Zea mays

Regd. No. IB 14052-28

ISSN 0970-4078 ONLINE ISSN 2229-4473

Society for Plant Research Official websites: www.vegetosindia.org, www.indianjournals.com

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Vegetos: An International Journal of Plant Research & Biotechnology [Thomson Reuter IF: 0.042 (2013-14); NAAS: 5.0 (2016)] (Journal is being indexed and abstracted in Thomson Reuter’s Science Citation Expanded (SciSearch® ), Journal Citation Reports/Science Edition, Biosis, Elsevier (SCOUPUS), CAB Abstracts, NLM, MAAPA Indian Science Abstracts, NAAS, UGC)

For online access and submission, log on to: www.vegetosindia.org or www.indianjournals.com Editor in Chief Prof SK Bhatnagar, Meerut Executive Editor Prof Abhay K Pandey, Allahabad Associate Editor Prof K S Rao, Delhi Prof M U Charaya, Meerut

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Editorial Board Dr Sangita Bansal, New Delhi; Prof T S Kahlon, USA; Dr Pratibha Singh, Delhi; Prof Y Vimala, Meerut; Prof Anjuli Agarwal, Majhera (U.A.); Dr Bhakti Rana, Meerut; Dr Manisha Mangal, New Delhi; Dr Stefan Kraan, Ireland, ; Dr Rajarshi K Gaur, Rajasthan; Prof Veena Gupta, New Delhi; Prof N Thajuddin, Tamil Nadu; Prof I B Prasher, Chandigarh; Prof P C Abhilash, Varanasi; Prof M S Khan, Seoul

Councillors Dr M C Sidhu, Chandigarh; Dr Anubhuti Sharma, Uttarakhand; Dr Brinchi Sarma, Varanasi; Dr K Ram Krishna, Jaipur; Dr Tabassum Afshan, New Delhi; Dr Purushottam, Meerut; Dr Pawan K Dadheech, Rajasthan; Dr S K M Basha, Nellore; Dr Hemant Kumar Gupta, Shimla; Dr Akash Tomar, Meerut ;Dr Rama Kant, Tripura; Note: Editorial Board of VEGETOS is not responsible for the truthfulness of scientific contents presented by the authors.

Vegetos: An International Journal of Plant Research & Biotechnology

Volume 31(4)

December, 2018

CONTENTS Sr.No.

Title

Page No.

1.

Biology and Biotechnology of Papaya, an important fruit crop of tropics: A Review

01-15

2.

Differential Calcium responsiveness in terms of Plant’s Growth and accumulation of Nutrient-Anti nutrient in two Finger Millet Genotypes differing in grain Calcium content using a designed Circulatory Hydroponics system

16-24

3.

Biochemical Characterization of Building Block of Condensed Tannin in Faba Bean (V icia faba L.)

25-27

4.

In vitro culturing and biochemical properties of green and blue-green algae in the laterite soil

28-34

5.

Structural Characterization of complete Chloroplast Genome of Sporobolus helvolus (Poaceae)

35-38

6.

Lignin Biodegradation in Nature and Significance

39-44

7.

Identification of the Constituents lacking in Bonner-Devirian Medium that makes it Incapable of supporting Flowering of the Duckweed, Lemna gibba L. G3

45-51

8.

Karyotype Stasis and Genetic Diversity in A momum spp. from Tripura, North-East India

52-58

9.

Blue Green Algal Flora of Alwar (Raj) India

59-61

10.

Establishment of a highly efficient Regeneration system in Tomato var. Pusa ruby amenable to Agrobacterium tumefaciens Mediated Plant Transformation system

62-67

11.

β-carotene Bioavailability and retention in Biofortified Maize (Zea mays L.) after Processing and Preparation of Indian foods

68-74

12.

Influence of Gibberellins on sink strength and Expression of Genes associated with Sucrose accumulation in Sugarcane (Saccharum spp. Hybrids)

75-81

13.

Agronomic Components of Drought Stressed Wheat Plants under Different Soil Properties

82-91

14.

In vitro Antioxidant and Sub-acute toxicity studies of Aqueous extract of White butterfly (Clerodendrum volubile) leaves

92-101

15.

Variability in some Physical and Chemical Properties of Soil along Atoposequence in Aba-Midan sub watershed in Bambasi Wereda, West Ethiopia

102-113

16.

Alleviating Salinity Stress in Wheat using Selenite Seed Primed

114-118

17.

