental analysis involving spectroscopy. Microscopy emphasises on the unique cellular structure characteristics of the alga, to achieve a visual screening only. In iodine staining (a histochemical method), imitation alga sample will turn dark blue or black in the presence of iodine solution, indicating presence of amylase (a natural component of starch) while the authentic alga will remain dull greenish. However, the latter offers no discriminatory advantage for adulterated samples containing both authentic and fake algae. Elemental analysis techniques often involve a sample preparation step, such as acid digestion, before performing analysis. Such techniques include atomic absorption spectroscopy (AAS) and inductively-coupled plasma mass spectrometry (ICP/ MS). This paper describes the application of dispersive X-ray fluorescence spectroscopy, which can effectively exclude contamination sources originating from apparatus, reagents and environment, since minimum sample preparation is required. Instead, direct (non-destructive) sample analysis can be performed with the added advantage of shorter analysis time. In this paper, we applied an alternative combination of microscopy and spectroscopic techniques, aimed at providing qualitative
A. Li et al. / Food Chemistry 129 (2011) 528–532
529
and quantitative (relative abundance) fingerprints of alga samples obtained from different sources, to establish a systematic workflow tailored for their unambiguous positive identification and classification. For comparison purposes, microscopy was applied, to obtain the morphological features of the algae, operating under transmission mode to distinguish between the alga samples. With the alternative analytical approach, elemental compositions were first obtained by applying energy dispersive X-ray fluorescence spectroscopy, followed by structural elucidation using Fouriertransform infrared (FTIR) spectroscopy.
2. Materials and methods 2.1. N. flagelliforme samples Due to the rarity of the N. flagelliforme, only seven samples could be obtained for this study. The authentic samples were sourced from reliable wholesalers while the remaining five samples were purchased from the retail markets. A portion of the dry samples of N. flagelliforme were first left to soak in water overnight and then examined under a polarising microscope the next day. Another portion of the as-received dry sample of N. flagelliforme was cut into small pieces and transferred into a disposable sample holder (about 0.5 g each), which was then placed in the analysis chamber for analysis using the ED-XRF. The remaining dry samples of N. flagelliforme were analysed directly using the FTIR. The same set of samples was used to perform all three types of analysis throughout this study.
Fig. 1a. Bead-like cell filaments of the authentic N. flagelliforme.
2.2. Polarising microscope analysis A DMRP cross-polarising microscope (Leica, Wetzlar, Germany) was used to study the cellular characteristics using the authentic sample micrograph as a reference standard. The soaked strands of the N. flagelliforme were drained and cut diagonally or ground with mortar and pestle to expose the underlying bead-like filament of prokaryotic cells, visible through the loosening of the gelatinous sheath on the alga before mounting on glass slides for examination.
2.3. ED-XRF analysis Elemental analysis was performed by using an energy dispersive X-ray spectrometer EDX-720 (Shimadzu, Tokyo, Japan). Quantitative analysis was carried out using the Standard-less Quantitative Analysis Software, by setting the accelerating voltage to 50 kV for elements from titanium to uranium, and 15 kV for elements from sodium to scandium. To achieve higher sensitivity for analysis, primary X-ray filters were used for elements from zinc to arsenic and lead, as well as from sulphur to potassium. Two detection time settings were used for data acquisitions. All scans were set to a detection time of 100 s while scans acquired using the primary filter, zinc to arsenic and lead, were set to a detection time of 200 s. The number of Ka or La counts per second (cps/lA) was measured for each of the elements present and quantified using the preset algorithm built into the software. Due to the unavailability of a calibration standard using elements detected in the same sample matrix, the values obtained were further computed to reflect their relative abundance with respect to the iron content present in the individual samples. An average for each sample category (authentic, imitation and adulterated) was then taken. This form of data treatment takes into account variation in the proportions of macromolecular pools among algae samples and their respective nutrient availability.
Fig. 1b. Adulterated sample containing some authentic N. flagelliforme strands.
Fig. 1c. Non-cellular material with masses of brown and black pigments.
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Fig. 2a. Starch granules found in the imitation N. flagelliforme samples.
Fig. 2b. Starch granules observed under polarised light.
