Vol 10 | Special Issue

ISSN: 2182-1054

International Journal of Food Studies

Progress and Optimization of Food Products Quality – Special Issue

Volume 10 | February 2021 International Journal of Food Studies

The International Journal of Food Studies (IJFS), a journal of the ISEKI_Food Association, is an international peerreviewed open-access journal featuring scientific articles on the world of Food in Education, Research and Industry. This journal is a forum created specifically to improve the dissemination of Food Science and Technology knowledge between Education, Research and Industry stakeholders. Manuscripts focusing on Food related Education topics are particularly welcome. The IJFS also accepts original research works dealing with food processing, design, storage and distribution, including effects on product’s safety and quality, and food chain sustainability. The journal is also open to other food-related topics such as food security and food policy.

Editor-in-Chief

PROFESSOR CRISTINA L. M. SILVA Catholic University of Portugal - College of Biotechnology, Rua Arquiteto Lobão Vital 172, 4200-374 Porto, Portugal

Vice Editors-in-Chief Professor Margarida Cortez Vieira Professor Paulo José do Amaral Sobral High Institute of Engineering of University of Algarve, University of São Paulo - Faculty of Animal Science and Estrada da Penha 139, 8005-139 Faro, Portugal Food Engineering, Av. Duque de Caxias Norte, 225, Campus Fernando Costa – USP, CEP 13635-900 Pirassununga, São Paulo, Brazil

Associate Editors Professor Liliana Tudoreanu Professor Rui Costa University of Agronomic Sciences and Veterinary College of Agriculture - Polytechnic Institute of Coimbra, Portugal Medicine Bucharest, Romania Dr. Rui Cruz Professor Margarida Cortez Vieira University of Algarve, Portugal University of Algarve, Portugal Professor Tanaboon Sajjaanantakul Professor Paulo José do Amaral Sobral Kasetsart University, Thailand University of São Paulo - Faculty of Animal Science and Food Engineering, Brazil Professor Victoria Jideani Cape Peninsula University of Technology, South Professor Petras Rimantas Venskutonis Kaunas University of Technology, Lithuania Professor Fabio Napolitano Università degli Studi della Basilicata, Italy

Advisory Board

Afam Jideani Gustavo Gutiérrez-López Peter Ho University of Venda, South Africa National School of Biological Sciences, University of Leeds, United Kingdom António Vicente Mexico Pilar Buera Universidade do Minho, Portugal José António Teixeira School of Exact and Natural Sciences, Brian McKenna Universidade do Minho, Portugal Argentina University College Dublin, Ireland Kristberg Kristbergsson Sam Saguy Elisabeth Dumoulin University of Iceland, Iceland Hebrew University of Jerusalem, Israel Paris Institute of Technology for Life, Mustapha El Idrissi Teresa Brandão France Mohamed Premier University, Morocco Catholic University of Portugal, Ferruh Erdogdu Pablo Ribotta Portugal Ankara University, Turkey School of Exact Physics and Natural V. Prakash Gerhard Schleining Sciences, Argentina Central Food Technological Research BOKU, Austria Paul Singh Institute, India Gustavo V. Barbosa-Canovas University of California - Davis, Venkatesh Meda Washington State University, United States University of Saskatchewan, Canada United States of America Paola Pittia University of Teramo, Italy

INTERNATIONAL JOURNAL OF FOOD STUDIES

Volume 10, SPECIAL ISSUE (2021) (Published 24 February 2021)

CONTENTS

1 Chemical Composition, Anti-Nutritional Factors and Pasting Properties of Cassava-African Yam Bean Flour Blends for Noodle Preparation GHANIYAH O. AJIBOLA AND ABIODUN A. OLAPADE

14 Effect of Taro (Colocasia esculenta) Enrichment on Physicochemical and Textural Properties of Cake ANUJ SAKLANI, RAVINDER KAUSHIK, PRINCE CHAWLA, NAVEEN KUMAR AND MUKUL KUMAR

26 Effect of Emulsifier Diacetyl Tartaric Acid Ester of Mono- and Diglycerides (DATEM) and Enzyme Transglutaminase on Quality Characteristics of Rice Bran Croissants WAN ZUNAIRAH WAN-IBADULLAH, AW YING HONG, M.A.R. NOR-KHAIZURA, NOR AFIZAH MUSTAPHA, Z. A. NUR HANANI, MOHAMMAD RASHEDI ISMAIL-FITRY AND ANIS SHOBIRIN MEOR HUSIN

41 Optimization of a Process for a Microgreen and Fruit Based Ready to Serve Beverage ANJALI SHARMA, PRASAD RASANE, ANIRBAN DEY, JYOTI SINGH, SAWINDER KAUR, KAJAL DHAWAN, ASHWANI KUMAR AND HARI SHANKAR JOSHI

57 Chickpea Protein Isolation, Characterization and Application in Muffin Enrichment SOBHY A. EL-SOHAIMY, MARAGETA A. BRENNAN, AMIRA M. G. DARWISH AND CHARLES S. BRENNAN

72 Nutritional Evaluation of Unripe Plantain, Moringa Seed and Defatted Sesame Seed Cookies MOPELOLA A. SODIPO, MATTHEW O. OLUWAMUKOMI, ZIANAB A. ODERINDE AND OLUGBENGA O. AWOLU

82 Chemical Characteristics and Sensory Properties of Novel Snacks Produced with Okara Fortified with Omega-3 from Fish Oil TRINIDAD SANDRA ÁLVAREZ AND DANIELA LORENA LAMAS

95 Study of Antimicrobial, Antioxidant and Cytotoxicity Properties of Selected Plant Extracts for Food Preservative Applications TANIA ISLAM, MD NAZRUL ISLAM, WAHIDU ZZAMAN AND MD MORSALINE BILLAH

112 The Effect of Seaweed (Eucheuma cottonii) Flour Addition on Physicochemical and Sensory Characteristics of an Indonesian-Style Beef Meatball ARIS SRI WIDATI, DJALAL ROSYIDI, LILIK EKA RADIATI AND HAPPY NURSYAM

121 Potential for Development of Novel Food Products from Azanza garckeana Tree Fruit: A Review JOHNSON MWOVE

International Journal of Food Studies IJFS February 2021 Volume 10 pages SI1–SI13

Chemical Composition, Anti-Nutritional Factors and Pasting Properties of Cassava-African Yam Bean Flour Blends for Noodle Preparation

Ghaniyah O. Ajibolaa* and Abiodun A. Olapadea

a Department of Food Technology, Faculty of Technology, University of Ibadan, Ibadan, Nigeria. *Corresponding author [email protected] Tel.: +234-8056348979

Received: 14 November 2017; Published online: 24 February 2021

Abstract

Noodle consumption has been increasing in Nigeria as a result of rapid urbanization, increase in population growth, and desire for convenience food. Noodles are produced from wheat flour which is not grown in Nigeria. In order to reduce wheat imports and improve utilization of local crops, various options have been developed to replace wheat flour partially or wholly in noodle production. This study was aimed at optimizing the level of major ingredients to obtain the best flour blend for noodle preparation with optimum nutritional quality. Pro-vitamin A cassava roots (Manihot esculenta Crantz) and African yam bean seeds (AYB) (Sphenostylis stenocarpa) were processed into flours. The ranges of these flours, based on preliminary findings, were computed into a central composite design of Response Surface Methodology (RSM) to obtain 13 flour blends with five central points. The chemical compositions, anti-nutritional factors, and pasting properties of these flour blends were analyzed and measured. By maximizing total β-carotene, protein content, and minimizing fat content, the predicted model indicated the optimum blend of 70.52% cassava flour to 29.48% AYB flour. The best flour blend sample of 69.23% cassava flour to 30.77% AYB flour gave the actual value of total β-carotene 6.76 µg/g, with proximate analysis composition of protein 6.17%, fat 0.82%, moisture 8.95%, ash 1.77%, crude fiber 5.09%, and carbohydrate 82.30%. The anti-nutritional factors of the best blend were 8.21 mg HCNeqv/kg, 1.69 mg phytate/g and 0.37 mg tannin/g. Keywords: Flour blends; Noodles; Nutritional quality; Pro-vitamin A cassava; Response Surface Methodology

1 Introduction potato. Seibel (2006) opined that no other crop can accomplish the baking properties of wheat. Many developing countries spend an exorbitant Hence, composite flour has become the emphasis amount of their foreign exchange on importation of various studies. There has arisen the need to of wheat (Ohimain, 2014). This is due to use affordable indigenous crops that still provide the high rate of wheat consumption in those optimum nutritive value and good processing countries. According to Oyeku et al. (2008), attributes to substitute for wheat flour in the it would be of great economic importance if food industry. wheat importation is minimized drastically by Local raw material replacement for wheat flour replacing it with other locally available raw is increasingly essential due to the rising market materials such as cassava, rice, cowpea, maize,

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a1 SI2 Ajibola and Olapade for confectioneries (Noor Aziah & Komathi, food. 2009) and convenience foods in emerging coun- The anti-nutrients like trypsin inhibitors, phytic tries. Thus, many developing countries have acid, saponins, heamagglutinins and tannins are encouraged the commencement of programmes some of the undesirable components in legumes to assess the feasibility of alternative and locally that could hinder utilization of important accessible flours as a substitute for wheat flour minerals such as calcium, magnesium, iron and (Abdelghafor, Mustafa, Ibrahim & Krishnan, zinc. They interfere with the absorption and 2011). Therefore, utilization of composite flour utilization of minerals and thereby contribute is considered beneficial in emerging nations to mineral deficiency (Qayyum, Butt, Anjum as it decreases the imported wheat flour and & Nawaz, 2012; Vasagam & Rajkumar, 2011). boosts the use of indigenous crops as flour They are generally toxic and may negatively (Hugo, Rooney & Taylor, 2000; Mamat et al., affect the nutrient value of legume seeds by 2014). This will generate income for farmers, impairing protein digestibility and mineral motivate cultivation of indigenous crops, create availability. However, they are heat labile and employment opportunities and thereby improve hence may be inactivated by processing methods local economies. involving heat (Ndidi et al., 2014). Babatunde (2012) opined that large components According to Bouchenak and Lamri-Senhadji of helpless populations, women of child-bearing (2013), legumes represent essential parts of age and children in regions where cassava the human diet in several areas of the world, consumption is high, were vulnerable to vitamin particularly in the developing countries, where A deficiency. According to UNICEF (2004) and they compensate for the lack of protein from World Bank (2009), Nigeria loses over US$1.5 diets rich in cereals, roots and tubers. Nwokeke, billion per annum in Gross Domestic Product Adedokun and Osuji (2013) opined that African (GDP) to vitamin and mineral deficiencies as yam bean seed (AYB) (Sphenostylis steno- many staple foods are very low in essential carpa) is one of the best sources for protein micronutrients. Therefore, adopting strategies supplementation of starchy foods. It has been to reduce these problems is highly essential. chosen to fortify starch-based food due to its In view of this, biofortification of staple crops nutritional potential to enrich food. The use with pro-vitamin A carotenoid is an evolving of AYB will also promote the utilization of approach to address the low vitamin A status this lesser-known and under-utilized legume in of the people (Tanumihardjo, Bouis, Hotz, a number of food preparations in developing Meenakshi & McClafferty, 2008). This has countries. The general objective of this study is promoted the biofortification of cassava. to evaluate the nutritional quality of pro-vitamin Yellow-fleshed cassava (Manihot esculenta A cassava-AYB flour blends for noodle prepara- Crantz) cultivar 070593 are being propagated tion. The specific objectives include assessment in Nigeria to aid in fighting dietary vitamin A of chemical, anti-nutritional factors, and pasting deficiency due to their high content in β-carotene properties of pro-vitamin A cassava and AYB (a precursor of vitamin A) (Omodamiro et al., flour blends. 2012). Since cassava is a major staple food crop in Nigeria, consumption of this pro-vitamin A cassava would help in combating vitamin A deficiency, a serious public health problem in many parts of the world including Nigeria (Omodamiro et al., 2011; Thakkar, Huo, Maziya-Dixon & Failla, 2009; Vimala, Thushara, Nambisan & Sreekumar, 2011). One of the restrictive factors to the utilization of cassava flour (CF) in food production is its low protein content. Therefore, fortification with protein-rich flour is required to improve the nutritional value of CF formulated

