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Home , Betanin, Pitaya

ANALYTICAL TECHNOLOGIES

LUISALDO SANDATE-FLORES1, JOSÉ RODRÍGUEZ-RODRÍGUEZ1, SAMANTHA CALVO-SEGURA2, ARTURO MAYORGA-MARTÍNEZ2, ROBERTO PARRA-SALDÍVAR1, CRISTINA CHUCK-HERNÁNDEZ2* *Corresponding author 1. Centro de Biotecnología FEMSA, School of Engineering and Sciences, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, CP 64849, Monterrey, N.L., México 2. Research Centre for Protein Development (CIDPRO), School of Engineering and Sciences, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, CP 64849, Monterrey, N.L., México

Luisaldo Sandate-Flores Evaluation of different methods for quantification in ( spp.)

KEYWORDS: Betanin, pitaya, Stenocereus, quantification, HPLC.

Spectrophotometry UV-Vis, with calibration curve (SWCC), High-performance liquid chromatography Abstract (HPLC), and Spectrophotometry with an extinction coefficient (SWEC) were used for betanin quantification in pulp and of pitaya (Stenocereus spp). The concentrations of pulp betanin measured by the three analytical methods did not differ significantly, as assessed by the Scheffe method (P > 0.05). The mean of pulp betanin was 2.092 mg/g dry sample. With respect to the peel samples, there was a significant difference: a lower betanin concentration was obtained by HPLC. Since the SWCC method required a standard solution, and that HPLC was time-consuming, the SWEC method represented the best choice for betanin quantification in pulp in order to reduce cost, time, and wastes.

INTRODUCTION despite the fact that its peel, pulp and have very attractive colours that go from white, pinkish, yellow to red In the food industry, the use of synthetic dyes (especially and even purplish, hues related to the presence of the two the red and yellow ones) has been restricted due to main types of : the red violet betacyanins and the their potential risks for human health (1). Consequently, yellow betaxanthines (4). there has been a growing interest in the development of However, it is important to take into account that these natural additives. Besides, consumers have been lately pigments are susceptible to temperature, , UV light, inclined to prefer natural products because of their eco- pH, and different cofactors during processing or storage (13). friendliness, apparent lack of toxicity, and nutraceutical The method to quantify this component should be therefore properties (2, 3). Some natural colours, as betalains, are easy, quick and with a minimum sample preparation. ; these help reduce cardiovascular diseases, Traditionally, spectrophotometric methods are used to cancer, and disorders associated to ageing (4, 5). quantify betacyanins and betaxanthins in food products. Betalains have been used as food colourants in gelatines, The former absorbs light at 540nm and the latter at 480nm desserts, confectioneries, dry mixes, poultry, dairy, and (15, 16-22). Nilsson (1970) established a spectrophotometric products (6). Betalains are divided into two groups: method for fresh red beets (23). Nevertheless, Schwartz (24) betacyanins,that provide red hues and betaxanthins that reported that this method was made for small amounts of provide yellow colours. Betanin is the dye that contributes interfering substances, and that it was not either intended from 75 to 95% to red colour in betacyanins. In order to to measure pigment content in samples that had been obtain the desired hue in the product, the amount of subjected to heat treatments (24, 25). Schwartz proposed a to be added, calculated as betanin, does not time-consuming isolation of crystalline reference substances exceed 150 ppm (7). for quantifi cation purposes. Betalains are water-soluble polyphenolic pigments, found in High-performance liquid chromatography (HPLC) is a plants related to the order Caryophyllales, which includes separation method widely used due to its high sensitivity, members as carnations, leaf succulents, but most of all and many other advantages. The spectrophotometry species that can tolerate dry environments as Cactaceae. UV-vis with calibration curve (SWCC) is another common In cacti, betalains provide the colours in fl owers and technique for betalain quantifi cation. Both HPLC and (8). Recently, the study of these pigments and their SWCC, by using a calibration curve, can be helpful to properties in fruits, such as (9, 10, 11) and determine the potential of a product from which the (12, 13, 14), has been increasing. The Pitaya de pigment could be extracted in order to use it as a food Mayo (Stenocereus griseus) has not been studied as much, colourant. However, HPLC technique is expensive and can