2.4. FTIR analysis The chemical bonding or molecular structure of the N. flagelliforme was analysed using a Nicolet 6700 FT-IR spectrometer Smart Durascope (Thermo Scientific Inc., Waltham, MA) equipped with a deuterated triglycine sulphate (DTGS) detector and a KBr beamsplitter. The OMNIC software was used to perform spectrum acquisition and data processing. The spectrometer was fitted with a single reflection diamond horizontal attenuated total reflection (ATR) module, to facilitate direct sample analysis without the need for sample preparation. The incident beam has a probing depth of 1 lm at 1000 cm 1. For each sample analysed, sixty-eight scans were acquired for the frequency region of 4000–650 cm 1 at a base resolution of 4 cm 1. An average of these scans was then taken.
3. Results and discussion N. flagelliforme showed unique cellular characteristics, represented by the presence of filaments bound together as macroscopically recognisable strands by a gelatinous sheath (But, Cheng, Chan, Lau, & But, 2002). By utilising a cross-polarising microscope operating under non-polarising transmission mode, filaments of bead-like cells can be observed for specimens containing the authen-
tic N. flagelliforme. Amongst the seven N. flagelliforme samples examined, the two authentic specimens exhibited the typical bead-like cell filaments characteristic of genuine N. flagelliforme. The micrographs of three other samples revealed the presence of additional other cell-like structures, suggesting an adulteration being made to the original alga matrix. In contrast, micrographs of the remaining two samples showed complete absence of bead-like cell filament, indicating that the samples were not of N. flagelliforme origin. Representative micrographs showing the authentic, adulterated and imitation algae are illustrated in Figs. 1a–c, respectively. In both Figs. 1a and b, cell walls may be observed. This suggested that the N. flagelliforme is closer to bacteria in terms of cellular composition than an alga (Murdock & Wetzel, 2009). Of interest, the non-cellular material present in the adulterated and imitation algae samples appeared as aggregates of brown and black pigments, together with some starch granules as shown in Fig. 2a. These starch granules take on complex geometries, from transparent disc-like and spherical shapes, to polyhedral, rounded or elongated irregular filaments. Within the framework of two-dimensional microscopy, the morphology of these starch granules bears resemblance to the morphological features of starch granules present in major plant sources, such as wheat and maize, as reported by Buléon, Colonna, Planchot, and Ball (1998). Under polarised light illumination, these starch granules displayed the characteristic dark crosses centred at the hilum (Fig. 2b). Although microscopy provided a relatively simple method of differentiating the authentic, adulterated and imitation algae, a unique approach was employed to provide a different perspective in discriminating these samples. Elemental analysis results from the ED-XRF showed that the authentic N. flagelliforme contained a series of elements, namely silicon, magnesium, sulphur, potassium, phosphorus, barium, chlorine, strontium and iron. These elements were also detected in alga samples containing adulterations. A summary of these ratios is shown in Table 1. With reference to Table 1, a striking difference was observed for the imitation alga samples, with the absence of magnesium, phosphorus and barium, thus forming a unique fingerprint for the identification of the N. flagelliforme samples. Another interesting finding was that the relative abundance for chlorine was significantly higher for the imitation samples, as compared to the others, suggesting the presence of common food additives, such as sodium chloride. Potassium was found to be present in all three types of alga samples studied, with the imitation alga samples having the lowest relative abundance and the authentic samples having the highest. Potassium is a major macronutrient associated with vital roles, such as stomatal behaviour, osmoregulation, enzyme activity, cell expansion, neutralisation of nondiffusible negatively-charged ions and membrane polarisation. The majority of the potassium intake by plants from the soil is used to maintain growth and development (Eisenman, 1961; Epstein, 1972; Clarkson & Hanson, 1980; Flowers & Läuchli, 1983; Kochian & Lucas, 1988; Schroeder, Ward, & Gassmann, 1994; Maathuis & Sanders, 1996; Elumalai, Nagpal, & Reed, 2002). In addition, high potassium concentrations are closely associated with drought resistance in plants. Clearly, potassium is an element that is fundamental to micro-organism sustenance under adverse conditions. However, its presence or absence alone is insufficient to be used as a marker to indicate if the alga samples analysed were authentic or to facilitate further classification. As a terrestrial alga that thrives in arid or semi-arid areas, the N. flagelliforme would probably contain elements whose concentrations are intimately associated with soils under different agricultural environments. Indeed, in a study conducted by Su and Yang (2008), comparing twenty-four chemical elements present in the surface layer of natural desert soils and cultivated farmland soils at a desert-oasis ecotone in north-west China, concentrations of strontium and silica were reported to be significantly lower in
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A. Li et al. / Food Chemistry 129 (2011) 528–532 Table 1 ED-XRF results showing the relative abundance of elements detected with respect to iron for each sample category. Identification by microscope
Authentic Imitation Adulterated a
Relative abundance to iron Silicon
Magnesium
Sulphur
Potassium
Phosphorus
Barium
Chlorine
Strontium
8.80 2.36 7.05
7.39 NDa 6.12
7.12 1.67 5.80
5.08 0.26 4.13
1.65 ND 1.49
2.25 ND 1.24
2.40 10.78 2.28
0.42 0.03 0.30
ND: not detected.