IJFS February 2021 Volume 10 pages SI1–SI13 Nutritional Quality of Cassava-African Yam Bean Flour Blends SI3

2 Materials and Methods ingredient factors, pro-vitamin A cassava flour and AYB flour, ranged from 60-90% and 10-40%, 2.1 Sources of materials respectively. These ranges were chosen based on preliminary findings on noodle preparation in our Pro-vitamin A (yellow-fleshed) cassava (Manihot laboratory. This generated 13 combinations with esculenta Crantz) of cultivar 070593 and African five central points as depicted in Table1. The yam bean (AYB) (Sphenostylis stenocarpa) of ac- central point was repeated five times to analyse cession (TSs 94) were obtained from the Cassava the reproducibility of the method. Processing Unit and Genetic Resources Centre (GRC) of the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. Flour blends preparation Pro-vitamin A cassava and AYB flours were 2.2 Preparation of flours weighed and thoroughly mixed at predetermined ratios using a Bajaj grinder mixer (model GX 10 Pro-vitamin A cassava roots were harvested 10- DLX, Bajaj Electrical Limited Mumbai, India) 12 months after planting, sorted and processed for 1 min to obtain a homogenous flour blend. into flours based on the method described by Aniedu and Omodamiro (2012). Cassava roots 2.4 Chemical analyses of flours were peeled manually, washed in clean water, grated and dewatered with the use of 50 ton and flour blends pressing equipment. The wet cake obtained was pulverised to decrease the size and increase the β-carotene determination surface area. The pulverised wet cake was dried The method of Carvalho et al. (2012) was fol- employing a Niji Lukas Flash dryer located at lowed with slight modification to determine β- IITA, Ibadan at a temperature of 130 oC for 20 carotene and its isomers. Ten grams of each s residence time. The dried cassava flour was sample were weighed and crushed with mortar milled, cooled to room temperature and sieved to aid β-carotene extraction. Distilled water was with 250 µm sieve prior to packaging. AYB added to soak the sample for 5 min, then 30 mL seeds were processed into flour according to the of acetone (C H O, Merck, KGaA, Germany) method described by Nwosu, Ahaotu, Ayozie, 3 6 was added. This was left for 20 min and 3 g of Udeozor and Ahaotu (2011) with a slight modi- celite (SiO , FOSS, North America) was added fication. The seeds were cleaned, sorted and 2 to aid the extraction. A pestle was used to crush soaked in water with seed to water ratio of 1:3 the sample 3 times for about 5 min each time and for 24 h. The modification involved boiling of acetone was added to the sample at each crush- soaked seeds at 100 oC for 10 min. These were ing time until it was completely extracted. This drained, cooled and manually dehulled. The de- was filtered in a Buchner funnel containing filter hulled seeds were washed to separate the seed paper connected to a pump to aid the filtration. coats and oven dried at 60 oC for 6 h. The dried The extract was transferred to a 500 mL separa- seeds were milled, cooled, and sieved through a tion funnel containing 40 mL of petroleum ether 250 µm sieve to obtain AYB flour and packaged (C H , Thermo Fisher Scientific, Waltham, in a low-density polyethylene bag until analysis. 6 14 US). The acetone was used to rinse the container to ensure all the extracts were removed. This 2.3 Experimental design acetone was carefully removed by washing via slow addition of distilled water into the extract. This study adopted Response Surface Method- This procedure was repeated four times until no ology (RSM) according to the central compos- residual solvent remained. The lower phase, be- ite rotatable design (CCD) for the establishment ing water was discarded and the upper phase was of test flour blends using Design Expert version carefully transferred via a small funnel contain- 6.0.6 (Stat-Ease Inc., Minneapolis). Two major ing cotton wool and anhydrous sodium sulphate

IJFS February 2021 Volume 10 pages SI1–SI13 SI4 Ajibola and Olapade

Table 1: Experimental design for flour blend optimization of pro-vitamin A cassava flour (CF) and African yam bean flour (AYBF), and actual experimental values of the blends

Coded values Actual experimental values *CF *AYBF CF AYBF β-carotene Protein Fat Trials (%) (%) (µg/g) (%) (%) 1 0 -1.414 94.49 5.51 7.61 2.31 0.73 2 1 -1 90.00 10.00 6.68 2.88 0.75 3 1.414 0 80.06 19.94 6.37 5.08 0.86 4 -1 -1 80.00 20.00 6.82 5.03 1.00 5 0 0 72.22 27.78 6.57 6.54 0.58 6 0 0 72.22 27.78 6.59 6.48 0.58 7 0 0 72.22 27.78 6.58 6.02 0.58 8 0 0 72.22 27.78 6.58 6.35 0.58 9 0 0 72.22 27.78 6.61 5.08 0.56 10 1 1 69.23 30.77 6.76 6.17 0.82 11 0 1.414 58.45 41.55 3.50 9.17 1.04 12 -1.414 0 54.25 45.75 5.90 9.77 1.40 13 -1 1 50.00 50.00 4.69 10.52 1.05 *CF = Pro-vitamin A cassava ﬂour, *AYBF = African yam bean ﬂour.

(Na2SO4, BDH laboratory supplies, England) to where Ax = carotenoid peak area, Cs(µg/ml) = remove residual water. This was made up to 25 standard concentration, As = standard area, V= mL with petroleum ether and allowed to settle. total extract volume (ml) and P = sample weight Fifteen millimetres of the extract were pipet- (g). ted into a concentrator tube and concentrated at o ® 40 C for 25 min. in a TurboVap LV concentra- Proximate composition of flour blends tion workstation (Caliper Life Sciences, U.S.A.). The concentrate was diluted with 1 mL of di- Moisture and ash contents of the flour blends chloroethane (CH2Cl2, BDH, Bristol, UK) and 1 were determined as described in AOAC (2005). mL of methanol (CH3OH, Sigma Aldrich, Ger- The crude protein was determined by the many). This was mixed in a vortex mixer and Kjeldahl method using a FOSS Kjeltec 2300 transferred to a 2-mL amber flask for subsequent model protein analyser as described by Alamu, analysis using High Performance Liquid Chroma- Maziya-Dixon, Popoola, Gondwe and Chikoye tography (HPLC) (Agilent 1200 series, Perkin (2016) with slight modifications. The modific- Elmer, USA, β-carotene standard: CAS: 7235- ations involved the addition of 12 mL of concen- 40-7, Sigma Aldrich, Germany, column: YMC trated H2S04 instead of 4 mL. 0.15 g of sample carotenoids column 250x4.6x 5 µm, detector: was weighed into a digestion tube, a tablet of PDA Waters 2695, Solvents: dichloromethane: Kjeldahl catalyst and 12 mL of concentrated cat no 67-56-1 and methyl tert-butyl ether, cat H2S04 were added. The crude fat contents of no 1634-04-4, Fisher Loughborough, UK.). Em- the flour blends were determined as described power 3 chromatography data software was used in AACC (2005) using an automated method to scan each sample and values obtained were in- (FOSS, Soxtec system HT2). The crude fibre put into equation (1) to determine the content of contents of the flour blends were determined as β-carotene and its isomers (µg/g). described in AOAC (1990) using FOSS Fibertec 2010 equipment.

Ax × Cs × V The total carbohydrate contents were calculated C = (1) by diﬀerence as reported by Koua et al. (2012). As × P

IJFS February 2021 Volume 10 pages SI1–SI13 Nutritional Quality of Cassava-African Yam Bean Flour Blends SI5

Anti-nutritional composition of ﬂour evaluate the model ﬁtness as indicated in Table blends 2.

The method of alkaline picrate as described by Onwuka (2005) was adopted in determining hy- 2.7 Statistical analysis drogen cyanide contents, while the phytate contents of flour blends were determined based on Data from analysis of flour blends were subjec- the method described by Maga (1982). Tannin ted to one-way analysis of variance (ANOVA) us- content was determined based on the method ing Statistical Package for Social Sciences (SPSS) described by Fagbemi, Oshodi and Ipinmoroti (version 20, 2013). Means were equated and sep- (2005) with slight amendments. arated using Duncan’s New Multiple Range Test (DMRT) and LSD at (p≤0.05). Design expert was also used for data analysis. 2.5 Determination of pasting characteristics of flour blends 3 Results and Discussion Pasting properties of the flour blends were determined as described in AACC (2005) using a 3.1 Chemical composition of flour Rapid Visco Analyser Super 4 (Model RVA-4C, blends Newport Scientific, Warriewood, Australia). The pasting properties: peak viscosity, trough, break- The total β-carotene contents of flour blends down viscosity, setback value, final viscosity in ranged from 3.50 to 7.61 µg/g flour. The flour centipoise (cP), pasting temperature (oC) and blend 94.49CF:5.51AYBF had the highest β- peak time (min) were obtained. These viscos- carotene, while sample 58.45CF:41.55AYBF had ities were converted to RVU (Rapid Visco Unit) the least. There were significant (p≤0.05) dif- with the conversion factor 12 cP = 1 RVU. ferences among the flour blends based on their total β-carotene contents as shown in Table3. It was observed that as AYB flour increased in the 2.6 Optimization of flour blends blend, the β-carotene content decreased. The proximate composition of flour blends is The optimisation procedure as described by shown in Table4. There were significant dif- Delgado, Paim, Pereira, Casal and Ramalhosa ferences (p≤0.05) in proximate composition of (2018) was followed. The Design expert software the flour blends due to different composition of version 6.0.6 (Stat-Ease Inc., Minneapolis) was the flours. The moisture contents of the flour used in this study. The ranges of pro-vitamin A blends ranged from 8.12 to 10.13% with the blend cassava flour (CF) and AYB flours (AYBF) as ob- 54.25CF:45.75AYBF having the highest moisture tained from preliminary study were inserted into content, while the blend 90CF:10AYB had the the design expert to generate the number of flour lowest. These values fell within the ranges ob- blends. These flour blends were optimized based tained by Iwe, Onyeukwu, Agiriga and Yildiz on the chosen quality parameters, i.e., maximiz- (2016) with values of 8-14% and compared with ing total β-carotene and protein contents to im- 13% maximum recommended moisture content prove the nutritional value, while minimizing fat for edible cassava flour by the Standard Organ- content to avoid rancidity of flours. The mean of ization of Nigeria (SON) as reported by Sanni et duplicate analysis values (see below) of total β- al. (2005). carotene, protein, and fat content were employed The fat contents of the flour blends ranged from to find an optimum flour blend value by fitting 0.56 to 1.4%. The blend 54.25CF:45.75AYBF polynomial models. The coefficients of polyno- had the highest fat content, while the blend mial models, coefficient of determination (R2), 72.22CF:27.78AYBF had the least. The reason adjusted R2 and adequate precision values of the for this low fat content might be that cereals and chemical properties of the blends were used to tubers accumulate energy in the form of starch