20 Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016 be highly contaminant because of the solvents used as the filter was washed with 30mL of acidified water. Then, the mobile phase. A third method for betalain quantification volumetric flask was filled to the mark with acidified water. is the spectrophotometry with the molar extinction Finally, the samples were diluted by a factor of 2 for HPLC coefficient (SWEC). In this method the concentration analysis. is obtained solving the expression of Beer´s law for concentration, dividing the measured absorbance of Pigment extraction from pitaya peel betalain solution by the molar extinction coefficient, a In order to optimize the betanin extraction from the peel of calibration curve is not required for the SWEC method, pitaya, a leaching system was conducted. Fifteen samples there is no need to get a chemical standard because were extracted using acidified water (1% v/v acetic acid) Beer’s Law states that molar absorptivity is constant (and as the solvent. The pitaya´s peel was cut into squares of the absorbance is proportional to concentration) for a approximately 1cm2. The peel was weighed (20g) and given substance dissolved in a given solute and measured brewed in 60mL of solvent. The infusion was filtered through at a given wavelength (26). Besides, it is faster to run tests cheesecloth and then 25mL of acidified water were used with SWEC than with SWCC or HPLC. Nevertheless, the to rinse the peel through the cap. The resultant liquid was molar coefficient is an intrinsic property of the species; collected and centrifuged at 4°C and 10,000 g for 20 min. it has to be tested for each chemical compound. The The supernatant was then placed in 100mL volumetric objective of this work was to quantify betalain in both peel flasks, and acidified water was used to complete the and pulp of pitaya , using three different analytical volume. methods (SWCC, SWEC, and HPLC) and determine which one was a more viable option. pH pH was determined with Thermo Fisher Scientific, Orion 3 star, U.S.A. MATERIALS AND METHODS Calibration curves Chemicals A solution stock was prepared transferring 0.2g of the Acetic acid > 99.9% was purchased from Sigma Aldrich, Betanin standard to a 10mL volumetric flask and making USA. Acetonitrile 99.9% was obtained from TEDIA high up to volume with acidified water (1% acetic acid/water purity solvents, USA. Betanin (red beet extract diluted with v/v). The concentration of betacyanin was calculated dextrin) standard was procured from ALDRICH chemistry, by the extinction coefficient for betanin (ε=60,000) the USA. However the concentration was not reported by the absorption maxima at 538, the equation used was [mg/l]= manufacturer. The betanin concentration was determined [(A*DF*MW*1000)/( ε*L)], where A is the absorption, DF is by spectrophotometry with the molar extinction coefficient the dilution factor, MW and ε are the molecular weight (23, 2). Water was provided by a Milli-Q water purification and extinction coefficient and L the pathlength of the system (Milipore, Germany). 1-cm cuvette, (25, 28). And it was 31.9mg/L. From the stock solution, 6 aliquots were taken to obtain six different Pitaya preparation concentrations: 1.595, 3.190, 5.582, 6.380, 7.975, and Pitaya (Stenocereus spp) was collected from a field of 12.760 mg/L. Ahuatlán, which belongs to the state of Puebla, México, on April 28, 2015. The were processed at the Fruit Estimation of and Vegetable Processing laboratory, in the Biotechnology SWCC Centre of FEMSA at the Campus Monterrey at ITESM, on The concentration of betacyanin pigment in extracts April 29, 2015. The fruits were washed and disinfected, from pulp and peel of pitaya, was determinated using a and then the prickles were manually removed. Peel and calibration curve (25, 28). The spectrophotometer used pulp were separated and the former was processed in an was HACH DR 500, Germany and Beckman Coulter DU extractor (Turmix, TU05, USA) in order to remove the seeds. 800, USA for spectrum. The absorbance of the samples was Peel, pulp and seeds were frozen and stored at -18ºC for further analyses. The pulp and peel were defrosted at 4°C one day before the analysis, moisture was determined by gravimetric method (27). Since light has been documented as a deteriorative factor for betalains (18), all extraction procedures were carried out in darkness.

Pigment extraction from pitaya pulp Three betanin quantification methods were evaluated: Spectrophotometry UV-vis with calibration curve (SWCC), High-performance liquid chromatography (HPLC), and Spectrophotometry with the molar extinction coefficient (SWEC). Fifteen replicates of pigment extraction per method were prepared. The pulp was defrosted. For each sample, 5g were weighed and placed in 40mL vials; 10mL of acidified water (1% acetic acid/deionized water v/v) were added; and then the vials were agitated for 15 min. The resultant mix was filtered through a paper filter No. 4 (150mm, Whatman, China) to a 50mL volumetric flask. The

Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016 21 measured at 540nm. The calibration curve was constructed by measuring the absorbance rate of betanin in six solutions. The linear equation derived from the calibration curve was used to determine the concentration of the pigment extraction from pulp and peel of pitaya.