23
Band C
Arbitrary Absorbance Units
Band A Band B
(c)
(b) (a) 0 1000
1500
2000
2500
3000
3500
Wavenumber (cm-1) Fig. 3. FTIR spectra showing vibration modes of alga samples containing (a) authentic strand, (b) adulteration and (c) no N. flagelliforme characteristics (imitation). Band A is assigned to water while Band B is assigned to ‘‘lipids’’. Band C is assigned to carbohydrates.
agricultural soils than in desert soils. Referring to Table 1, we found that the relative abundance for strontium and silicon to be higher for the authentic N. flagelliforme samples, thus supporting the reported findings by Su and Yang (2008). Furthermore, the relative abundances of silicon and sulphur present in both the authentic alga sample and the adulterated alga sample are significantly higher than those observed for the imitation alga sample. This observation is interesting in that it shows a clear demarcation, differentiating the authentic alga from its counterfeit. In particular, silicon is reported to be the building block fundamental to some types of cell walls (Murdock & Wetzel, 2009). We do not have a clear understanding on the role of sulphur in our alga samples, but propose that these compounds (along with chlorine) are fundamental towards establishing a complete elemental profile of the authentic alga. It must be noted that depending on the degree of adulteration in the N. flagelliforme samples, the elemental relative abundance in these samples may display trends that are similar to the authentic or the imitation alga. Clearly, by performing non-destructive energy-dispersive X-ray fluorescence spectroscopy, we are able to create an unambiguous elemental profile characteristic of the N. flagelliforme and use it as a reference criterion to facilitate further alga sample discrimination. Structural elucidation achieved by performing FTIR revealed that all alga samples analysed contained some similar group frequencies. These frequencies are represented by a broad absorption band from 3570–3100 cm 1 (A), a weak absorption centred at 2920 cm 1 (B) and a strong absorption centred at 1000 cm 1 (C). We attribute the bands at A, B and C to be due to hydroxyl group OAH stretching, alcohol related CAO stretching and aliphatic organic compound related CAH stretching, respectively, as illustrated in Fig. 3. Common to all spectra, characteristic absorption
bands in the region of 1870–1650 cm 1 were absent. This region is commonly populated by compounds containing carbonyl functional group. Its absence may possibly suggest that monosaccharides, such as simple sugars, are not present in the alga samples. Comparing the spectra of the reference alga in Fig. 3a with alga samples containing adulteration (Fig. 3b) and samples not of N. flagelliforme origin (Fig. 3c), there exist significant differences in absorption bands at some localised regions. The imitation alga sample lacked vibration modes at an absorption band from 1700 to 1400 cm 1. This broad absorption band is populated by two dominant peaks centred at about 1600 and 1540 cm 1, respectively. From Fig. 3a and b, the separation between these peaks became more pronounced and disappeared totally in Fig. 3c. To understand the change in intensity of these vibration modes, we refer to the micrographs representing the morphology of samples containing adulteration and the imitation samples. We found evidence of an increase in starch content represented by the observation of increasing amount of starch-related morphology from Fig. 3a–c, respectively. This observation suggested that vibration modes related to carbohydrates such as starch may be more pronounced for alga samples containing fewer N. flagelliforme characteristics. Indeed, Fig. 3c showed a feature-rich absorption band located at 1200–1100 cm 1 band, which is commonly assigned to the CAO stretching of carbohydrates, such as starch (Coates, 2000). Together with the micrographs showing presence of cellwalls in alga samples from Figs. 1a and b, we propose the absorption bands centred at 1600 and 1540 cm 1 to be group frequencies, due to protein related amide I and amide II functional groups. Clearly by surveying the absorption bands located at 1700– 1400 cm 1 and 1200–1100 cm 1, we are able to perform further discrimatory classification of authentic alga from those which are not of N. flagelliforme origin. 4. Conclusions In conclusion, we have applied a combination of two analytical techniques, namely energy-dispersive X-ray fluorescence and FTIR spectroscopy to perform alga samples discrimination, aimed at differentiating the authentic alga from its counterfeit. We found that apart from the presence of bead-like filament of prokaryotic cells observed under the microscope, authentic alga samples actually showed representative elemental fingerprints, as revealed by using energy dispersive X-ray fluorescence spectroscopy. Together with the unambig uous functional group, identified by using FTIR, we have demonstrated a systematic, rapid and innovative approach to authentic N. flagelliforme identification and discrimination, while maintaining sample integrity. These methods thus applied perhaps offer statefunded technical programs focused on the biology and cultivation techniques of N. flagelliforme (aimed at meeting market demand for the alga (Gao, 1998)), a solution to achieve true alga verification. Acknowledgements The authors would like to acknowledge the technical support from Shimadzu (Asia Pacific) Pte Ltd. for the use of their ED-XRF