Hemibiotrophic foliar Fungal Diseases and their Dynamism in GPS system in Soybean growing areas of Karnataka

119-122

18.

Extraction, Purification and Characterization of Peroxidase form Bacopa monnieri from Ranchi

123-125

19.

Electrophoretic Characterization of the Cowpea Mutants for Seed Storage Proteins [V igna unguiculata (L.)Walp]

126-129

20.

Assessment of the Osmotolerant Endophytic Bacteria from Mustard and Nagphani for their Plant Growth Promoting activities under Osmotic stress

130-134

21.

The Genus Eulophia R. Brown ex Lindl. (Orchidaceae) in Tripura State, India

135-137

22.

Adopted Cropping Systems, Tillage Practices and Subsistence of Shire to May-Dimu Area Residing Farmers

138-141

All articles published in Vegetos: International Journal of Plant Research & Biotechnology are the property of SPR, and is protected by copyright laws. Copyright © 2018, SPR, All Rights Reserved.

Vegetos An International Journal of Plant Research & Biotechnology

Research Article

A SOCIETY FOR PLANT RESEARCH PUBLICATION

β-carotene Bioavailability and retention in Biofortified Maize (Zea mays L.) after Processing and Preparation of Indian foods Sandesh G. M.1 , C. Saran Kumar1 , P. Bharathi1 , M. Dhasarathan2, A. Karthikeyan2, V. Meenakshi2, K. Thangaraj1, S. Vellaikumar3, V. Baskaran4 and N. Senthil5* Abstract β-carotene rich maize hybrids were developed to target vitamin A deficient populations in developing countries. However, processing the maize into food products may reduce its β-carotene content. Thus, understanding β-carotene retention is important for assessing efficacy of biofortified foods. The objectives of this study were to determine the concentration of β-carotene in biofortified maize hybrids as well as to evaluate their retention during processing of popular maize foods consumed in India. The β-carotene content in maize hybrids and their food products were estimated through high-performance liquid chromatography (HPLC). Results showed that β-carotene content ranged from 0.61-6.35µg/g in raw maize inbreds, after invitro digestion the bioavailabity of βcarotene ranged 0.33-4.03 µg/g. In food products the retention of βcarotene was 24.14-58.78 % as the β-carotene degradation will be influenced by the processing methods and external environmental conditions like light, temperature, pH, oxidation and storage. In conclusion, the degradation of β-carotene occurs during digestion storage and cooking thus for development of biofortified maize hybrids the bioavailability and their retention plays a significant role. This study demonstrates, there is a need to optimize and recommend maize processing methods in which there will be maximum retention of provitamin A, β-carotene to ensure optimum delivery of β-carotene to consumer. Keywords β-carotene, maize, Bioavailability

β

carotene

Sandesh G.M. et al. Vegetos 31(4):68-74, 2018 Doi:10.5958/22294473.2018.00096.4

retention,

invitro

digestion,

Introduction Vitamin A- deficiency (VAD) is a global burden affecting an estimated 190 million preschoolaged children and associated with blindness and increased mortality. Biofortification of staple crops with provitamin A carotenoids is an alternative approach to promote optimal VAD status. Maize is consumed by more than a billion people in sub-Saharan Africa, Latin America and in many countries in Asia. Thus, maize is targeted for biofortification with provitamin A Corresponding author: Department of Biotechnology, Agricultural College and Research Institute, Madurai-625104, TamilNadu Agricultural University, Tamil Nadu, India Email: [email protected] Received:10.09.2018 Revised:24.10.2018 Published: 31.12.2018