SWEC Table 1. Results of betanin concentration (mg/g dry sample) Only the pigment extractions from pulp and peel of pitaya measured from both pulp and peel of pitaya with the different analytical methods. were analysed in spectrophotometer (for quantifying HACH DR 500, Germany). The absorbance of the samples The mean values not sharing a letter in common are significantly different was measured at 540nm (2, 23). The concentrations according to the Scheffe method (α = 0.05). were obtained solving the expression of Beer´s law for concentration, dividing the measured absorbances of (16, 11) that the pulp of () and betalain solutions by the molar extinction coefficient cactacea fruit, such as the varieties of Rojo San Martín and (60,000). The spectrophotometric method used was (25, 28). Rojo Cenizo of Opuntia ficus indica, have about twice or even three times the amount of betacyanins as the ones of HPLC pulp of Stenocereus spp. In HPLC technique the pigment extractions from pulp and Despite the fact that there were slight differences between peel of pitaya, as well as the standards for the calibration the mean values of betanin concentration in pitaya pulp curve were analysed by HPLC/UV-Vis (Agilent Technologies, obtained with the three different quantification techniques, 1200 Series, Germany) at the same wavelength. A they were not statistically significant (P > 0.05) (Table 1). column Eclipse XDBC18, 5µm, 150mm × 4.6mm (Agilent Therefore, the choice of the best method for betanin Technologies, Germany) was used. The method of Vergara quantification in pulp could be based on the analytical (22) was adjusted to a mobile phase: 85% v/v A (water- requirements. For instance, HPLC is not very convenient for acetic acid 1%), and 15% B (acetonitrile-acetic acid 1% industrial applications because the equipment is expensive v/v). The flow rate was 1 mL/min. The column temperature and requires highly specialized training for its use. Besides, was 25°C, and the run time was 10 min. the solvents utilized are costly and contribute significantly to the environmental pollution. The SWCC technique is Experimental design and statistical analyses easy to implement, but it requires preparation of chemical The experiment comprised two fixed factors: quantification standards and the evaluation of a calibration curve. On the method, with three levels (SWCC, HPLC, and SWEC), and other hand, the SWEC method could be the most suitable betanin source, with two levels (peel and pulp of pitaya). in terms of time, sample preparation and use of resources A total of 90 samples were analysed (15 replicates per for measuring betanin in pitaya pulp. treatment). In spite of SWEC, this method has already been used in The data analysis was carried out by SAS (SAS 9.3, SAS other references (2, 15, 23, 28) but no statistical evaluation Institute Inc., Cary, NC, USA). was found to support this procedure replacing the HPLC The results of betanin concentration were not normally technique. distributed, according to the Kolmogorov-Smirnov test. In order to normalize it, a power transformation (λ = 0.25) was Betanin in peel performed. The results were then subjected to an ANOVA, In pitaya peel, the SWCC, SWEC, and HPLC methods and differences of Least Square Means were obtained by measured, respectively, betanin concentrations of 0.116 the Scheffe method (α = 0.05), which estimates all possible ± 0.033, 0.118 ± 0.036, and 0.056 ± 0.011mg/g dry sample. contrasts among the factor level means, and not just As seen on Table 1, the results obtained from the SWCC the pairwise differences considered by the Tukey-Kramer and SWEC techniques were not significantly different from method. each other, but the betanin concentration measured with HPLC was significantly lower (P < 0.05). The significant difference of HPLC was probably due to the fact that RESULTS AND DISCUSSION this method measured a specific compound at the specified wavelength. The other two methods might have Betanin in pulp measured all the similar compounds in a wavelength (total As is shown on Table 1, the concentration obtained of betacyanins). Furthermore, betanin concentration was betanin by the SWEC analytical method, in the pitaya higher in the pulp than in the peel, compared to other pulp was 2.048± 0.089mg/g dry sample, García-Cruz (16) betacyanins. determined that red fruits of Stenocereus pruinosus had a The varieties Rojo San Martín and Rojo Cenizo of prickly concentration of 2.860 ± 0.038mg/g of betacyanins in dry pear (Opuntia ficus-indica) are reported to have pulp. It has been documented (15) that the red pulp of betacyanin concentrations of 0.241 and 1.346mg/g dry Stenocereus griseus had a betacyanin concentration of sample of peel, respectively (11). Betanin concentration 1.996 ± 0.024mg/g dry sample. in prickly pear is 4-24 fold times higher than in pitaya peel In this work, only betanin concentration was determined. estimated by the HPLC method in this work. The similarities observed between the results obtained in On the other hand, the pigment extraction from the peel this study and those of betacyanins reported in literature showed an orange hue. The pigments that could have could be attributed to the fact that betanin is the major given such colouration to the pitaya peel are betaxanthins. red pigment among the group of betacyanins; it represents García-Cruz (16) determined that the red pulp of from 75 to 95% of the red colour (29). It has been reported Stenocereus pruinosus had a betaxanthin concentration of