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system, the Forensic Chemistry and Physics Laboratory at the Health Sciences Authority for the use of their polarising microscope as well as the Pharmaceutical Laboratory at the Health Sciences Authority for the use of their FTIR system. References Buléon, A., Colonna, A., Planchot, T., & Ball, S. (1998). Starch granules: Structure and biosynthesis. International Journal of Biological Macromolecules, 23, 85–112. But, P. P. H., Cheng, L., Chan, P. K., Lau, D. T. W., & But, J. W. H. (2002). Nostoc flagelliforme and fakes items retailed in Hong Kong. Journal of Applied Phycology, 14, 143–145. Clarkson, D. T., & Hanson, J. B. (1980). The mineral nutrition of higher plants. Annual Review of Plant Physiology, 31, 239–298. Coates, J. (2000). Interpretation of infrared spectra, a practical approach. In R. A. Meyers (Ed.), Encyclopedia of analytical chemistry (pp. 10815–10837). Chichester: John Wiley and Sons Ltd. Diao, X. M. (1996). Study of natural conditions and ecological physiology characteristics for growth of Nostoc flagelliforme in Qinghai province. Chinese Journal of Ecology, 15, 8–13 (in Chinese with English summary). Eisenman, G. (1961). On the elementary atomic origin of equilibrium ionic specificity. In A. Kleinzeller & A. Kotyk (Eds.), Symposium on membrane transport and metabolism (pp. 163–179). New York: Academic Press.
Elumalai, R. P., Nagpal, P., & Reed, J. W. (2002). A mutation in the arabidopsis KT2/ KUP2 potassium transported gene affects shoot cell expansion. The Plant Cell, 14, 119–131. Epstein, E. (1972). Mineral nutrition of plants: Principles and perspectives. New York: Wiley. Flowers, T. J., & Läuchli, A. (1983). Sodium versus potassium: Substitution and compartmentalization. In A. Läuchli & R. L. Bieleski (Eds.), Inorganic plant nutrition (pp. 651–681). Berlin: Springer. Gao, K. (1998). Chinese studies on the edible blue-green alga, Nostoc flagelliforme: A review. Journal of Applied Phycology, 10, 37–49. Kochian, L. V., & Lucas, W. J. (1988). Potassium transport in roots. In J. A. Callow (Ed.), Advances in botanical research (pp. 93–178). London: Academic Press Ltd. Maathuis, F. J. M., & Sanders, D. (1996). Mechanisms of potassium absorption by higher plant roots. Physiologia Plantarum, 96, 158–168. Murdock, J. N., & Wetzel, D. L. (2009). FT-IR microscopy enhances biological and ecological analysis of algae. Applied Spectroscopy Reviews, 44, 335–361. Schroeder, J. I., Ward, J. M., & Gassmann, W. (1994). Perspectives on the physiology and structure of inward-rectifying K+ channels in higher plants: Biophysical implications for K+ uptake. Annual Review of Biophysics and Biomolecular Structure, 23, 441–471. Su, Y. Z., & Yang, R. (2008). Background concentrations of elements in surface soils and their changes as affected by agriculture use in the desert-oasis ecotone in the middle of Heihe River Basin, North-west China. Journal of Geochemical Exploration, 98, 57–64.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Discriminating authentic Nostoc flagelliforme from its counterfeits by applying alternative ED-XRF and FTIR techniques Angela Li ⇑, Yun Wei Yat, Wee Kim Yap, Chee Wei Lim, Sheot Harn Chan Food Safety Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram Road, Singapore 169078, Singapore
a r t i c l e
i n f o
Article history: Received 20 April 2010 Received in revised form 7 January 2011 Accepted 16 January 2011 Available online 21 January 2011 Keywords: Nostoc flagelliforme Counterfeits Microscopy X-ray fluorescence Infrared spectroscopy
a b s t r a c t Nostoc flagelliforme is an edible blue-green algae belonging to the Nostocaceae family. It is recognised as a Chinese delicacy in south-eastern Asia and is widely consumed. Due to its high economic value and diminishing supply, as a result of overharvesting, counterfeits have often been found in the retail markets. Methods involving microscopy and histochemistry were conventionally applied to differentiate the authentic N. flagelliforme from its counterfeits. In this paper, we report an alternative analytical approach, using a combination of non-destructive energy dispersive X-ray fluorescence (ED-XRF) and Fourier-transform infrared (FTIR) spectroscopy, to achieve the objective of authentic N. flagelliforme verification. In view of the scarcity of this Chinese delicacy, such a non-destructive methodology would be ideal to preserve the integrity of the sample and yet provide a means to discriminate between authentic and counterfeit samples. Ó 2011 Published by Elsevier Ltd.