carotenoids by the HarvestPlus Challenge Programme. Biofortified maize has been selectively bred to have a greater carotenoid content, with special emphasis on βcarotene, and are being disseminated in many Asian countries including India. Compared to the other provitamin A carotenoids, β-carotene has the highest vitamin A activity, and it is widely accepted when compared to as alpha carotene or β-cryptoxanthin the βcarotene has twice the vitamin A activity (Haskell 2012; Weber and Grune 2012). One molecule of β-carotene can theoretically be converted to two molecules of vitamin A this is primarily because only β-carotene have the symmetrical provitamin A carotenoid and is unique (Grune et al. 2010). Stating that β-carotene is a better source of vitamin A due to its superior conversion and provitamin activity. β-carotene accumulation is not toxic, so it is considered a safe source of vitamin A. Today’s debate concerns a major aspect to consider when looking at biofortification strategy is the potential effect of food processing on the provitamin A carotenoid content of the biofortified food products. Recent studies have demonstrated that the Provitamin A carotenoids retention in biofortified maize up to 75% (Muzhingi et al. 2016), when processing foods products under traditional methods. The provitamin A carotenoids may be lost in the by-products due to the method of processing. Li et al. (2007) reported that only modest loss of provitamin A carotenoids in high β- carotene maize could be directly attributed to household practices of cooking fermented maize porridges. Pasteurization and blanching at 60 are the initial treatments during food processing that is employed to minimize or inactivate enzymes (Cruz et al. 2009) and vegetative microorganisms (Maiani et al. 2009). Therefore, the enzymatic action may decrease and provitamin A retention will be more in the products. According to Lozano-Alejo et al. (2007) a biofortified maize on frying and nixtamalization which are normal procedure to prepare food products of the average retention of 64 % of provitamin A was noticed. Factors such as heat, light, chemical treatments, and oxygen exposure may have detrimental effect on several bioactive constituents (Sanjuan et al. 2000). In India maize is processed in several ways into a wide variety of food products, the processing steps involve the milling of maize into different particle size, followed by cooking of milled products. Currently there

All articles published in Vegetos: International Journal of Plant Research & Biotechnology are the property of SPR, and is protected by copyright laws. Copyright © 2018, SPR, All Rights Reserved.

Citation: Sandesh G.M. et al. β-carotene bioavailability and retention in biofortified maize (Zea mays L.) after processing and preparation of Indian foods. Vegetos 31(4): 68-74, 2018. Doi:10.5958/2229-4473.2018.00096.4

is a lack of data on the retention of Provitamin A carotenoid β-carotene when it is subjected to different food products. It is important to quantify the losses of βcarotene during processing of provitamin A-biofortified maize. These losses should then be taken into account when setting targets for the provitamin A content of the maize. Therefore, these studies suggest that cooking methods have a significant effect on carotenoids retention. Thus, this study aims to evaluate the β-carotene content in the normal and biofortified maize hybrids in raw maize flour and invitro digested samples to know the bio accessibility of the β-carotene and to assess the retention of β-carotene during the preparation of major maize foods consumed in Southern India. The food products studied were roti and chapati which are the most common food that are consumed in Tamil Nadu and some of the commercial product like noodles and popcorn to explore the feasibility of utilization of biofortified maize as a source of provitamin A carotenoid β-carotene. Materials and Methods Plant genetic material The Experimental F1 maize hybrids were normal yellow maize UMI 1200, provitamin A biofortified UMI 1200 β and F1 hybrid UMI 1200β × UMI 1230β. The maize hybrids were developed through marker assisted breeding at Agricultural College and Research Institute, Madurai, Tamil Nadu. UMI 1200 and UMI 1230 are University Maize Inbred, collected from the gene pool of Tamil Nadu Agricultural University are the parents of the popular Maize hybrid Co6, which is superior in yield and other agronomical characters but with poor β-carotene content. The exotic PV Donor of International Maize and Wheat Improvement Centre (CIMMYT) was used for backcrossing and the β- converted lines were developed at AC & RI, Madurai. These hybrids were used for this study. Chemicals and Standards β-carotene was extracted from normal yellow maize and bio-fortified maize for experimental purpose. Standard β-carotene (99%), butylated hydroxyl toluene (BHT), porcine pepsin (P2500 units/mg protein, P7012), bovine/ovine bile acid mixture (BB381) and porcine pancreatin standards were purchased from Sigma-Aldrich (St. Louis, USA). All solvents used in the analysis were acetonitrile, dichloromethane, methanol, ammonium acetate, hexane of High-performance liquid chromatography (HPLC) grade were purchased from Vegetos 31 (4) December, 2018

Sisco Research Laboratories (Mumbai, India) in CFTRI, Mysore. Extraction of β- carotene from raw maize flour β-carotene was extracted from maize and food samples according to the procedure described by Lakshminarayana et al. (2005) with slight modification. In brief finely ground 10 g maize flour was ground again using pestle and mortar along with sodium sulfate (5g) and 0.1% BHT. β-carotene was extracted using ice-cold acetone by keeping overnight in a shaking water bath (Scigenics Orbitek, India) at 4 C and the extract was dried over anhydrous sodium sulphate (5 g) and filtered through Whatman No.1 filter paper. The extraction with acetone continued until the sample turned colorless (crude extract). The polled extract (volume 250 ml) was dried over anhydrous sodium sulphate (15 g) and filtered through Whatman No.1 filter paper. The crude extract was saponified with methanolic KOH (30%) at room temperature (27 ) for 3 h in dark to extract β- carotene. The samples extracted were concentrated in rotary evaporator at 30ºC in 70 rpm made up to 4 ml by resuspending using methanol and concentrated to 2 ml with nitrogen gas, followed by quantification of the βcarotene was carried out using HPLC analysis will be given in this chapter elsewhere next.