IJFS February 2021 Volume 10 pages SI1–SI13 SI6 Ajibola and Olapade

Table 2: Coefficient of polynomial models of RSM for pro-vitamin A cassava flour and African yam bean flour blends Variables β-carotene Protein Fat Terms Coefficient P Coefficient P Coefficient P Constant 6.59 0.0005 6.38 0.0001 0.58 0.008 A1 0.54 0.0032 -1.64 0.0001 -0.17 0.0059 B2 -0.76 0.0005 2.31 0.0001 0.05 0.3039 2 A1 0.18 0.2105 0.42 0.009 0.2 0.0038 2 B2 -0.73 0.0009 -0.43 0.008 0.13 0.0263 A1B2 0.56 0.0155 -0.55 0.0089 0.06 0.3953 Lack of fit s 0.0001 ns 0.1199 s 0.0001 R2 0.9360 0.9904 0.8525 Adjusted R2 0.8902 0.9836 0.7491 Adequate precision 15.463 41.266 7.611 Model Quadratic Quadratic Quadratic 2 A1 denotes Pro-vitamin A cassava flour, B2 denotes African yam bean flour, R denotes coefficient of determination, s denotes significant, ns signifies non-significant.

rather than lipids. Moreover, these low-fat con- cowpea seeds. The carbohydrate content of the tents are beneficial as they ensure longer shelf life flour blends ranged from 76.60-86.86%. The of the flours (Beebe, Viviana Gonzalez & Ren- blend 94.49CF:5.51AYBF had the highest carbo- gifo, 2000). hydrate, while sample coded 50CF:50AYBF had The protein content of the flour blends varied the lowest. It was observed that as the quantity from 2.31 to 10.52%. The blend 50CF:50AYBF of AYB flour increased in the flour blends, the had the highest protein content, while the blend carbohydrate content decreased (Table4). 94.49CF:5.51AYBF had the lowest. A significant increase in protein content was noted among the blends as the quantity of AYB flour increased. 3.2 Anti-nutritional compositions This increase was expected because AYB flour of flour blends is rich in protein and was in agreement with results obtained from Idowu (2015) for a snack made Table5 shows the anti-nutritional composi- from yellow maize and AYB flour blends. Pro- tion of the flour blends. The hydrogen cy- duction of high-quality noodles utilises flour of 7 anide (HCN) composition ranged from 6.22 to 9.5% protein content (Choy, 2011). to 10.32 mg HCNeqv/kg with sample coded The ash contents of the flour blends ranged 94.49CF:5.51AYBF having the highest, while from 1.66 to 2.06% with sample coded 58.45CF:41.55AYBF had the least. It was ob- 50CF:50AYBF having the highest while the served that hydrogen cyanide content of the blend 80CF:20AYBF had the lowest. These val- blend decreased as the quantity of AYB flour ues fell within the range reported by Omeire, Ka- increased in the blend. The phytate contents buo, Nwosu, Peter-Ikechukwu and Nwosu (2015) of the flour blends ranged from 1.08 to 2.44 for wheat cassava enriched noodles, values of mg/g with sample coded 54.25CF:45.75AYBF 1.39-2.84%. The crude fibre contents of the flour and 94.49CF:5.51AYBF having the highest and blends ranged from 3.19 to 5.09%. Sample coded least, respectively. The tannin contents of the 69.23CF:30.77AYBF had the highest crude fibre. flour blends ranged from 0.11 to 0.63 mg/g- These values were lower than those obtained with sample coded 50CF:50AYBF having the from Iwe et al. (2016), 0.95-6.27% for composite highest, while sample 94.49CF:5.51AYBF had flours made from FARO 44 rice, AYB and brown the least. There were significant (p≤0.05) dif-

IJFS February 2021 Volume 10 pages SI1–SI13 Nutritional Quality of Cassava-African Yam Bean Flour Blends SI7

Table 3: Total β-carotene of pro-vitamin A cassava ﬂour (CF) and African yam bean ﬂour blends

Sample code Coded value Coded value β- 13-cis Trans 9-cis Total CF: AYBF (%) A B cryptoxanthin (µg/g) (µg/g) (µg/g) β-Carotene (µg/g) (µg/g) 94.49CF:5.51AYBF 0 -1.414 0.86d 1.71d 3.76e 2.13c 7.61d 90CF:10AYBF 1 -1 0.76e 1.51e 3.28f 1.89d 6.67ghi 80.06CF:19.94AYBF 1.414 0 0.97c 1.46ef 2.97g 1.93d 6.36j 80CF:20AYBF -1 -1 0.74ef 1.54e 3.34f 1.93d 6.82fg 72.22CF:27.78AYBF 0 0 0.63g 1.39f 3.29f 1.89d 6.57hi 72.22CF:27.78AYBF 0 0 0.64g 1.39f 3.29f 1.91d 6.59hi 72.22CF:27.78AYBF 0 0 0.63g 1.39f 3.28f 1.91d 6.58hi 72.22CF:27.78AYBF 0 0 0.64g 1.39f 3.29f 1.90d 6.57hi 72.22CF:27.78AYBF 0 0 0.63g 1.39f 3.30f 1.91d 6.61hi 69.23CF:30.77AYBF 1 1 0.73f 1.50e 3.36f 1.91d 6.76fgh 58.45CF:41.55AYBF 0 1.414 0.41j 0.78i 1.73j 0.99h 3.50m 54.25CF:45.75AYBF -1.414 0 0.59h 1.25g 2.99g 1.53f 5.90k 50CF:50AYBF -1 1 0.49i 0.99h 2.36i 1.32g 4.69l CF = Pro-vitamin A cassava flour, AYBF = African yam bean flour. Mean value from triplicate determinations. Mean values with different superscripts within a column are significantly (P≤0.05) different.

Table 4: Proximate composition of pro-vitamin A cassava flour (CF) and African yam bean flour (AYBF) and their flour blends

Sample Coded Coded Moisture Fat Protein Ash Crude Carbohydrate code value value fibre CF: AYBF (%) A B (%) (%) (%) (%) (%) (%) 94.49CF:5.51AYBF 0 -1.414 8.35±0.24fg 0.73±0.07ef 2.31±0.13j 1.75±0.02bcd 4.01±0.18cde 86.86±0.34a 90CF:10AYBF 1 -1 8.12±0.09g 0.75±0.04de 2.88±0.06i 1.77±0.06bc 4.26±0.19bc 86.48±0.19b 80.06CF:19.94AYBF 1.414 0 8.61±0.16fg 0.86±0.04d 5.08±0.10h 1.80±0.01b 3.98±0.06cde 83.65±0.15c 80CF:20AYBF -1 -1 9.55±0.09cd 1.00±0.05c 5.03±0.16h 1.66±0.01d 3.76±0.27def 82.76±0.15d 72.22CF:27.78AYBF 0 0 9.77±0.18bc 0.59±0.04h 6.54±0.10e 1.70±0.01bcd 4.53±0.33b 81.39±0.21g 72.22CF:27.78AYBF 0 0 9.53±0.27cd 0.57±0.03h 6.48±0.19e 1.68±0.06cd 4.20±0.34bcd 81.75±0.41fg 72.22CF:27.78AYBF 0 0 9.62±0.09bc 0.58±0.01h 6.02±0.10g 1.70±0.02bcd 3.61±0.50ef 82.09±0.18ef 72.22CF:27.78AYBF 0 0 9.79±0.07bc 0.61±0.05gh 6.35±0.16ef 1.72±0.01bcd 4.35±0.30bc 81.53±0.13g 72.22CF:27.78AYBF 0 0 9.17±0.02de 0.56±0.03h 6.48±0.10e 1.78±0.01b 3.19±0.14g 82.01±0.08ef 69.23CF:30.77AYBF 1 1 8.95±0.04ef 0.82±0.06d 6.17±0.10fg 1.77±0.01bc 5.09±0.06a 82.30±0.16e 58.45CF:41.55AYBF 0 1.414 10.13±0.08b 1.04±0.03c 9.17±0.13d 1.98±0.03a 4.17±0.13bcd 77.86±0.13i 54.25CF:45.75AYBF -1.414 0 8.87±0.41ef 1.40±0.06b 9.77±0.10c 1.76±0.02bc 4.32±0.10bc 78.20±0.47h 50CF:50AYBF -1 1 9.77±0.18bc 1.05±0.04c 10.52±0.10b 2.06±0.04a 3.47±0.15fg 76.60±0.17j 100:0 9.87±0.05bc 0.54±0.02i 1.89±0.03k 1.29±0.05e 0.80±0.04i 86.98±0.16a 0:100 11.72±0.63a 1.99±0.02a 19.6±0.15a 2.07±0.02a 2.90±0.18h 64.62±0.27k CF = Pro-vitamin A cassava flour, AYBF = African yam bean flour. Mean triplicate determinations ± standard deviation. Mean values with different superscripts within a column are significantly (P≤0.05) different.