22 Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016 10 REASONS TO ATTEND OF INTERNATIONAL SCIENTIFIC 3.21 ± 0.56μg/g dry sample. However, betaxanthins have CONFERENCE ON PROBIOTICS never been determined in the peel of pitaya. Another AND PREBIOTICS - IPC2016 important thing to consider was that probably in the peel, the betaxanthins concentration was going to be larger Probiotics are nonpathogenic live microorganism that can provide a diverse than the betacyanin concentration, exactly like what happened in the red pulp that García-Cruz et al. (15, health benefits on the host. Prebiotics is a non-digestible fiber compounds 16) reported. Therefore, the standard (betanin solutions), that induce the growth or activity of microorganisms. Taking probiotics plus and the pulp and peel extractions were subjected to a prebiotics could bring diverse types of health benefits. Probiotics are wavelength scan (300-600 nm) in the spectrophotometer (Figure 1). consumed in diverse ways including dairy product, food supplements and As seen on Figure 1, the chemical standard (betanin) functional foods with specific health claims. Recently, many reports suggest had a signal only at 540nm, whereas both pulp and peel that certain probiotic strains or multi strain mixture have potent showed a remarkable signal at 480nm, and a slight signal immunomodulatory activity in diverse disorders including allergic asthma, at 540nm. It is known that betaxanthins absorb light at 480nm (22). So, based on this qualitative test of wavelength atopic dermatitis and rheumatoid arthritis. However some of the probiotic scan, it can be inferred that despite the attractive red strains have an adverse effect on people with underlying health problems or colouration, the presence of betaxanthins (responsible immuno-compromised individuals. In addition, however, efficacy of probiotic for the orange colour) might be signifi cant in peel of the Stenocereus spp. administration is quite different depending on the type of strains and the amounts of doses and underlying mechanism of action is still unclear. By Standard solution, and pulp and peel extractions attending the International Scientific Conference on Probiotics and were analysed in HPLC at 540nm. Figure 2 shows the chromatograms obtained. For the peel extraction, there Prebiotics - IPC2016, you will have a chance to expand your ideas on recent were signals at different retention times, each representing advances made in the science of probiotics, highlighting their current and a different type of betacyanin. They all had about the future roles in maintaining health and preventing diseases. The next IPC will same height. This could have been the reason why the be held during 21st - 23rd June 2016 in Budapest, Hungary. betacyanin concentration measured with HPLC was

THE MAIN TOPICS OF IPC2016:

1. Prebiotics - How does it work and how to develop a novel prebiotics?

2. Human milk oligosaccharides as a prebiotics and immune modulator.

3. How to get an approval for probiotics with health claims? Approaches to get approval from EFSA and other countries.

4. How to identify functional probiotics? Development of novel screening system and evaluation procedure to identify functional probiotics.

5. How probiotics work? Elucidation of underlying mechanism of actions.

6. What are the effector molecules mediate health benefits of probiotics? How to identify and how to evaluate the functionality?

Figure 1. Overlapped spectrums from pulp and peel samples, and 7. How to characterize probiotic strains? Genetics, functional the betanin standard, within a wavelength range of 300-600 nm. genomics of probiotic strains. The pulp was diluted by a factor of 10. 8. How to formulate the probiotics to keep maximum effectiveness?

9. Clinical trial of probiotics; What are the current statues of health benefit of probiotics in diverse disorders?

10. Future probiotics? Novel function of probiotics?

By attending IPC2016, you will meet the top class scientists who influenced

the past, influence the present, and most importantly emerging young

scientists who will shape the future of probiotics. Facilitating a global

co-operation between scientists and institutions is another benefit.