In vitro Digestion To estimate the bio accessibility of β- carotene from maize samples in-vitro digestion was done. The schematic representation of the experimental protocol describing the in vitro digestion of β- carotene (Fig 1). In brief, in a 15-ml screw cap test tube, 1 gm of ground sample subjected to in vitro digestion simulating the

Fig 1. Schematic r epr esentation of Invitr o digestion for micellization of beta carotene

69


gastric and intestinal phase of digestion according to the method proposed by Garret et al. (1999) with slight modification. In brief, 3 ml of 0.5% pepsin (porcine gastric mucosa 88-2500 units/mg protein) in phosphate buffer (3.6 mmol/L CaCl2, 104 mmol/L MgCl2.6H2O, 49 mmol/L NaCl, 12 mmol/L KCl, 6.4 mmol/L KH2PO4) was added to the sample. The pH was adjusted to 2.02 with 2 mol/L HCl. The tubes were screw capped under a stream of nitrogen and incubated for 1 h t 37 C in a shaking water bath (Scigenics Orbitek, India) at 120 strokes/min (gastric phase). On cooling, the pH was raised to 5.0 with 1 mol/L NaHCO 3 followed by the addition of 6 ml 0.1 mol/L NaHCO3 containing 16g/L pancreatin (porcine pancreas8 x U.S.P specifications) and 25.38 g/L bile extract (porcine). Then the pH of the digesta was further adjusted to 7.5 by 1 N NaOH. The test tubes were blanketed with a stream of nitrogen and subjected to incubation at 37 C with shaking at 120 strokes/min for 2 h (intestinal phase). After incubation, an aliquot of digesta (1 ml) was withdrawn from each sample, centrifuged (Z 360 K, BHG Hermle, Gosheim) at 12,000 x g at 4 C for 120 min to separate the aqueous fraction (digesta) that contain micelles and according to the procedure of Lakshminarayana et al. (2009), βcarotene extracted from the digesta was used for quantification by HPLC. HPLC analysis of β-carotene High performance liquid chromatography is used to separate components of a mixture by using a variety of chemicals interaction between substance being analyzed (analyte) and the chromatography column (C 30). Βcarotene extracted from both raw maize flours and digesta (micellizable β- carotene) was quantified with an HPLC system (LC-10AVP; Shimadzu, Kyoto, Japan) equipped with Shimadzu photodiode array (PDA) detector (SPD-M20A). β- carotene extract (10µl) injected was separated on a Princeton SPHER C-30 (ODS) column (250 mm x 4.6 mm; 5µm) by isocratically eluting with the mobile phase of Acetonitrile: dichloromethane: methanol (60:20:20, v/v/v) containing 0.1% ammonium acetate at 1 ml/min. Β- carotene was monitored at 450 nm using software (Shimadzu Class VP version 6.14 SP1 software). The β- carotene peak identified was confirmed by the characteristic spectrum of it and quantified by comparing the authentic β- carotene standard peak with the peak area of samples. Preparation of maize food products Raw Maize flour: The maize gr ains wer e dr y milled in a Cyclone miller (Retsch cyclone mill, India) and passed through 60 BS sieve to get a fine maize flour of 250 µm Vegetos 31 (4) December, 2018