IJFS February 2021 Volume 10 pages SI1–SI13 SI8 Ajibola and Olapade

Table 5: Anti-nutritional compositions of pro-vitamin A cassava flour (CF) and African yam bean flour (AYBF) and their flour blends

Sample code Coded value Coded value HCN Phytate Tannin CF: AYBF (%) A B (mg/kg) (mg/g) (mg/g) 94.49CF:5.51AYBF 0 -1.414 10.32±0.14a 1.08±0.10i 0.11±0.01h 90CF:10AYBF 1 -1 10.18±0.35a 1.37±0.07gh 0.22±0.01g 80.06CF:19.94AYBF 1.414 0 9.41±0.22b 1.42±0.12g 0.24±0.01fg 80CF:20AYBF -1 -1 9.39±0.15b 1.55±0.17fg 0.26±0.01f 72.22CF:27.78AYBF 0 0 8.81±0.35c 1.95±0.10e 0.26±0.03f 72.22CF:27.78AYBF 0 0 9.45±0.47b 2.00±0.10de 0.24±0.01fg 72.22CF:27.78AYBF 0 0 8.88±0.38c 2.09±0.20cde 0.22±0.01g 72.22CF:27.78AYBF 0 0 9.51±0.46b 2.07±0.14cde 0.21±0.01g 72.22CF:27.78AYBF 0 0 8.69±0.40c 1.89±0.15e 0.25±0.02f 69.23CF:30.77AYBF 1 1 8.21±0.22d 1.69±0.10f 0.37±0.02d 58.45CF:41.55AYBF 0 1.414 6.22±0.27gh 2.24±0.07bc 0.34±0.01e 54.25CF:45.75AYBF -1.414 0 7.31±0.27ef 2.44±0.06b 0.45±0.03c 50CF:50AYBF -1 1 7.99±0.14e 2.20±0.10cd 0.63±0.02b 100:0 6.76±0.28g 1.38±0.07gh 0.34±0.01e 0:100 3.47±0.05i 2.55±0.11a 0.82±0.01a CF = Pro-vitamin A cassava flour, AYBF = African yam bean flour, HCN = hydrogen cyanide. Mean triplicate determinations ± standard deviation. Mean values with different superscripts within a column are significantly (P≤0.05) different.

ferences among the samples in terms of their from 214.66 to 514.5 RVU with sample coded phytate and tannin contents. The chosen 94.49CF:5.51AYBF having the highest, while blend sample 69.23CF:30.77AYBF contained sample coded 50CF:50AYBF having the lowest. 8.21 mg HCNeqv/kg of hydrogen cyanide, 1.69 The trough viscosity varied from 157.58 RVU to mg phytate/g and 0.37 mg tannin/g. This hy- 206.5 RVU for flour blends. This measures the drogen cyanide fell within a safe level of no more ability of the paste to withstand breakdown dur- than 10 mg HCNeqv/kg of cassava flour as re- ing cooling (Danbaba et al., 2012). A signific- commended by the Food and Agriculture Organ- ant decrease was noted in peak and breakdown isation of the United Nations and World Health viscosities as the level of AYB flour in the mix- Organisation (FAO/WHO, 2013). The hydrogen tures increased. This is as a result of protein in cyanide, phytate and tannin contents in the flour AYB flour forming an insoluble system that traps blends were lower than the allowable level of 50 swollen starch granules thus, preventing disinteg- mg HCNeqv/kg dry mass, 250-500 mg/100 g dry ration of the noodle surface which would make it mass, and 20 mg/g dry mass, respectively for soft and sticky (Harvey & Morgenstern, 2001). AYB seed as reported by Ndidi et al. (2014). Such a decrease was similarly reported by Dhull and Sandhu (2018) for wheat-fenugreek composite flour noodles. The peak and trough viscos- 3.3 Pasting properties of flour ities in this study were higher than those stated blends by Adegunwa, Bakare and Akinola (2012) for enriched noodles with values of 104.33-165.08 RVU There were significant differences in the past- and 51.67-109 RVU accordingly. The chosen ing properties of flour blends, except for their blend (69.23CF:30.77AYBF) had high peak and pasting temperatures as depicted in Table6. trough viscosities which is good for noodle mak- The peak viscosities of the blends ranged

IJFS February 2021 Volume 10 pages SI1–SI13 Nutritional Quality of Cassava-African Yam Bean Flour Blends SI9 b b a f bc de de de de ef bcd cde cde cde cde 0.07 0.00 0.00 0.71 0.53 0.28 0.00 0.07 0.00 0.71 0.07 0.64 0.28 0.60 0.60 C ± ± ± ± ± ± ± ± ± o ± ± ± ± ± ± 74.30 74.25 82.33 71.35 73.83 72.80 72.55 72.55 72.65 72.20 73.40 73.05 73.20 73.03 72.92 i i b c a h cd de de ef ef ef ef gh fg 0.01 0.05 0.05 0.14 0.02 0.00 0.00 0.00 0.05 0.05 0.01 0.05 0.00 0.00 0.03 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.23 4.23 5.50 5.23 7.33 4.47 5.33 5.20 5.23 5.10 5.13 5.17 5.13 4.53 5.00 b b c a a a bc bc bc cd e g h f f 3.83 0.47 3.01 2.48 2.12 2.95 1.06 5.95 2.95 0.94 3.54 2.48 0.71 2.47 5.89 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 89.50 45.58 10.50 62.25 63.58 111.46 111.42 103.63 120.92 121.17 119.25 110.33 109.21 105.67 102.75 h i b c e e e g d d d a f cd j 4.01 5.66 7.01 3.65 1.53 0.30 3.48 1.65 0.77 3.54 2.00 1.12 1971 1.47 1.41 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 20.00 203.17 320.92 307.13 285.33 281.83 287.21 245.04 296.25 296.96 296.50 346.75 266.04 229.63 303.04 standard deviation. Mean values with different ± b b c e e e g d a f f i h h j 3.30 0.00 4.77 3.59 0.83 5.30 4.07 5.24 0.06 0.41 0.65 1.30 4.71 4.01 0.00 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.00 57.08 82.83 86.00 310.58 308.00 232.13 190.13 188.50 186.83 162.88 221.46 343.54 170.71 174.88 i c c e e g g d a h h de b fg j 1.53 1.06 3.54 1.00 1.47 0.18 0.59 0.24 0.47 4.18 1.89 0.47 0.71 0.18 1.41 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 9.00 206.5 157.58 197.92 199.75 182.79 183.79 173.88 179.08 188.50 225.83 162.42 185.92 175.79 i b c j e g d a k h h f m n 0.18l 166.04 4.66 0.71 1.83 2.30 4.95 1.47 8.78 0.53 5.72 3.42 0.88 6.36 0.24 1.41 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05) different. ≤ Table 6: Pasting properties of pro-vitamin A cassava flour (CF), African yam bean flour and their flour blends 54.25CF:45.75AYBF -1.414 0 265.63 90CF:10AYBF 1 -1 494.38 Sample codeCF: AYBF (%)94.49CF:5.51AYBF Coded values Coded values A Peak 0 viscosity Trough -1.414 B Breakdown Final Viscosity 514.50 Setback (RVU)CF = Peak Pro-vitamin A (RVU) cassava flour, AYBF = African yam bean flour. Mean triplicate Pasting determinations (RVU) (RVU) (RVU) Time (min) Temperature superscripts within a column are significantly (p 50CF:50AYBF100:00:100 -1 1 214.66 569.37 10.00 58.45CF:41.55AYBF 0 1.414 252.04 69.23CF:30.77AYBF 1 1 333.13 72.22CF:27.78AYBF 0 0 364.00 72.22CF:27.78AYBF 0 0 374.38 72.22CF:27.78AYBF 0 0 384.75 72.22CF:27.78AYBF 0 0 363.38 72.22CF:27.78AYBF 0 0 341.96 80CF:20AYBF -1 -1 421.21 80.06CF:19.94AYF 1.414 0 407.92

IJFS February 2021 Volume 10 pages SI1–SI13 SI10 Ajibola and Olapade ing as high peak viscosity indicates that starch the responses: total β-carotene, protein and fat will not collapse as long as noodles are appro- contents were modelled using the polynomial re- priately cooked. Likewise, Bhattacharya, Zee sponse surface models of the Design expert soft- and Corke (1999) stated that high trough viscos- ware. This provided equations that predict re- ity signifies low cooking loss and superior eating lationships between independent and response quality. variables within a specified range as represented Breakdown viscosity of the flour blends ranged in equations (2) to (4). The equations (2) and (3) from 57.08 to 310.58 RVU with sample coded were for maximizing total β-carotene and protein 90CF:10AYBF having the highest breakdown contents, while equation (4) was for minimizing viscosity and sample 50CF:50AYBF the lowest. fat content of the flour blend. This is an index of the stability of the starch and measures the ease at which the gelatinized starch Total β − carotene : can be collapsed (Maninder, Sandhu & Singh, 2 2007). Protein traps gelatinised starch thus re- Y = 6.59 + 0.54A1 − 0.76B2 + 0.18A1 (2) 2 ducing the breakdown viscosity and improving − 0.73B2 + 0.56A1B2 starch stability. Hence, low breakdown viscosity is desirable for good noodle quality. Protein: The final viscosity of the flour blends ranged from 2 Y = 6.38 − 1.64A1 + 2.31B2 + 0.42A1 (3) 203.17 to 320.92 RVU. These values were higher − 0.43B2 − 0.55A B2 than those stated by Adegunwa et al. (2012) 2 1 with values of 126.42-217.08 RVU for noodles en- Fat: riched with soy flour and carrot powder. The 2 Y = 0.58 − 0.17A1 + 0.049B2 + 0.2A1 (4) setback viscosity varied from 45.58 to 119.25 2 RVU with sample 94.49CF:5.51AYBF having + 0.13B2 + 0.057A1B2 the highest setback values. The chosen blend Where A1= Pro-vitamin A cassava flour and B2 (69.23CF:30.77AYBF) had a moderate setback = African yam bean flour. value of 103.63 RVU, good for producing qual- The estimation of how well the model fits the ity noodles, and this value fell within the range data can be assessed by the value of R2, adjusted stated by Adegunwa et al. (2012) with values of R2 and adequate precision. The R2 ranges from 0 73.25-118.42 RVU. It was noted that the sample to 1; when it is nearer to 1 the regression equation with the lowest AYB flour inclusion had the estimation fits the sample data very well. The highest setback value, thus undergoing faster composite desirability has a range of zero to one. syneresis. This faster syneresis causes rapid qual- One signifies the ideal case, while zero denotes ity degradation which is not good for noodles. that one or more responses are not within their Peak time and pasting temperatures of flour satisfactory boundaries. It was observed that R2 blends ranged from 4.23 to 5.50 min and 72.20 and adjusted R2 for the chosen models for β- to 74.30 oC accordingly. Adebowale, Sanni and carotene, protein and fat contents were greater Oladapo (2009) stated that a pasting temperat- than 0.80 and therefore indicated good fit models ure lower than the boiling temperature of water (Table2). The models for total β-carotene, pro- suggests that samples will form paste in hot wa- tein and fat contents were quadratic, and found ter below its boiling point, thus potentially pro- to be significant for total β-carotene and fat with moting cost saving. ‘Prob >F’ less than 0.05. The predicted optimum blend from the RSM calculation was 70.52CF:29.48AYBF which yiel- 3.4 Predictive models and ded response values for total β-carotene, protein optimum flour blend and fat content as 6.46 µg/g, 6.68% and 0.58%, conditions respectively. The desirability value of this optimum blend as obtained from the Design expert The effects of two independent variables A1 (pro- software was 0.725. The actual experimental val- vitamin A cassava flour) and B2 (AYB flour) on ues of 69.23CF:30.77AYBF flour blend (Table1