1. Introduction Nostoc flagelliforme is a terrestrial blue-green alga, grown in the arid or semi-arid geographic regions, including Algeria, China, Czechoslovakia, France, Mexico, Mongolia, Morocco, Russia, Somalia and USA. The alga adapts well to extreme environmental conditions, such as dramatic daily and yearly temperature variations and frequent wind, thereby demonstrating its ecological drought-adaptation and physiological heat-resistant capabilities (Gao, 1998). In China, it has been reported to be found in the northern and north-western regions: Qinghai, Xinjiang, Ningxia, Gansu, Shanxi and Shaanxi, Inner Mongolia and Hebei (Diao, 1996). N. flagelliforme is known to the southern Chinese population as ‘‘Facai’’, due to its black, hair-like appearance. This alga is widely used in Chinese cuisine especially during the festive seasons as its name ‘‘Facai’’ is a homonym with prosperity. The use of N. flagelliforme as a delicacy can be dated back to the Jin dynasty (A.D. 265– 316). In addition, it has also been recognised to possess herbal properties, as documented in the Compendium of Materia Medica, recorded more than 400 years ago (Gao, 1998). Rising demand for N. flagelliforme during the last century has led to extensive damage to the vegetation residing in northern and north-western China, thereby giving rise to desertification. As a result, the State Council of the People’s Republic of China imposed a
⇑ Corresponding author. Tel.: +65 6213 0735; fax: +65 6213 0749. E-mail address: [email protected] (A. Li). 0308-8146/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.foodchem.2011.01.054
ban on the collection and trading of N. flagelliforme in 2000, in an effort to begin the ecological conservation of northern China. This conservation strategy created a domino effect on the supply and demand food chain for ‘‘Facai’’. Specifically, it triggered a sharp reduction of supplies in countries outside China, causing a resultant price hike. Being a premium foodstuff, it has been transformed into a prime target for adulteration by profit-driven producers. Conventional methods used to differentiate the authentic alga from its counterfeits include microscopy, iodine staining colour test and elemental analysis involving spectroscopy. Microscopy emphasises on the unique cellular structure characteristics of the alga, to achieve a visual screening only. In iodine staining (a histochemical method), imitation alga sample will turn dark blue or black in the presence of iodine solution, indicating presence of amylase (a natural component of starch) while the authentic alga will remain dull greenish. However, the latter offers no discriminatory advantage for adulterated samples containing both authentic and fake algae. Elemental analysis techniques often involve a sample preparation step, such as acid digestion, before performing analysis. Such techniques include atomic absorption spectroscopy (AAS) and inductively-coupled plasma mass spectrometry (ICP/ MS). This paper describes the application of dispersive X-ray fluorescence spectroscopy, which can effectively exclude contamination sources originating from apparatus, reagents and environment, since minimum sample preparation is required. Instead, direct (non-destructive) sample analysis can be performed with the added advantage of shorter analysis time. In this paper, we applied an alternative combination of microscopy and spectroscopic techniques, aimed at providing qualitative
A. Li et al. / Food Chemistry 129 (2011) 528–532
529
and quantitative (relative abundance) fingerprints of alga samples obtained from different sources, to establish a systematic workflow tailored for their unambiguous positive identification and classification. For comparison purposes, microscopy was applied, to obtain the morphological features of the algae, operating under transmission mode to distinguish between the alga samples. With the alternative analytical approach, elemental compositions were first obtained by applying energy dispersive X-ray fluorescence spectroscopy, followed by structural elucidation using Fouriertransform infrared (FTIR) spectroscopy.