(MF). The flour used as a control sample and stored in 20 ̊ C for 45 days to check beta carotene retention after storage for a certain period. Maize Roti: The r oti was pr epar ed accor ding to the procedure normally that were carried out by local women in Tamil Nadu without adding any supplements except salt, water which were used to prepare dough. Sunflower oil was used for baking which is having low provitamin A in it. Maize chapati: Maize chapati was pr epar ed in a lab by mixing flour of Maize: wheat (1:1), the good quality commercially available wheat flour was added to get consistency for chapati and required quantity of salt and water was used to prepare it and baked in a low flame with little amount of sunflower oil for roasting. Noodles: Based on the available liter atur es, noodles were prepared with other ingredients required for it .500 gm maize flour, water (30%), salt (1% and oil (5ml) constant was used. The method of preparation was a modification of method described by (Kruger et al. 1994). Flour mixtures was mixed with salt and steamed for 10 min then transferred to single dolly machine (La Monferrina Italy) and kneaded for 10 min. Then water was added intermediately at the rate of 30 % proper dough consistency. After 10 min the dough was extruded using dies having perforation of 1.6 mm and the noodles were steamed on a boiling water bath for 10 min (102105). the steamed noodles were allowed to cool at room temperature (25 ± 3 ) and were dried in a hot air oven at 45 for 3 h until the moisture percentage reaches 8 to 9 %. The noodles were used for β- carotene estimation. Popcorn: Maize seeds wer e r insed for 1 h in water , it was drained and then seeds were added to dry popcorn maker and heated for 1-2 min at 100 . the popped corn was collected and grinded in cyclone grinder and used for extraction of β- carotene. Apparent retention of β-carotene Apparent retention can be defined as the ratio of the nutrient content in the processed food to the nutrient content in the raw food, expressed on a dry weight basis (Murphy et al. 1975). Previous researchers (Muzhingi et al. 2008; Li et al. 2007) have found the calculation of apparent retention to be straight forward, as it on dry matter basis. Apparent retention was calculated, using the equation given by Murphy et al. (1975). Nutrient content per g of cooked food (dry basis) % Apparent retention= ---------------------------------------------------------------x 100 Nutrient content per g of raw food (dry basis)

70


Statistical analysis All the experiments were conducted in triplicate for raw maize samples, invitro digested and food products. The digestive stability or bio accessibility is the ratio of Β- carotene recovered in the digesta after the oral and intestinal phase of digestion to the β- carotene content in the normal maize samples. STATA statistics version (statacorp 14.2) was used to analyze the data. Standard descriptive statistics including mean and SD from triplicate analysis were calculated for the retention of β- carotenoids from processed maize. Univariate analysis of variance and Tukey post-hoc multiple comparisons of means were used to calculate the influence of β- carotene from processed maize. Differences were considered significant at P < 0.05.

1200β (4.03±0.292 µg/g) having significant variation in β-carotene content. β-carotene content was found. The βcarotenoid reduction between the maize hybrids and their in vitro digestion (Fig 2). The recovery of β-carotene after stimulated gastrointestinal digestion ranged from 54.10 % to 69.51 % in both normal and biofortified maize hybrids. The bioaccesibity of β-carotene after in

Results and Discussion Biofortification of staple food crops enriched in pro-VA carotenoids is being considered to alleviate the prevalence of VAD in India and many other developing nations (Bouis et al. 2014). In addition to the increased density of nutrient, ensuring higher bioavailability is required for these foods to be nutritionally superior. The current study is divided into two parts, in first part βcarotene content was estimated in normal and invitro digested samples In second part the food products were prepared from selected biofortified maize sample and the retention of beta carotene after cooking was estimated.

Fig 2. Compar ison of beta car otene content between r aw maize flour and invitro digested samples

vitro digestion (Fig 3). Where in the genotype UMI 1200β × UMI 1230β (69.51 %) β-carotene is bioaccessible, in UMI 1200 (54.10 %), UMI 1200β (63.47 %) of β-carotene bioavailable after digestion.

UMI 1200 gives less β-carotene after digestion as its content was low in flour. The genotype UMI 1200β tends to give more bio accessible β-carotene, even though

The β-carotene content of biofortified maize before and after in vitro digestion (Table 1). The highest β-carotene content (7.41±0.040 µg/g) was found in F1 hybrid of provitamin A biofortified maize inbred lines involving, UMI 1200β and UMI 1230β. While the content of β-carotene in normal maize inbred UMI 1200 Table 1. β-carotene content in raw maize flour samples and in vitro digested samples.

S. No

Samples

1 2

UMI 1200 UMI 1200 β UMI 1200 β x UMI 1230β

3

β-carotene (µg/g) Raw Maize In vitro flour accessibility Mean ± SD Mean ± SD 0.61± 0.155* 0.33±0.033* 6.35 ± 0.016* 4.03 ± 0.292* 7.41 ± 0.040*

5.15 ± 0.220*

Value of means ± SD, each analysis was carried out in triplicate. *significant at P