IJFS February 2021 Volume 10 pages SI1–SI13 Nutritional Quality of Cassava-African Yam Bean Flour Blends SI11 trial 10) exhibited total β-carotene, protein and Adegunwa, M., Bakare, H. A. & Akinola, O. F. fat content values of 6.76 µg/g, 6.17% and 0.82%, (2012). Enrichment of noodles with soy respectively, which was in agreement with pre- flour and carrot powder. Nigerian Food dicted response values. Journal, 30, 74–81. doi:10 . 1016 / S0189 - 7241(15)30016-3 4 Conclusion Alamu, E., Maziya-Dixon, B., Popoola, I., Gondwe, T. & Chikoye, D. (2016). Nutri- Response surface methodology was adopted tional evaluation and consumer preference in optimizing the pro-vitamin A cassava and of legume fortified maize-meal porridge. 4, African yam bean flour blends. The blends were 664–670. doi:10.12691/jfnr-4-10-6 formulated to maximize total β carotene and pro- Aniedu, C. & Omodamiro, R. M. (2012). Use of tein content while minimizing fat content which newly bred beta-carotene cassava in pro- resulted in recommended (predicted) values of duction of value-added products: Implic- the blend 70.52CF: 29.48AYBF. The blend coded ation for food security in nigeria. Global 69.23CF:30.77AYBF was closest to the predicted Journal of Science Frontier Research Ag- value and thus, the best chosen blend for noodle riculture and Veterinary Sciences, 12 (10), preparation. This optimum blend would be suit- 1–10. able for developing cassava-African yam bean AOAC. (1990). Official Methods of Analysis, noodles. The protein content of pro-vitamin A 15th Ed. Association of Official Analytical cassava flour improved as the quantity of AYB Chemists, Ed. Kenneth Helrich. flour increased in the blend. Our findings should AOAC. (2005). Official Methods of Analysis, promote the utilization of both local crops. 18th Ed. Association of Official Analytical Chemists. Washington D.C., USA. Babatunde, J. (2012). Journey to consumption Acknowledgements of pro-vitamin A cassava varieties in Ni- geria. Vanguard. Retrieved from www . The authors appreciated the efforts of GRC and vanguardngr.com the Cassava Processing Unit of the International Beebe, S., Viviana Gonzalez, A. & Rengifo, J. Institute of Tropical Agriculture for the provision (2000). Research on trace minerals in the of AYB seeds and cassava roots used for this re- common bean. Food & Nutrition Bulletin, search. 21. doi:10.1177/156482650002100408 Bhattacharya, M., Zee, S. Y. & Corke, H. (1999). References Physicochemical properties related to quality of rice noodles. Cereal Chemistry, AACC. (2005). Approved methods of the Amer- 76 (6), 861–867. doi:10 . 1094 / CCHEM . ican Association of Cereal Chemists, 11th 1999.76.6.861 Ed. St.Paul, MN. Bouchenak, M. & Lamri-Senhadji, M. (2013). Abdelghafor, R. F., Mustafa, A. I., Ibrahim, Nutritional quality of legumes, and their A. M. H. & Krishnan, P. (2011). Quality of role in cardiometabolic risk prevention: A bread from composite flour of sorghum and review. Journal of Medicinal Food, 16 (3), hard white winter wheat. Advance Journal 185–198. doi:10.1089/jmf.2011.0238 of Food Science and Technology, 3, 9–15. Carvalho, L., Oliveira, A., Godoy, R., Pacheco, Adebowale, A.-R., Sanni, S. A. & Oladapo, F. O. S., Nutti, M., Carvalho, J., . . . G Fukuda, (2009). Chemical, functional and sensory W. (2012). Retention of total carotenoid properties of instant yam - breadfruit flour. and beta-carotene in yellow sweet cassava Nigerian Food Journal, 26. doi:10 . 4314 / (manihot esculenta crantz) after domestic nifoj.v26i1.47417 cooking. Food & Nutrition Research, 56. doi:10.3402/fnr.v56i0.15788 Choy, A. (2011). Enhancing the quality of instant noodles: The impact of low protein wheat

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flour, ingredients and processing conditions Idowu, A. O. (2015). Nutrient composition and (Doctoral dissertation, School of Applied sensory properties of kokoro (a nigerian Sciences, Science, Engineering and Health snack) made from maize and african yam Portfolio RMIT University, Malaysia). bean flour blends. International Food Re- Danbaba, N., Anounye, J. C., Gana, A. S., Abo, search Journal, 22 (2), 739–744. M. E., Ukwungwu, M. N. & Maji, A. T. Iwe, M., Onyeukwu, U., Agiriga, A. & Yildiz, F. (2012). Physical and pasting properties of (2016). Proximate, functional and pasting ‘ofada’rice (Oryza sativa L.) varieties. Ni- properties of faro 44 rice, african yam bean gerian Food Journal, 30 (1), 18–25. and brown cowpea seeds composite flour. Delgado, T., Paim, B., Pereira, J., Casal, S. & Cogent Food & Agriculture, 2, 1142409. Ramalhosa, E. (2018). Optimization of os- doi:10.1080/23311932.2016.1142409 motic dehydration of chestnut (castanea Koua, G. Y. A., Megnanou, R., Kouassi, A., sativa mill.) slices using response sur- Séverin Kra, K., N’zue, B., Bi, D. & face methodology. International Journal of Niamké,S. L. (2012). Biochemical charac- Food Studies, 7, 52–68. doi:10.7455/ijfs/7. terization of new varieties of yellow colored 1.2018.a5 pulp cassava flours from côte d’ivoire. Dhull, S. B. & Sandhu, K. S. (2018). Wheat- Journal of Applied Biosciences, 53, 3760– fenugreek composite flour noodles: Effect 3772. on functional, pasting, cooking and sensory Maga, J. A. (1982). Phytate - its chemistry, oc- properties. Current Research in Nutrition currence, food interactions, nutritional sig- and Food Science, 6 (1), 174–182. doi:10 . nificance, and methods of analysis. Journal 12944/CRNFSJ.6.1.20 of Agricultural and Food Chemistry, 30 (1), Fagbemi, T. N., Oshodi, A. A. & Ipin- 1–9. doi:10.1021/jf00109a001 moroti, K. O. (2005). Processing effects on Mamat, H., Matanjun, P., Ibrahim, S., Amin, some antinutritional factors and in vitro S. F. M., Hamid, M. A. & Rameli, A. S. multienzyme protein digestibility (ivpd) (2014). The effect of seaweed composite of three tropical seeds: Breadnut (arto- flour on the textural properties of dough carpus altilis), cashewnut (anacardium oc- and bread. Journal of Applied Phycology, cidentale) and fluted pumpkin (telfairia oc- 26 (2), 1057–1062. doi:10.1007/s10811-013- cidentalis). Pakistan Journal of Nutrition, 0082-8 4 (4), 250–256. Maninder, K., Sandhu, K. & Singh, N. (2007). FAO/WHO. (2013). Proposed draft maximum Comparative study of the functional, levels for hydrocyanic acid in cassava and thermal and pasting properties of flours cassava products. Joint FAO/WHO food from different field pea (pisum sativum l.) standards programme Codex committee on and pigeon pea (cajanus cajan l.) cultivars. contaminants in foods. CX/CF.13/7/10. Food Chemistry, 104, 259–267. doi:10 . Codex Alimentarius Commission E, Rome, 1016/j.foodchem.2006.11.037 Italy. Ndidi, U., Unekwuojo Ndidi, C., Abbas, O., Harvey, W. & Morgenstern, M. (2001). Bak- Muhammad, A., Graham Billy, F. & Oche, ing Industry Research Trust (BIRT). Flour O. (2014). Research article proximate, an- properties for pasta manufacture. Crop and tinutrients and mineral composition of Food Research confidential report no. 419. raw and processed (boiled and roasted) Retrieved from https : / / bakeinfo . co . nz / sphenostylis stenocarpa seeds from south- files/file/118/issue14.pdf ern kaduna, northwest nigeria. ISRN nutri- Hugo, L. F., Rooney, L. W. & Taylor, J. R. N. tion, 2014. doi:10.1155/2014/280837 (2000). Malted sorghum as a functional Noor Aziah, A. & Komathi, C. A. (2009). ingredient in composite bread. Cereal Acceptability attributes of crackers made Chemistry, 77 (4), 428–432. doi:10 . 1094 / from different types of composite flour. CCHEM.2000.77.4.428 International Food Research Journal, 16, 479–482.

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Nwokeke, B., Adedokun, I. & Osuji, C. (2013). ates recovery. Journal of Animal and Plant Effect of blending on the proximate, past- Sciences, 22 (4), 1156–1162. ing and sensory attributes of cassava- Sanni, L., Maziya-Dixon, B., Akanya, J., african yam bean fufu flour. International I. Okoro, C., Alaya, Y., V. Egwuonwu, C., Journal of Scientific and Research Public- . . . Dixon, A. (2005). Standards for cassava ations, 3 (8), 2250–3153. products and guidelines for export. Nwosu, J. N., Ahaotu, I., Ayozie, C., Udeozor, Seibel, W. (2006). Future of flour: A compen- L. O. & Ahaotu, N. N. (2011). The prox- dium of flour improvement. In L. Popper imate and functional properties of african (Ed.), (Chap. Composite flours, pp. 193– yam bean (sphenostylis sternocarpa) seeds 198). Bergen: Verlag AgriMedia. as affected by processing. Nigerian Food Tanumihardjo, S. A., Bouis, H., Hotz, C., Journal, 29 (2). Meenakshi, J. V. & McClafferty, B. (2008). Ohimain, E. I. (2014). The prospects and chal- Biofortification of staple crops: An emer- lenges of composite flour for bread produc- ging strategy to combat hidden hunger. tion in Nigeria. Global Journal of Human- Comprehensive Reviews in Food Science Social Science, 14 (3), 1–11. and Food Safety, 7 (4), 329–334. Omeire, G. C., Kabuo, N. O., Nwosu, J. N., Thakkar, S., Huo, T., Maziya-Dixon, B. & Peter-Ikechukwu, A. & Nwosu, M. O. Failla, M. (2009). Impact of style of pro- (2015). Enrichment of wheat/cassava cessing on retention and bioaccessibility noodles with partially deffated protein- of beta-carotene in cassava (manihot escu- rich flour. Journal of Environmental lanta, crantz). Journal of Agricultural and Science, Toxicology and Food Technology Food Chemistry, 57, 1344–8. doi:10.1021/ (IOSR-JESTFT), 9 (5), 121–125. jf803053d Omodamiro, R. M., Oti, E., Egesi, C. N., Uk- UNICEF. (2004). Vitamin and mineral defi- pabi, U. J., Etudaiye, H. A. & Chijioke, ciency: A global progress report. U. (2011). Sensory evaluation of fufu pro- Vasagam, K. P. K. & Rajkumar, M. (2011). duced from high beta-carotenoid cassava. Beneficial influences of germination and In 35th annual conference and agm of ni- subsequent autoclaving of grain legumes geria institute of food science and techno- on proximate composition, antinutritional logy, markudi. factors and apparent digestibility in black Omodamiro, R. M., Oti, E., Etudaiye, H. A., tiger shrimp, penaeus monodon fabricius. Egesi, C., Olasanmi, B. & Ukpabi, U. J. Aquaculture Nutrition, 17 (2), E188–E195. (2012). Production of fufu from yellow cas- doi:10.1111/j.1365-2095.2009.00748.x sava roots using the odourless flour tech- Vimala, B., Thushara, R., Nambisan, B. & nique and the traditional method: Evalu- Sreekumar, J. (2011). Effect of processing ation of carotenoids retention in the fufu. on the retention of carotenoids in yellow- Adv. Appl. Sci. Res, 3 (5), 2566–2572. fleshed cassava (manihot esculenta crantz) Onwuka, G. I. (2005). Food analysis and instru- roots. International Journal of Food Sci- mentation: Theory and practice. Napthali ence and Technology, 46 (1), 166–169. prints. doi:10.1111/j.1365-2621.2010.02478.x Oyeku, O. M., Kupoluyi, C. F., Osibanjo, H. A., World Bank. (2009). Nutrition at a glance. Orji, C. U., Ajuebor, F. N., Ajiboshin, world development indicators database. I. O. & Asiru, W. B. (2008). An economic Retrieved from http : / / siteresources . assessment of commercial production of worldbank.org/NUTRITION/Resources/ 10% cassava-wheat composite flour bread. 281846-%201271963823772/nigeria.pdf Journal of Industrial Research & Techno- logy, 2, 20–30. Qayyum, M. M. N., Butt, M. S., Anjum, F. M. & Nawaz, H. (2012). Composition analysis of some selected legumes for protein isol-

