β-carotene Bioaccessibility from Biofortified Maize (Zea mays L.) is Related to its Density and is Negatively Influenced by Lutein and Zeaxanthin

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1 β-carotene Bioaccessibility from Biofortified Maize (Zea mays L.) is Related to its Density and is Negatively Influenced by Lutein and Zeaxanthin Nivedita Dube, Purna Chandra Mashurabad, Firoz Hossain, Raghu Pullakhandam, Dinesh Kumar Bhardwaj, Rajendran Ananthan and Thingnganing Longvah National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, Telangana, India

2 INCIDENCE OF VITAMIN A DEFICIENCY IN INDIA Clinical VAD RDA of VA & VA Intake Trends (Source: Muthayya et al, 2013) Incidence of clinical signs of vitamin A deficiency such as Bitot s spots (0.8%) and conjunctival sclerosis (1.8%) still exist in India (Laxmaiah et al., 2012, NNMB report 2005) Sub-clinical VAD Subclinical VAD- defined by serum retinol concentration of <20 µg/dl or <0.7µmol/L Prevalence of >20% severe public health problem; 60% Incidence in Indian population The average intake of VA in 75% Indian rural households is <50% of RDA. Intake deficit is major etiological factor for the observed high prevalence of clinical and sub-clinical VAD among Indian population. 2

3 INTERVENTION STRATEGIES TO MITIGATE VAD Existing Strategies Supplementation of Mega dose of VA through VA prophylaxis program Industrial fortification of processed foods Promotion of consumption of carotene rich food sources through nutrition education Advantages of Biofortification Food Biofortification with pro-va carotenoids as long term sustainable strategy to combat clinical & sub-clinical VAD Ensures regular daily intake of nutrients across all age groups Self-sustainable Cost-effective Potentially wide-reaching Under Agri-CRP targeted crop biofortification to enhance nutritional content of major food crops of India-targeting 7 crops, including maize for improving provitamin A (pro-va) carotenoids. 3

4 CAROTENOIDS IN PLANT FOODS Carotenoids : C40 tetraterpenoid yellow pigments found in plants Based on provitamin A activity they have been classified into: Non-pro-VA carotenoids Pro-VA carotenoids BC, AC, and BCX- sources of VAget transformed into retinol in human body Retinol activity equivalents (RAEs)- 1µg RAE = 8 µg of all-trans-βcarotene from food (8:1) and1µg RAE = 16 µg for 16:1 for other pro- VA carotenoids Unlike non-vegetarians, pro-va only source of VA for obligatory vegetarians- major part of Indian population.

5 PRO-VITAMIN A BIOFORTIFIED MAIZE Maize- rich source of macro, micronutrients and carotenoids. Orange maize has very high amount of total carotenoids, but non-pro-va xanthophylls (lutein & zeaxanthin) > pro-va carotenoids viz., β-carotene. Pro-VA enrichment of maize developed by IARI plant breeders targeting crtrb1 allele and lcye alleles yielding high conc. of lysine, tryptophan & pro-va carotenoids. The purpose of maize biofortification is to improve pro-va content- when fed to target population shows an impact in biochemical status in short-term and functional status in long run. Therefore, ensuring bioavailability of pro-va in biofortified maize is the first necessary nutritional investigation.

6 STUDY RATIONALE A relatively high bioavailability of enhanced nutrients required to be marketed as nutritionally superior sources of micronutrients. Increased nutrient density Increased Bioavailibility? Biofortification of maize by selective breeding for enhancement of β-carotene content, also enhances its bioavailability OBJECTIVES i. To estimate the proximate and carotenoid profile of selected biofortified maize hybrids. ii. To estimate the digestive stability of carotenoids in selected biofortified maize hybrids. iii.to assess the efficiency of micellarization of carotenoids in selected biofortified food crops. iv.to assess the effect of non-pro VA carotenoids on β-carotene micellarization. 6

7 METHODOLOGY Sample Collection A. B. A. Mature maize crops prior to harvest C. Post-harvest maize kernels dried under shade without removing husk. Once dried, kernels were kept in gunny bags for one month under normal storage conditions. C. B. Harvested, dried maize crops Identification & procurement of test materials β-carotene biofortified staple food crops developed in India- ICAR institutions: Professor Jayashankar Telangana State Agricultural University (PJTSAU), Hyderabad and Indian Institute of Agriculture Research (IARI), New Delhi. 38 maize samples (18 normal+ 5 pro-va+ 7 QPM+ 8 pro-va-qpm) were procured from Maize Research Centre, PJTSAU Hyderabad and IARI, New Delhi respectively. The pro-va maize and QPM + pro-va maize hybrids were selectively bred to enhance the β-carotene content in their kernels. 7

8 METHODOLOGY Nutrient Composition Analysis All maize samples were subjected for proximate analysis which includes moisture, crude protein, crude lipids, carbohydrate and fiber (total, soluble and insoluble) by AOAC (1999) methods. Total carotenoids - determined in powdered maize samples by modified method of Kurilich & Juvik (1999) using HPLC with a sensitivity of 1 ng/µl. 8