Sin-Hyeog Im, Ph.D. President of IPC2015

Figure 2. Chromatogram for peel, pulp, and the standard betanin solution at 540nm.

Komenského 1337 024 01 Kysucké Nové Mesto Slovakia 2016 Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016 Tel.: +421 917 194 367 E-mail: [email protected] www.probiotic-conference.net lower than SWCC and SWEC in peel extraction samples. 1212 (2001). Therefore, the spectrophotometric techniques might have 9. Screekantha D., Arunasree M.K., Roy R.K., et al. “Betanin a overestimated the betanin concentration in the pitaya betacyanin pigment purified from fruits of Opuntia ficus-indica peel, similarly to what Schwarts (30) reported with respect induces apoptosis in human chronic myeloid leukemia Cell” Phytomedicine, 14(11), 739-746 (2007). to beetroot. 10. Osorio-Esquivel O., Ortiz-Moreno A., Álvarez V.B., et al. “Phenolics, betacyanins and activity in Opuntia joconostle fruits” Food Res. Int., 44(7), 2160-2168 (2011). CONCLUSIONS 11. Jiménez-Aguilar D. M., López-Martínez J. M., Hernández- Brenes C., et al. “, composition Three different methods for betanin quantification were and antioxidant activity of Mexican commercial varieties of tested in peel and pulp of pitaya (Stenocereus spp). cactus pear”, J. Food Compos. Anal., 41, 66–73 (2015). Spectrophotometry with a molar extinction coefficient 12. Wu L.C., Hsiu-Wen H., Yun-Chen C., et al. “Antioxidant and (SWEC) was reported for this purpose for the first time in antiproliferative activities of red pitaya”, Food Chem., 95(2), this work. The mean of betanin concentration found in 319-327 (2006). pitaya pulp was 2.092mg/g dry sample, which is within the 13. Herbach K., Maier C., Stintzing F.C. et al. “Effects of processing and storage on juice color and betacyanin stability of purple range previously reported by other authors. The betanin pitaya (Hylocereus polyrhizus)”, Eur. Food Res. Technol. 224, concentration in the peel was 0.117mg/g dry sample, 649-658 (2007). as measured by the SWCC and SWEC methods, and 14. Suh D.H., Lee S., Heo do Y., et al. “Metabolite profiling of red 0.056mg/g by HPLC. The SWEC method represented the and white pitayas (Hylocereus polyrhizus and Hylocereus best choice for betanin quantification in pulp in order to undatus) for comparing betalain biosynthesis and antioxidant reduce cost, time, and wastes. activity”. J. Agri. Food Chem. 62 (34), 8764-8771 (2014). Since betaxanthins might be important components in 15. García-Cruz L., Salinas-Moreno Y.,Valle-Guadarrama S. the colour of the pitaya peel, a recommendation for “Betalains, phenolic compounds and antioxidant activity in future works would be to measure those compounds with pitaya de Mayo (Stenocereus griseus H.)”, Rev. Fitotec. Mex., a quantitative method. For that purpose, an extract or 35(3), 1–5 (2012). 16. García-Cruz L., Valle-Guadarrama S., Salinas-Moreno standard of betaxanthin is required. Y. et al. “Physical , Chemical , and Antioxidant Activity Characterization of Pitaya ( Stenocereus pruinosus ) Fruits”, Plant Food Hum. Nutr., 68(4), 403–410 (2013). ACKNOWLEDGEMENTS 17. G. Calogero, J.-H. Yum, A. Sinopoli, et al. “ and betalains as light-harvesting pigments for dye-sensitized solar The authors express their gratitude to Legado Base de la cells” Sol. Energy, 86(5),1563–1575 (2012). Pirámide and Chair of Environmental Bioprocesses from 18. Herbach K. M., Stintzing F. C., Carle R., “Betalain stability and Tecnológico de Monterrey, for all the financial support. degradation structural and chromatic aspects”, J. Food Sci., We are thankful to Dr. Carlos Brambila for his leadership in 71 (4), R41–R50 (2006). a multidisciplinary team. We would also like to thank Dr. 19. Repositorio Digital:http://hdl.handle.net/10317/769 (accessed Timothy Schwinghamer for helping us with the statistical on Abril 5th 2015). 