IJFS February 2021 Volume 10 pages SI1–SI13 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI14–SI25

Eﬀect of Taro (Colocasia esculenta) Enrichment on Physicochemical and Textural Properties of Cake

Anuj Saklania, Ravinder Kaushikad*, Prince Chawlab, Naveen Kumarc, and Mukul Kumarb

a School of Bioengineering and Food Technology, Shoolini University, Solan, India, 125001 b Department of Food Technology, Lovely Professional University, Phagwada, Punjab, India c Amity University, Jaipur, Rajasthan, India d University of Petroleum and Energy Studies, Dehradun, Uttrakhand, India-248007 *Corresponding author ravinder foodtech2007@rediﬀmail.com Tel: +91-9416962729

Received: 3 July 2018; Published online: 24 February 2021

Abstract

Taro is a plant widely produced in tropical areas for its underground corms and it is used mainly as a vegetable. Its physicochemical, sensory properties and health benefits led to its use in value-added products. The cake is a high value-added bakery product and it needs a lower amount of gluten protein, therefore, taro flour (TF) was supplemented in wheat flour (WF) at different levels (10, 20, 30 and 40%). In connection with this, the incorporation of taro flour into wheat-based products has been reported to increase their nutritional and textural quality. The taro-supplemented cake showed higher mineral and fiber content, however, reduced caloric value. It was observed that taro supplementation reduced gluten and protein content and had higher oil and water binding capacity, therefore suitable for cake preparation. Taro cake had improved texture and sensory characteristics in comparison to wheat cake. It can be concluded that addition of taro for cake preparation improves nutrition and quality characteristics, therefore, it can be recommended to use taro for cake preparation. Keywords: Cake; Taro; Textural properties; Sensory properties; Flour enrichment; Composite flour

1 Introduction day. Taro is also a good source of thiamin, ri- boflavin, iron, phosphorus and zinc and a very When a crop is being considered for food, nu- good source of vitamin B6, vitamin C, niacin, tritional value and consumer acceptance are the potassium, copper, and manganese. Taro also key factors for selection or rejection. The nu- contains greater amounts of vitamin B-complex tritional value of food depends upon its nutri- than whole milk (Soudy, Delatour & Grancher, tional contents. As far as consumer acceptance is 2010). In addition, taro is especially useful to concerned, tubers of (Colocasia esculenta, com- persons allergic to cereals and can be consumed monly known as taro or cocoyam, are an import- by children who are sensitive to milk. For sup- ant food of several developing countries in Africa, plying nutrients, the taro tubers are considered West Indies, Pacific region and Asia (Nip, Whi- a good source of carbohydrates and potassium. taker & Vargo, 1994). Large servings of taro Taro is also a tuber that is very rich in carbo- corms can become a significant source of diet- hydrates, ranging between 73 to 80% which is ary protein, especially if taken more than once a mainly starch, 77.9%, and 1.4% crude fiber, dry

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a2 Cake prepared from taro (Colocasia esculenta) SI15 matter basis. Because of its high carbohydrate tribution to a broader supply of food products content, this tuber represents one of the main with beneficial health effects. In addition, cakes sources of energy in many parts of the tropics have a relatively constant place in our diet for and sub-tropics providing about a third of the a long time and their continuous popularity has food intake of more than 400 million people in encouraged the development of newer and more these areas (Huang, Chen & Wang, 2007). attractive products that are available on the mar- Nowadays, zinc deficiency is widespread and af- ket today. It is often a dessert of choice for meals fects the health and well-being of populations at ceremonial occasions, particularly wedding an- worldwide; since taro is one of the few non- niversaries and birthdays (Srivastava, Sanjeev & animal sources of zinc, its utilization should, Kumar, 2002). Taro has wide medicinal proper- therefore, be pursued to help in the alleviation ties, significant amounts of vitamin A, vitamin of zinc deficiency which is associated with stunt- C, and other phenolic cell reinforcements. The ing (Rana et al., 2018). However, despite the present study was undertaken to develop and wide application and great potential of taro as evaluate cake enriched with taro in different pro- a chief dietary source of carbohydrate and other portionate mixtures with wheat (10, 20, 30 and essential nutrients, its usage is often limited by 40 %). The physical characterization and sens- its anti-nutrient contents which are either po- ory qualities of taro flour and wheat flour cake tentially toxic or may limit the bioavailability of are reported in this work. nutrients (Hambidge, 2000). Processing of food like noodles and cookies from taro helps in re- 2 Materials and Methods ducing its anti-nutrient content which can help improve its production and utilization potential The taro was obtained from the local market of (SPC, 2006). Solan, Himachal Pradesh, India. A fine part of The market of novel plant-based foods Is gain- the taro was scraped, washed, shredded into 1 ing popularity, such as spices and herbs (Sharma, cm3 cubes and dried in a mini tray drying oven Kaushik, Sharma, Sharma et al., 2016; Sharma, (Maro Scientific Works Pvt. Ltd., New Delhi, In- Kaushik, Sharma, Chouhan & Kumar, 2016) dia) for 48 h at 50 oC. Dried cubes were milled us- and also serve as source of vitamins (Kaushik, ing a roller-mill (Chopin Laboratory CD-1 mill, Chawla, Kumar & Kumar, 2017; Kaushik, France) as per the method described by Kaushik Sachdeva, Arora & Gupta, 2015; Sachdeva, et al. (2017). To obtain uniform particle size, Kaushik, Arora & Palaniswamy, 2015) and min- taro flour was sieved through a 60 mm mesh erals (Kaushik, Sachdeva & Arora, 2015; Sach- sieve. The taro flour was then packed in an air- deva, Kaushik, Arora & Kapila, 2015). Cer- tight plastic container for future use. All chem- eals are the main economic crops from plants icals used were AR grade. and several bakery products are prepared from them like snacks (Sharma, Khatkar, Kaushik, Sharma, Sharma et al., 2017), noodles (Kaushik, 2.1 Chemical composition of TF Chawla, Kumar, Janghu & Lohan, 2018) rusk and WF and their (Lohan, Kaushik, Bansal & Gandhi, 2020), cook- proportionate mixture ies (Kumar, Khatkar & Kaushik, 2014) and so on. Bakery products are widely consumed and Standard methods of AOAC (2005) were used are becoming a major component of the global to determine Moisture (AOAC - 925.10), fat food market (Kaushik et al., 2018). Kumar et (AOAC - 2003.05) by soxhlet extraction and al. (2014) utilize taro as a potential substitute ash (AOAC - 923.03) by combustion. Protein for wheat flour in cake. Cake is one of the most (AOAC - 960.52) was determined by the micro common bakery products consumed by nearly all Kjeldahl method. Petroleum ether, sodium hy- levels of society. This is mainly due to its ready- droxide, sodium hydroxide, and ammonium bor- to-eat nature, availability in different varieties ate chemicals were used in the preparation of TF. and affordable cost. The development of bakery All chemicals were from Loba Chemical Com- foods enriched with fiber is an important con- pany.

IJFS February 2021 Volume 10 pages SI14–SI25 SI16 Saklani et al.

Table 1: Proportions of ingredients in cake formulations

Formulations Ingredients Sample 1 Sample 2 Sample 3 Sample 4 Egg (g) 80 80 80 80 Butter (g) 40 40 40 40 Water (ml) 18 18 18 18 Sugar (g) 24 24 24 24 Taro Flour (g) 10 20 30 40 Wheat Flour(g) 90 80 70 60

2.2 Physical characterization of probe was used to compress the sample to 50% TF and WF of its original height (40 mm) at a speed of 10 mm/s. The equipment was interfaced with a The characterization of TF and WF bulk dens- computer, which controls the instruments and ity was determined by the method described by analyzes the data, using the software supplied by Kaur and Singh (2005). Water solubility in- Texture Technologies Corp. Textural paramet- dex was determined by the method described by ers (hardness, cohesion, springiness, adhesion, Anderson, Conway, Pfeifer and Griffin (1969). and chewiness) were calculated from the TPA Water-holding capacity and fat-binding capacity curves. The hardness is the peak power of the were determined by the method described by primary pressure cycle, stickiness is the separa- Kaushik, Kumar, Sihag and Ray (2015), sodium tion of the recognized tallness of the item on the dodecyl sulfate sedimentation was determined second pressure determined by the first pressure by the method described by Axford, Mcdermott separation, cohesiveness and adhesiveness were and Redman (1978), wet and dry gluten con- resolved. tent were determined by the method described by Kaushik, Kumar et al. (2015). Color parameters of flour were measured with a Lovibond, Tintometer Colorimeter MODEL F (The Tinto- 2.4 Preparation of Cake meter Ltd., United Kingdom) on the basis of L*, a* and b* values (Bouaziz et al., 2016). The TF and WF were mixed in four ratios 10:90, 20:80, The cake was prepared according to Ceserani, 30:70 and 40:60 and named them as sample 1, Kinkton and Foskett (1995), flour blend 100 g, sample 2, sample 3 and sample 4, respectively. fine powder sugar 24 g, egg 80 g, butter 40 g, and water 18 ml Table1. The sugar was added to the butter and beaten for 3 min. Eggs were 2.3 Textural analysis of dough beaten and added gradually to the mixture and and extracted gluten whipped for 2 min. The TF was incorporated in WF at different concentrations (10, 20, 30 and Dough samples were calculated by the texture 40%) over a period of 7 min. with good cream- profile analysis (TPA) method using a TMS Tex- ing between the additions. Cake samples were ture Analyzer (Food Technology Corporation, put in the baking oven (Electric 2 Deck 4 Tray Sterling, Virginia, USA) equipped with a 1000 Oven, TME-2D-4) at a temperature of 170 oC for (N) load cell, and a 0.05 (N) detection range. 20-25 min. The cakes were cooled and removed A sample of dough was removed into a molded from the pan after 1h. The size of the pan was Nalgene polypropylene tube (5 cm height) that 5.5×3.5×2 inches. The cakes were packaged in was placed in a fixture to hold it in place un- aluminum foil and kept on the shelf until sample der the texture analyzer. An acrylic cylindrical analysis in the food laboratory.