9 METHODOLOGY In vitro Simulated Digestion Simulated in vitro digestion carried out as described by Garret et al., 1999; Pullakhandam et al., 2007 except that an oral phase of digestion was included in study to account for high starch content in maize. Digestion reactions were undertaken in maize porridge, with peanut oil (3%, w/w), which was added prior to initiation of oral phase of digestion. Carotenoids in digesta and micellar fractions - analysed using HPLC (Kurilich & Juvik, 1999) Garrett et al. (1999) J. Agric. Food Chem. 47:4301

10 Hybrid MSC Trait Proximates in Maize Samples Proximates Moisture Protein Ash Fat IDF SDF TDF CHO DHM CML BML7 Normal BML BML UMI VH27-PV RESULTS Pusa-PV Pusa-PV-16-2 Pro- VA Pusa-PV Pusa-PV

11 RESULTS Proximates in Maize Samples Hybrid HQPM1-PV Trait Proximates Moisture Protein Ash Fat IDF SDF TDF CHO HQPM4-PV HQPM5-PV HQPM7-PV pro-va+ Pusa-APQH8 QPM HM9Q-PV Vivek QPM9- PV Pusa-APQH

12 RESULTS Proximates in Maize Samples Hybrid Trait Proximates Moisture Protein Ash Fat IDF SDF TDF CHO HM4Q HM8Q HM9Q HM10Q HM11Q QPM HQPM HQPM HQPM HQPM

13 Hybrid Trait RESULTS Carotenoid Profile of Maize Hybrids Carotenoids (µg/100g) ± SD Lutein Zeaxanthin β-crypto α-carotene β-carotene BML ± ± ± ± ±6.46 HM10Q 50.2± ± ± ± ±18.91 HM11Q 59.9± ± ± ± ±10.60 DHM ± ± ± ± ±4.95 LM11Q 236.1± ± ± ± ±3.98 MSC ± ± ± ± ±7.37 CML ± ± ± ± ±0.00 UMI1200 Normal 220.4± ± ± ± ±2.57 CM ± ± ±16.73 ND 46.4±6.89 BML ± ± ± ± ±5.43 LM ± ± ±13.89 ND 31.4±3.56 BML ± ± ± ± ±1.24 PSC 214.9± ± ± ± ±3.45 KDMI ± ± ±11.64 ND 12.8±1.54 CML ± ±22.15 ND 34.4± ±2.34

14 RESULTS Carotenoid Profile of Maize Hybrids Hybrid Trait Carotenoids (µg/100g) ± SD Lutein Zeaxanthin β-crypto α-carotene β-carotene Pusa-PV ± ± ± ± ±13.91 Pusa-PV ± ± ± ± ±10.68 Pusa-PV-16-4 Pro-VA 152.0± ± ± ± ±32.44 VH27-PV 444.5± ± ± ± ±5.61 Pusa-PV ± ± ± ± ±12.67 Pusa-HQPM ± ± ± ± ±10.45 HM8Q 704.0± ± ± ± ±0.90 Pusa-AQH ± ± ± ± ±3.56 QPM HQPM ± ± ± ± ±1.98 HQPM ± ± ± ± ±2.67 HQPM ± ± ± ± ±1.67 HM9Q 211.0± ± ± ± ±0.56 HQPM5-PV 125.0± ± ± ± ±18.63 Pusa-APQH ± ± ± ± ±21.42 Pro-VA HQPM1-PV 259.2± ± ± ± ± QPM Vivek QPM9-PV 209.2± ± ± ± ±22.46 PUSAAPQH ± ± ± ± ±

15 Retinol Equivalents (RE) of pro-va Carotenoids & %Contribution To RDA Hybrid Trait RE β- cryptoxanthin RESULTS RE α- carotene RE β- carotene Total RE from pro- VA/ 200g % RDA of VA/ 200g MSC DHM 121 Normal BML Pusa-AQH QPM Pusa-HQPM Pusa-PV Pusa-PV-16-3 Pro-VA Pusa-PV Pusa-APQH4 Pro-VA PUSA-APQH8 QPM The conversion ratio used for calculation of RE= 8:1 for β-carotene & 16:1 for α-carotene and β-cryptoxanthin. RDA calculation was done using RDA for Indians, Indian Council of Medical Research, 2010, India. 15

16 RESULTS Digestive Stability & % Micellerization of Carotenoids From Maize The digestive stability is the ratio of carotenoids recovered in the digesta after the oral, gastric and intestinal phases of digestion to the carotenoid content in the maize porridge. The recovery of both non pro-va and pro-va carotenoids after simulated digestion was always >80% in normal & biofortified maize hybrids. The percent micellarization is the ratio of carotenoids recovered in the aqueous micellar fraction compared to that of carotenoid content in digesta. %Micellerization of carotenoids, on addition of 3% dietary fat prior to simulated 16 digestion. Values are Mean±SD.