2. Materials and methods 2.1. N. flagelliforme samples Due to the rarity of the N. flagelliforme, only seven samples could be obtained for this study. The authentic samples were sourced from reliable wholesalers while the remaining five samples were purchased from the retail markets. A portion of the dry samples of N. flagelliforme were first left to soak in water overnight and then examined under a polarising microscope the next day. Another portion of the as-received dry sample of N. flagelliforme was cut into small pieces and transferred into a disposable sample holder (about 0.5 g each), which was then placed in the analysis chamber for analysis using the ED-XRF. The remaining dry samples of N. flagelliforme were analysed directly using the FTIR. The same set of samples was used to perform all three types of analysis throughout this study.
Fig. 1a. Bead-like cell filaments of the authentic N. flagelliforme.
2.2. Polarising microscope analysis A DMRP cross-polarising microscope (Leica, Wetzlar, Germany) was used to study the cellular characteristics using the authentic sample micrograph as a reference standard. The soaked strands of the N. flagelliforme were drained and cut diagonally or ground with mortar and pestle to expose the underlying bead-like filament of prokaryotic cells, visible through the loosening of the gelatinous sheath on the alga before mounting on glass slides for examination.
2.3. ED-XRF analysis Elemental analysis was performed by using an energy dispersive X-ray spectrometer EDX-720 (Shimadzu, Tokyo, Japan). Quantitative analysis was carried out using the Standard-less Quantitative Analysis Software, by setting the accelerating voltage to 50 kV for elements from titanium to uranium, and 15 kV for elements from sodium to scandium. To achieve higher sensitivity for analysis, primary X-ray filters were used for elements from zinc to arsenic and lead, as well as from sulphur to potassium. Two detection time settings were used for data acquisitions. All scans were set to a detection time of 100 s while scans acquired using the primary filter, zinc to arsenic and lead, were set to a detection time of 200 s. The number of Ka or La counts per second (cps/lA) was measured for each of the elements present and quantified using the preset algorithm built into the software. Due to the unavailability of a calibration standard using elements detected in the same sample matrix, the values obtained were further computed to reflect their relative abundance with respect to the iron content present in the individual samples. An average for each sample category (authentic, imitation and adulterated) was then taken. This form of data treatment takes into account variation in the proportions of macromolecular pools among algae samples and their respective nutrient availability.
Fig. 1b. Adulterated sample containing some authentic N. flagelliforme strands.
Fig. 1c. Non-cellular material with masses of brown and black pigments.
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A. Li et al. / Food Chemistry 129 (2011) 528–532
Fig. 2a. Starch granules found in the imitation N. flagelliforme samples.
Fig. 2b. Starch granules observed under polarised light.
2.4. FTIR analysis The chemical bonding or molecular structure of the N. flagelliforme was analysed using a Nicolet 6700 FT-IR spectrometer Smart Durascope (Thermo Scientific Inc., Waltham, MA) equipped with a deuterated triglycine sulphate (DTGS) detector and a KBr beamsplitter. The OMNIC software was used to perform spectrum acquisition and data processing. The spectrometer was fitted with a single reflection diamond horizontal attenuated total reflection (ATR) module, to facilitate direct sample analysis without the need for sample preparation. The incident beam has a probing depth of 1 lm at 1000 cm 1. For each sample analysed, sixty-eight scans were acquired for the frequency region of 4000–650 cm 1 at a base resolution of 4 cm 1. An average of these scans was then taken.