20. Wrolstad R. E., Acree E., Decker E. A., et al.: Betalains, in analysis and interpretation of this work. Handbook of food analytical chemistry, Edited by Wrolstad R. E., Ed. John Wiley & Sons, New Jersey, U.S.A.(2005). 21. Repositorio Academico de la Universidad de Chile: http:// REFERENCES AND NOTES www.repositorio.uchile.cl/handle/2250/11486 (accessed on Abril 4th 2015). 1. Jiménez-Aparicio A., Gutiérrez-López G. “Production of 22. Castellanos-Santiago E. , Yahia E. M., “Identification and food related colorants by culture of plant cells. The case of quantification of betalains from the fruits of 10 Mexican prickly betalains”, Adv. Exp. Med. Biol., 464, 195–210 (1999). pear cultivars by high-performance liquid chromatography 2. Chethana S., Nayak C. A., Raghavarao K. S. M. S. “Aqueous and electrospray ionization mass spectrometry.” J. Agric. Food two phase extraction for purification and concentration of Chem., 56(14), 5758–64 (2008). betalains”, J. Food Eng., 81, 679–687 (2007). 23. Nilsson T. “Studies into the pigments in beetroot”, 3. Fernández-López J. A., Almela L. “Application of high- Lantbrukshögskolans Ann., 36, 179–219 (1970). performance liquid chromatography to the characterization 24. Schwartz S.J.,Elbe J.H., “Quantitative determination of of the betalain pigments in prickly pear fruits”, J. Chromatogr. individual betacyanin pigments by High Performance Liquid A, 913(1), 415–420 (2001). Chromatography”, J. Agric. Food Chem. 28(3) ,540–543 4. Delgado-Vargas F., Jiménez A. R., Paredes-López O., “Natural (1980). pigments: , anthocyanins, and betalains- 25. Stintzing F.C., Carle R., “Betalains emerging prospects for food characteristics, biosynthesis, processing, and stability”, Crit. scientists”, Trends Food Sci. Technol. 18(10), 514–525 (2007). Rev. Food Sci. Nutr., 40(3), 173–289 (2000). 26. Handbook of Chemistry and Physics, Edited by Weast, R.C., 5. Cai Y., Sun M., Corke H., “Antioxidant activity of betalains from Ed. CRC Press Cleveland.Ohio, USA(1975). plants of the Amaranthaceae” J. Agric. Food Chem. 51(8), 27. Moisture gravimetric method. AOAC Official Method 934.06. 2288-2294 (2003). AOAC International (1990). 6. Counsell J. N., Jeffries G. S, Knewstubb C. J.”Some other 28. Stintzing F.C., Schieber A., Carle R.,” Evaluation of colour natural colors and their applications, Natural colors for foods properties and chemical quality parameters of cactus ”, and other uses”, Edited by Dunastable J.A., Ed. Applied Eur. Food Res. Technol., 216, 303-311(2003). Science London, England, United Kingdom (1979). 29. González E. M., Vigoa Y. G. “Colorantes naturales de origen 7. Francis. F.J. “Anthocyanins and Betalains”, in Colorants, Edited vegetal”, Cienc. y Tecnol. Aliment., 17(1), 68–75 (2007). by Francis. F.J., Ed. Eagan, Saint Paul, Minnesota, U.S.A. (1999). 30. Schwartz S. J., Hildenbrand B. E., Von Elbe J. H., “Comparison 8. Wybraniec S., Platzner I., Geresh S., et al. “Betacyanins from of spectrophotometric and HPLC methods to quantify vine cactus Hylocereus polyhizus” Phytochemistry, 58(3),1209- betacyanins”, J. Food Sci., 46(1), 296–297 (1981).

24 Agro FOOD Industry Hi Tech - vol. 27(1) - January/February 2016 EARLY BIRD RATES END 3/4 supplysideshow.com

Answering that This event is the one Make plans now to join question is the theme of place to gain the us at the only B2B event Ingredient Marketplace knowledge, connections for the nutraceutical 2016, Mind of the and suppliers for your space that analyzes Consumer. Why? next big innovation. how the mind of the Because the surest way Come learn from keynote consumer impacts to develop products speakers, Storefronts ingredient science that end up in the Live, Sports Nutrition and finished product consumers’ shopping Panel Discussion, innovation. carts is to analyze what’s briefs, networking on their minds. opportunities, Probiotics Marketplace, and the results of the inaugural “Mind of the Consumer” study.

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