17 RESULTS The Mean Efficiency of Micellarization of Xanthophylls and Carotenes from All Test Maize Hybrids The maize flour was subjected to simulated in vitro digestion, followed by measuring the carotenoid content in aqueous micellar fraction. The bars represent means ± SD for 6 observations generated in two independent experiments. Means without a common letter in a column differ significantly (p<0.05). 17

18 RESULTS Relationship between the Content of Carotenoids in Aqueous Micellar Fraction and the Content in the Digesta of Maize Hybrids Linear regression analysis of the non-pro-va carotenoids, viz. Lutein & Zeaxanthin and pro-va carotenoids, viz. BCX, AC & BC content in the micellar fraction to that of digesta of maize hybrids. 18

19 RESULTS Effect of Non-pro VA Carotenoids on β-carotene Micellarization Regression analysis of the micellar content of β-carotene vs. non-provitamin A xanthophylls (Lutein+Zeaxanthin) content in the digesta. 19

20 SALIENT FINDINGS Pro-VA carotenoids (β-cryptoxanthin, α-carotene and β-carotene) content increased up to 10 fold in biofortfied maize Pusa-APQH8; highest β- carotene Pusa-PV Consumption of 200 g/day of Pusa-APQH8 (pro-va+qpm) and Pusa-PV-16-3 (pro-va) would respectively contribute 76% and 67% of RDA of VA. Digestive stability of carotenoids (recovery) during in vitro digestion was always 89 to 94% from both normal and biofortified maize hybrids. Carotenoid Micellarization Efficiency is related to their degree of polarity. Mean efficiency of micellerization of carotenoids was in order of LUT=ZEA=BCX AC=BC. Content of LUT, ZEA, BCX, AC & BC in aqueous micellar fraction were also significantly correlated with their content in digesta of resp. maize hybrids. Micellarization of BC is -vely correlated with content of LUT and ZEA in digesta, but remained statistically insignificant. However, BC micellarization is - vely and significantly correlated with that of LUT+ZEA content in digesta. 20

21 >250 Million people world over affected by VAD 80% of total South Asian population (women and children) with clinical VAD reside in India PRO-VITAMIN A MAIZE Food + Nutrition Security come together in biofortification of cereals & can easily be utilized with PDS β-carotene hydroxylase allele introgression based breeding appears to be a promising strategy to increase the content and bioavailability of β-carotene from maize. This study would aid in understanding relative absorption of carotenoids from biofortified maize which may prove to be helpful in introducing such crops in the National Nutritional Intervention Strategy.

22 THANK YOU FOR YOUR ATTENTION

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24 Process of absorption of carotenoids from food matrix. 1, 2) Disruption of food matrix, 3) release of bile salts from common bile duct, 4) uptake of carotenoid molecule in lipid droplet and formation of micelle, 5) uptake of carotenoid molecule in enterocyte, and 6) release of carotenoid molecule in blood circulation.

25 IN VITRO SIMULATED DIGESTION ORAL PHASE Test Meal Oral Digesta GASTRIC PHASE Gastric Digesta INTESTINAL PHASE Aqueous Micellar Fraction Intestinal Digesta Total Carotenoid Fraction Garrett et al. (1999) J. Agric. Food Chem. 47:4301

26 CAROTENOID EXTRACTION Micellar fraction/ Whole Crotenoid alliquote + Petroleum ether & Acetone (2:1) + BHT (4.5 mm/l) Repeat 3 times 1. Vortex (X2) 2. Centrifugation (2000g;5min) Pipette out top most layer & collect in separate tube All 3 fractions are pooled & evaporated using C Resolubalise in ethyl acetate:methanol (1:1 v/v) & analyse immediately 26

27 ESTIMATION OF CELLULAR UPTAKE/TRANSPORT USING CACO-2 CELLS Carotenoid Uptake Test Media DMEM + Micellar Fraction Carotenoid Transport Apical CELL MONOLAYER Caco-2 cells on plastic well Basolateral Apical CELL MONOLAYER Caco-2 cells on trans-well insert 4 hours 0-10 hours Microporous Membrane Harvest Cells Analyze for Carotenoids Collect Basolateral Media Analyze for Carotenoids 27

28 ESTIMATION OF CELLULAR UPTAKE USING CACO-2 CELLS Wash Caco-2 cells (11-12 days post confluency) with basal med at 37 C Add DMEM containing 25% micellar fraction At incubation end point remove medium Wash monolayer with- 1. Ice cold PBS (ph 6.8)+Albumin(2g/L) 2. Ice cold PBS (ph 6.8) [X2] Collect cells, pellet by centrifugation & store at -80 C for max 2 week 28

29 CAROTENOID EXTRACTION FROM CACO-2 CELLS Thaw frozen cell pellets on ice Add 1mL PBS with 10% ethanol+bht (4.5 mm/l) Sonicate on ice (10-15 sec) Add 1 ml Petroleum ether:acetone (2:1 v/v) Follow same steps as for carotene (whole and micellarised fraction) extraction 29