3. Results and discussion N. flagelliforme showed unique cellular characteristics, represented by the presence of filaments bound together as macroscopically recognisable strands by a gelatinous sheath (But, Cheng, Chan, Lau, & But, 2002). By utilising a cross-polarising microscope operating under non-polarising transmission mode, filaments of bead-like cells can be observed for specimens containing the authen-
tic N. flagelliforme. Amongst the seven N. flagelliforme samples examined, the two authentic specimens exhibited the typical bead-like cell filaments characteristic of genuine N. flagelliforme. The micrographs of three other samples revealed the presence of additional other cell-like structures, suggesting an adulteration being made to the original alga matrix. In contrast, micrographs of the remaining two samples showed complete absence of bead-like cell filament, indicating that the samples were not of N. flagelliforme origin. Representative micrographs showing the authentic, adulterated and imitation algae are illustrated in Figs. 1a–c, respectively. In both Figs. 1a and b, cell walls may be observed. This suggested that the N. flagelliforme is closer to bacteria in terms of cellular composition than an alga (Murdock & Wetzel, 2009). Of interest, the non-cellular material present in the adulterated and imitation algae samples appeared as aggregates of brown and black pigments, together with some starch granules as shown in Fig. 2a. These starch granules take on complex geometries, from transparent disc-like and spherical shapes, to polyhedral, rounded or elongated irregular filaments. Within the framework of two-dimensional microscopy, the morphology of these starch granules bears resemblance to the morphological features of starch granules present in major plant sources, such as wheat and maize, as reported by Buléon, Colonna, Planchot, and Ball (1998). Under polarised light illumination, these starch granules displayed the characteristic dark crosses centred at the hilum (Fig. 2b). Although microscopy provided a relatively simple method of differentiating the authentic, adulterated and imitation algae, a unique approach was employed to provide a different perspective in discriminating these samples. Elemental analysis results from the ED-XRF showed that the authentic N. flagelliforme contained a series of elements, namely silicon, magnesium, sulphur, potassium, phosphorus, barium, chlorine, strontium and iron. These elements were also detected in alga samples containing adulterations. A summary of these ratios is shown in Table 1. With reference to Table 1, a striking difference was observed for the imitation alga samples, with the absence of magnesium, phosphorus and barium, thus forming a unique fingerprint for the identification of the N. flagelliforme samples. Another interesting finding was that the relative abundance for chlorine was significantly higher for the imitation samples, as compared to the others, suggesting the presence of common food additives, such as sodium chloride. Potassium was found to be present in all three types of alga samples studied, with the imitation alga samples having the lowest relative abundance and the authentic samples having the highest. Potassium is a major macronutrient associated with vital roles, such as stomatal behaviour, osmoregulation, enzyme activity, cell expansion, neutralisation of nondiffusible negatively-charged ions and membrane polarisation. The majority of the potassium intake by plants from the soil is used to maintain growth and development (Eisenman, 1961; Epstein, 1972; Clarkson & Hanson, 1980; Flowers & Läuchli, 1983; Kochian & Lucas, 1988; Schroeder, Ward, & Gassmann, 1994; Maathuis & Sanders, 1996; Elumalai, Nagpal, & Reed, 2002). In addition, high potassium concentrations are closely associated with drought resistance in plants. Clearly, potassium is an element that is fundamental to micro-organism sustenance under adverse conditions. However, its presence or absence alone is insufficient to be used as a marker to indicate if the alga samples analysed were authentic or to facilitate further classification. As a terrestrial alga that thrives in arid or semi-arid areas, the N. flagelliforme would probably contain elements whose concentrations are intimately associated with soils under different agricultural environments. Indeed, in a study conducted by Su and Yang (2008), comparing twenty-four chemical elements present in the surface layer of natural desert soils and cultivated farmland soils at a desert-oasis ecotone in north-west China, concentrations of strontium and silica were reported to be significantly lower in
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A. Li et al. / Food Chemistry 129 (2011) 528–532 Table 1 ED-XRF results showing the relative abundance of elements detected with respect to iron for each sample category. Identification by microscope
Authentic Imitation Adulterated a
Relative abundance to iron Silicon
Magnesium
Sulphur
Potassium
Phosphorus
Barium
Chlorine
Strontium
8.80 2.36 7.05
7.39 NDa 6.12
7.12 1.67 5.80
5.08 0.26 4.13
1.65 ND 1.49
2.25 ND 1.24
2.40 10.78 2.28
0.42 0.03 0.30
ND: not detected.