IJFS February 2021 Volume 10 pages SI14–SI25 Cake prepared from taro (Colocasia esculenta) SI17

2.5 Physical characteristics of et al. (2017), Sachdeva, Kaushik, Arora and Pa- cakes laniswamy (2015) and Sachdeva, Kaushik, Arora and Kapila (2015). Data were subjected to a The volume of cakes was measured using the one-way analysis of variance (ANOVA). raised displacement method (Rosell, Rojas & de Barber, 2001). Weight, length, width, and height 3 Results and Discussion of the cakes were estimated. The textural properties of cakes were estimated by a Texture Ana- lyzer. Cake slices (2.5 cm thick) were placed on 3.1 Physical and chemical the Texture analyzer platform. An acrylic cyl- characterization of TF and indrical probe was used to compress the cake WF sample up to 50% of its original height at a speed of 10 mm/s. Colors of crust and crumb Physical properties of granular solids play an im- were measured using the Portable Colorimeter portant role in manufacturing as they determ- (Lovibond, Colorimeter MODEL F). ine the application and use of food materials for several food products. Physical properties are 2.6 FTIR interpretation important in choosing the flow behavior of their proper handling during the several phases of pro- The microstructural changes were verified with cessing, conveying and storage (Ganesan, Rosen- the FTIR spectrometer (CARY 630 Agilent trater & Muthukurnarappan, 2008). The phys- Technologies, Santa Clara, California, USA). Mi- ical properties of the TF and WF are summar- crolab Software (Bozeman, Montana, USA) was ized in Table2. Bulk density is an indication of used to generate data with a resolution of 8 cm-1. the porosity of a product and a function of flour The graphs obtained using the FTIR were ana- wettability without the influence of any compres- lyzed using a method described by Lampman, sion. It is also a reflection of what the sample Kriz and Vyvyan (2001). can carry if rested directly on another. The bulk density of TF and WF was 0.658 and 0.652 (g/cm3), respectively. As the concentration of 2.7 Sensory evaluation TF increased in mixed flour the bulk density also increased. Njintang et al. (2007) determined a The sensory analysis was performed at the Food similar bulk density of taro flour (0.645 g/cm3), and Nutrition Laboratory by 35 students and however, somewhat higher bulk density (0.745 teachers of Shoolini University, from 19 to 37 g/cm3) was reported by Tagodoe and Nip (1994). years old. Judges evaluated the attributes: color, Water solubility index was 37.88 and 31.24% for odor, flavor, taste, texture and overall acceptab- TF and WF, respectively and the results revealed ility using a nine-point hedonic scale (where 1 = that with the increase in taro concentration, wa- liked extremely and 9 = disliked extremely). The ter solubility index also increased. Flours with sensory analysis was conducted at room temper- high water solubility index have been reported ature, and each judge received the samples in to be good constituents in bakery applications as white plastic dishes, numbered with random di- they improve solubility characteristics and lead gits (Lohan et al., 2020). Cake samples were left to the improved freshness of baked products (Ma to cool for 4h after baking then samples were cut et al., 2011). Water holding capacity of TF and and subjected to taste panel. WF was 7.91 and 6.75% and fat binding capacity of TF and WF was 7.09 and 5.87%, respect- 2.8 Statistical analysis ively and the different concentrations of TF and WF water holding capacity and fat binding ca- Means (n=3), standard error mean (SEM), linear pacity showed increasing order. The high syner- regression analysis and 95% confidence intervals gic value observed for wheat flour compared to were calculated using Microsoft Excel 2007 (Mi- taro flour may be attributed to the differences in crosoft Corp., Redmond, WA) as per Kaushik amylose content, given the fact that starch with

IJFS February 2021 Volume 10 pages SI14–SI25 SI18 Saklani et al.

Table 2: Physical and chemical characteristics of TF and WF

Taro flour Wheat flour TF/WF(%) TF/WF(%) TF/WF(%) TF/WF(%) Flour parameters (TF) (WF) 10:90 20:80 30:70 40:60 Bulk density(g/cm3) 0.658±0.015c 0.652±0.03a 0.652±0.028a 0.653±0.016ab 0.655±0.014b 0.656±0.026b Water solubility index (%) 37.88±0.59d 31.24±0.65a 31.54±0.49a 32.56±0.57b 33.86±0.46c 34.17±0.41c Water holding capacity (g/g) 7.91±0.20d 6.75±0.45a 6.84±0.36ab 7.08±0.29b 7.19±0.65bc 7.30±0.49c Fat-binding capacity (g/g) 7.09±0.21d 5.87±0.39a 6.12±0.28b 6.22±0.19bc 6.31±0.34c 6.40±0.25c Moisture content (%) 8.59±0.20a 11.42±0.27c 11.09±0.29c 10.86±0.24bc 10.62±0.34b 10.42±0.31b Crude Protein (%) 4.55±0.15a 9.56±0.89d 9.06±0.64cd 8.59±0.58c 8.11±0.44bc 7.62±0.59b Crude Fat (%) 0.65±0.006a 1.56±0.56b 1.48±0.26b 1.39±0.22b 1.25±0.27b 1.13±0.19b Ash (%) 4.38±0.35d 1.24±0.28a 1.49±0.22ab 1.88±0.18b 2.22±0.14b 2.59±0.24c Crude Fiber (%) 4.37±0.20d 3.94±0.34a 4.09±0.35ab 4.13±0.37b 4.19±0.31bc 4.23±0.28c Total Carbohydrates (%) 70.45±2.36a 74.67±2.85d 73.36±2.75c 72.79±2.48c 71.35±2.46b 70.74±2.44ab SDS Sedimentation (%) 13.45±0.85a 53.02±0.72f 51.35±0.89e 47.06±0.71d 43.28±0.69c 38.55±0.58b Wet gluten Content (%) 0.00a 30.45±0.28f 28.25±0.48e 26.64±0.59d 24.88±0.66c 21.74±0.51b Dry gluten Content (%) 0.00a 9.83±0.34d 8.53±0.38c 7.76±0.33c 7.15±0.28bc 6.64±0.24b Calories (Kilo Calories) 302.6±2.26a 342.48±2.27d 333.16±2.25cd 328.12±2.13c 320.44±2.18bc 315.08±2.14b Data are presented as means±SEM (n=3). a−bMeans within row with different lowercase superscript are significantly different (p<0.05) from each other. Taro flour (TF), Wheat flour (WF)

Table 3: Color characteristics of Taro, wheat and diﬀerent concentration mixes of ﬂour

Flour L* a* b* TF 74.59±0.78a 5.81±0.45c 17.22±0.66e WF 98.19±2.13e 0.49±0.09b 11.13±0.46a TF/WF (%) 10:90 96.43±2.19d 0.44±0.05ab 15.19±0.54d TF/WF (%) 20:80 94.71±2.11c 0.42±0.06ab 13.96±0.48c TF/WF (%) 30:70 93.22±2.16b 0.40±0.03a 12.59±0.41b TF/WF (%) 40:60 91.79±1.56b 0.38±0.02a 11.88±0.36ab Data are presented as means±SEM (n=3). a−bMeans within row with different lowercase superscript are significantly different (p<0.05) from each other. Taro flour (TF), Wheat flour (WF)

high amylopectin content has been reported to greater number of hydroxyl groups which exist retrograde slowly (Bamidele, Cardoso & Olaofe, in the fiber structure and allow more water in- 1990). The water absorption capacity indicates teractions through hydrogen bonding (Rosell et the water holding capacity of dough during the al., 2001). Fat binding capacity is an import- processing of different bakery products. The ant feature of polysaccharides. It is in part re- higher water absorption capacity of gluten indic- lated to the chemical composition, but it is more ates a high content of starch. Gluten samples closely linked to the porosity of the fiber struc- varied in all four wheat cultivars which may be ture than to the affinity of the fiber molecule due to the difference in drying conditions. The to oil (Biswas, Kumar, Bhosle, Sahoo & Chatli, water absorption capacity is considered a critical 2011). In different concentration of taro and WF function of the protein in viscous food like soups, the moisture content, crude protein, crude fat, gravies, dough, and baked products mainly bread and carbohydrates were present in decreasing or- and cakes (Singh & Singh, 2006). The differences der and ash and crude fiber was present in in- in water absorption are mainly attributed to the creasing order. Similar results were reported by

IJFS February 2021 Volume 10 pages SI14–SI25 Cake prepared from taro (Colocasia esculenta) SI19

Eke, Sanni and Owuno (2009) in banana cake, index were increased. and See, Noor Aziah and Wan Nadiah (2007) in bread cakes. SDS sedimentation varied between 3.45 to 33.02% respectively, and as the concentra- 3.2 Dough properties tion of TF increased, SDS value decreased. The SDS sedimentation volume is a good indicator Textural properties of dough, gluten of wheat flour quality. Present SDS values are extracted and cake in agreement with the values determined by Su- pekar, Patil and Munjal (2005). The SDS sedi- Textural properties (hardness, cohesiveness, mentation value of wheat flours is based on the stickiness, and adhesion) were analyzed to assess fact that gluten protein absorbs water and swells the effect of the addition of TF to WF on dough considerably when treated with lactic acid. The quality shown in Table4. As reported by Carson wet gluten content and the dry gluten content and Sun (2001), texture analysis is an object- were present in WF 30.45 and 9.83%, respective physical examination of baked products and ively and absent in TF. Gluten is an important gives direct information on the product quality, constituent of wheat because it provides strength while dough rheology tests provide information to dough and texture to baked wheat products. on the baking suitability of the flour as raw ma- Higher gluten content in WF is recommended for terial. Similar results showing increased hardness bread and lower gluten content is found better were found in bread prepared with 10%, 20%, for biscuits and cookies (Autran, Hamer, Plijter and 30% buckwheat flour (Torbica, Hadnadev & & Pogna, 1997). In a response surface study Dapcevic, 2010). Gluten has a significant effect on gluten extraction from low-grade flour and on the consistency of dough, and also affects the durum flour, it was found that the protein con- structure of molecules in the flour. According to centration in protein fractions increased as the Chang and Liu (1991), the level and structure of water content in the dough increased from 400 protein and starch have an effect on the hydra- to 710 g/Kg (Dik, Yondem-Makascioglu, Aytac tion pattern of flour and modify the consistency & Kincal, 2002). This provides the dry matter of of dough. In gluten-free muffins prepared with gluten protein present in flour and water absorp- teff, flour similar results were reported by Tess, tion capacity of gluten present in flour. However, Bhaduri, Ghatak and Navder (2015). results determined by Singh and Singh (2006) ranged between 5.9 and 10.1 % of dry gluten Physical characteristics of the cake yield. Supekar et al. (2005) determined the dry gluten content in the range of 9.4 to 12.7 %. Sim- The effect of TF incorporation on the physical ilar results were reported by Pharande, Dhotre properties of the cake is summarized in Table5. and Adsule (1988). Increase in the width and height of the taro cake Color is a significant parameter which in- and dough were affected by the addition of TF cludes the physical appearance of the product in WF. The weight and the volume of the cake (Bhandari, Patel & Dong Chen, 2008). Table3 were also affected by the addition of TF during shows the color values (L*, a* and b*) of TF formulation. In fact, the weight of the cake after was ranged between L* 74.59, a* 5.81, b* 17.22 cooking was increased with the addition of TF. and WF L* 98.19, a* 0.49, b* 11.13 and in dif- The highest weight was exhibited with TF ad- ferent concentrations of TF and WF color values dition at a level of 40%. The volume of cake ranged between L* 96.43, a* 0.44, b* 15.19 in increased with the addition of TF. TF/WF 10:90%, L* 94.71, a* 0.42, b* 13.96 in TF/WF 20:80%, L* 93.22, a* 0.44, b* 12.41 in TF/WF 30:70% and L* 91.79, a* 0.38, b* 11.88 3.3 FTIR interpretation in TF/WF 40:60%. Kumar et al. (2014) reported similar results for taro cake. They also repor- FTIR is a cheap, fast, reliable, correct and non- ted that with substitution of taro carbohydrate, destructive method, to find the functional groups fiber, water absorption index and water solubility present in a sample (Schwanninger, Rodrigues,