23
Band C
Arbitrary Absorbance Units
Band A Band B
(c)
(b) (a) 0 1000
1500
2000
2500
3000
3500
Wavenumber (cm-1) Fig. 3. FTIR spectra showing vibration modes of alga samples containing (a) authentic strand, (b) adulteration and (c) no N. flagelliforme characteristics (imitation). Band A is assigned to water while Band B is assigned to ‘‘lipids’’. Band C is assigned to carbohydrates.
agricultural soils than in desert soils. Referring to Table 1, we found that the relative abundance for strontium and silicon to be higher for the authentic N. flagelliforme samples, thus supporting the reported findings by Su and Yang (2008). Furthermore, the relative abundances of silicon and sulphur present in both the authentic alga sample and the adulterated alga sample are significantly higher than those observed for the imitation alga sample. This observation is interesting in that it shows a clear demarcation, differentiating the authentic alga from its counterfeit. In particular, silicon is reported to be the building block fundamental to some types of cell walls (Murdock & Wetzel, 2009). We do not have a clear understanding on the role of sulphur in our alga samples, but propose that these compounds (along with chlorine) are fundamental towards establishing a complete elemental profile of the authentic alga. It must be noted that depending on the degree of adulteration in the N. flagelliforme samples, the elemental relative abundance in these samples may display trends that are similar to the authentic or the imitation alga. Clearly, by performing non-destructive energy-dispersive X-ray fluorescence spectroscopy, we are able to create an unambiguous elemental profile characteristic of the N. flagelliforme and use it as a reference criterion to facilitate further alga sample discrimination. Structural elucidation achieved by performing FTIR revealed that all alga samples analysed contained some similar group frequencies. These frequencies are represented by a broad absorption band from 3570–3100 cm 1 (A), a weak absorption centred at 2920 cm 1 (B) and a strong absorption centred at 1000 cm 1 (C). We attribute the bands at A, B and C to be due to hydroxyl group OAH stretching, alcohol related CAO stretching and aliphatic organic compound related CAH stretching, respectively, as illustrated in Fig. 3. Common to all spectra, characteristic absorption
bands in the region of 1870–1650 cm 1 were absent. This region is commonly populated by compounds containing carbonyl functional group. Its absence may possibly suggest that monosaccharides, such as simple sugars, are not present in the alga samples. Comparing the spectra of the reference alga in Fig. 3a with alga samples containing adulteration (Fig. 3b) and samples not of N. flagelliforme origin (Fig. 3c), there exist significant differences in absorption bands at some localised regions. The imitation alga sample lacked vibration modes at an absorption band from 1700 to 1400 cm 1. This broad absorption band is populated by two dominant peaks centred at about 1600 and 1540 cm 1, respectively. From Fig. 3a and b, the separation between these peaks became more pronounced and disappeared totally in Fig. 3c. To understand the change in intensity of these vibration modes, we refer to the micrographs representing the morphology of samples containing adulteration and the imitation samples. We found evidence of an increase in starch content represented by the observation of increasing amount of starch-related morphology from Fig. 3a–c, respectively. This observation suggested that vibration modes related to carbohydrates such as starch may be more pronounced for alga samples containing fewer N. flagelliforme characteristics. Indeed, Fig. 3c showed a feature-rich absorption band located at 1200–1100 cm 1 band, which is commonly assigned to the CAO stretching of carbohydrates, such as starch (Coates, 2000). Together with the micrographs showing presence of cellwalls in alga samples from Figs. 1a and b, we propose the absorption bands centred at 1600 and 1540 cm 1 to be group frequencies, due to protein related amide I and amide II functional groups. Clearly by surveying the absorption bands located at 1700– 1400 cm 1 and 1200–1100 cm 1, we are able to perform further discrimatory classification of authentic alga from those which are not of N. flagelliforme origin. 4. Conclusions In conclusion, we have applied a combination of two analytical techniques, namely energy-dispersive X-ray fluorescence and FTIR spectroscopy to perform alga samples discrimination, aimed at differentiating the authentic alga from its counterfeit. We found that apart from the presence of bead-like filament of prokaryotic cells observed under the microscope, authentic alga samples actually showed representative elemental fingerprints, as revealed by using energy dispersive X-ray fluorescence spectroscopy. Together with the unambig uous functional group, identified by using FTIR, we have demonstrated a systematic, rapid and innovative approach to authentic N. flagelliforme identification and discrimination, while maintaining sample integrity. These methods thus applied perhaps offer statefunded technical programs focused on the biology and cultivation techniques of N. flagelliforme (aimed at meeting market demand for the alga (Gao, 1998)), a solution to achieve true alga verification. Acknowledgements The authors would like to acknowledge the technical support from Shimadzu (Asia Pacific) Pte Ltd. for the use of their ED-XRF
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