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Table 4: The textural properties of diﬀerent types of dough, gluten extract and cake

Cohesiveness (mm) Adhesion (N) Stickiness (N) Hardness (N) Textural properties of Dough TF (100%) 0.44±0.028a 0.26±0.027a 1.13±0.031a 0.89±0.027a WF (100%) 0.46±0.033a 0.20±0.026a 0.98±0.034a 0.63±0.029a TF/WF (%) 10:90 0.46±0.023a 0.22±0.035a 1.00±0.038a 0.67±0.036a TF/WF (%) 20:80 0.45±0.031a 0.24±0.031b 1.04±0.056a 0.71±0.044a TF/WF (%) 30:70 0.45±0.025a 0.25±0.029a 1.07±0.064a 0.76±0.042b TF/WF (%) 40:60 0.44±0.029a 0.26±0.025b 1.09±0.054a 0.81±0.033b Textural properties of gluten TF (100%) -NA- -NA- -NA- -NA- WF (100%) 0.18±0.019a 0.12±0.027a 0.41±0.31a 0.39±0.31a TF/WF (%) 10:90 0.17±0.022a 0.11±0.025 a 0.39±0.28b 0.37±0.29a TF/WF (%) 20:80 0.16±0.021a 0.10±0.021b 0.37±0.24a 0.36±0.27a TF/WF (%) 30:70 0.15±0.018a 0.09±0.023a 0.36±0.27a 0.35±0.28a TF/WF (%) 40:60 0.14±0.020a 0.08±0.022a 0.34±0.25b 0.33±0.26b Textural properties of cake TF (100%) 2.15±0.19b 1.19±0.21b 1.65±0.36b 1.13±0.36b WF (100%) 2.11±0.16a 1.11±0.19a 1.44±0.34a 0.98±0.27a TF/WF (%) 10:90 Cake 2.14±0.15a 1.12±0.24a 1.57±0.41a 1.03±0.28a TF/WF (%) 20:80 Cake 2.13±0.24a 1.14±0.17a 1.59±0.35b 1.05±0.45ab TF/WF (%) 30:70 Cake 2.12±0.25ab 1.17±0.11b 1.60±0.28a 1.09±0.33a TF/WF (%) 40:60 Cake 2.11±0.31b 1.18±0.25b 1.63±0.19ab 1.12±0.27b Data are presented as means±SEM (n=3). a−bMeans within columns with different lowercase superscript are significantly different (p<0.05) from each other. Taro flour (TF), Wheat flour (WF)

Table 5: Physical characteristics of cakes

Length Width Height Weight before Weight after Volume (cm) (cm) (cm) cooking (g) cooking (g) (cm3) TF (100%) 13.6±0.37a 8.5±0.44a 7.0±0.22a 150±0.98a 142±0.89a 809±1.13b WF (100%) 13.4±0.33a 8.5±0.41a 7.1±0.21a 150±0.92a 144±0.92b 808±1.11a TF/WF 10%/90% 13.4±0.36a 8.5±0.45a 7.0±0.20a 150±0.95a 143±0.94a 797±1.13a TF/WF 20%/80% 13.5±0.38a 8.5±0.51a 7.0±0.25a 150±0.87b 142±0.90b 803±1.21a TF/WF 30%/70% 13.4±0.39a 8.5±0.35a 7.0±0.27a 150±0.75b 141±0.80a 809±1.19b TF/WF 40%/60% 13.5±0.37a 8.5±0.47a 7.1±0.29b 150±0.90a 144±0.95a 814±1.15b Data are presented as means±SEM (n=3). a−bMeans within row with different lowercase superscript are significantly different (p<0.05) from each other. Taro flour (TF), Wheat flour (WF)

IJFS February 2021 Volume 10 pages SI14–SI25 Cake prepared from taro (Colocasia esculenta) SI21

Figure 1: Spectra representation of FTIR analysis of TF, WF and diﬀerent concentrations of TF and WF

Table 6: Liking scores of cakes prepared with TF

Characteristics Maximum TF WF TF/WF TF/WF TF/WF TF/WF score (100%) (100%) 10%/90% 20%/80% 30%/70% 40%/60% Color 10 7.5±0.24a 7.5±0.22a 8.5±0.35c 8.0±0.23b 8.0±0.33b 8.0±0.25b Texture 10 8.0±0.28a 8.0±0.24a 8.0±0.24a 8.0±0.30a 8.0±0.25a 8.0±0.18a Taste 10 7.5±0.23a 8.0±0.26b 8.5±0.28c 8.5±0.28c 8.0±0.29b 8.0±0.27b Odor 10 8.0±0.19b 7.5±0.27a 8.5±0.27c 8.5±0.26c 8.0±0.24b 8.5±0.25c Overall 10 8.0±0.22a 8.0±0.23a 8.5±0.21b 8.5±0.25b 8.0±0.31a 8.0±0.22a Acceptability Data are presented as means±SEM (n=3). a−bMeans within row with different lowercase superscript are significantly different (p<0.05) from each other. Taro flour (TF), Wheat flour (WF)

IJFS February 2021 Volume 10 pages SI14–SI25 SI22 Saklani et al.

Pereira & Hinterstoisser, 2004). To have inform- 4 Conclusions ation about the chemical structure of taro flour and wheat flour, the FTIR spectrum is given in The health benefits of taro are numerous and Figure1. The FTIR spectrum was recorded in reported by several papers. Therefore, taro- the transmission mode between 4000 cm-1 and enriched cake was developed. Most of the physic- 500 cm-1 for taro and wheat. The spectrum of chemical properties of taro were different from biopolyols had characteristic bands of cellulose WF. Enrichment with taro significantly affects at 3420, 1163, 1060, and 1033 cm-1. Addition- textural properties as taro lacks gluten. The ally, the peaks observed at 1714, 1660, 1117, and composite taro flour at the 40% replacement level 1013 cm-1 were related to the functional groups showed physical properties similar to wheat cake of lignin. The broad band at about 3600-3000 samples, especially for products from taro-wheat cm1 was related to the characteristic stretching composite flour. The taro-wheat composite flour vibration of -OH. The various functional groups had the lowest setback and processing stability, present in both TF and WF were (-C=C-O-C) which indicated low staling or aging of dough (Ether), (-C-OH) (Phenol) and (-C-OH) (1o and for cake replaced with taro flour. The composite 2o Alcohol) and (-C-H) (Alkyl). The presence of cake would serve as a functional food because of these functional groups represents the molecular the high trace element content. It can be sugges- composition of both TF and WF which further ted that other non-cereal products like elephant supports the conclusion of no interaction in char- foot yam and water caltrop will also be tried for acteristic functional groups of flour with the in- cake preparation. For the future, taro will be gredients of TF. tried for production of noodles, cookies, bread, buns, pizza base and pan bread.

Acknowledgements

This work was conducted in the School of Bioen- 3.4 Sensory Evaluation gineering and Food Technology, Shoolini Univer- sity, Solan, Himachal Pradesh. All chemicals, glass wares and Instruments were provided by Table6 shows effects of adding different con- the School of Bioengineering and Food Techno- centrations of taro on sensory evaluation of pre- logy, Shoolini University, Solan, Himachal Pra- pared cake. The current study applied the sens- desh. ory profiling method involving a panel and had a differential rank ordered perception for the four manufactured cake samples. In taste and odor References there were significant differences in control and Anderson, R. A., Conway, H. F., Pfeifer, V. F. & other concentrations. Karao˘gluand Kotancilar Griffin, E. L. (1969). Gelatinization of corn (2009) reported no significant difference among grits by roll- and extrusion-cooking. Cereal samples with and without cake preparation us- Science Today, 14 (1), 4–7. ing tapioca starch for liking scores of appear- AOAC. (2005). Official methods of analysis. The ance, color and odor. Samples 10 and 20% TF- Association of Official Analytical Chem- added showed the highest taste scores. In or- ists (18th ed.). 481. North Fredrick Avenue der, WF cake showed the lowest scores whereas, Gaithersburg, Maryland, USA. 10 and 20% TF-added cake showed highest taste Autran, J. C., Hamer, R. J., Plijter, J. J. & scores. The overall sensory acceptability of TF Pogna, N. E. (1997). Exploring and im- cake was lowest, whereas, 10% and 20% TF ad- proving the industrial use of wheats. Cereal ded cake showed the highest scores. According to Foods World, 42 (4), 216–227. these results, 10 and 20% TF-added cakes showed higher sensory scores, however, 10% TF-added cake was utilised for further studies.

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IJFS February 2021 Volume 10 pages SI14–SI25 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI26–SI40

Eﬀect of Emulsiﬁer Diacetyl Tartaric Acid Ester of Mono- and Diglycerides (DATEM) and Enzyme Transglutaminase on Quality Characteristics of Rice Bran Croissants

Wan Zunairah Wan-Ibadullaha*, Aw Ying Honga, M. A. R. Nor-Khaizuraa, Nor Afizah Mustaphab, Z. A. Nur Hananib, Mohammad Rashedi Ismail-Fitryb, and Anis Shobirin Meor Husinb

a Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia *Corresponding author [email protected] Tel: +603 8946 8352

Received: 3 August 2018; Published online: 24 February 2021

Abstract

Rice bran (RB) is a good source of dietary fibre. Addition of rice bran into croissant interferes with the gluten formation of dough and hence affect the physicochemical properties of croissant. The effect of RB addition on physicochemical properties of croissant were determined by using 0%, 10% and 15% RB. Besides, additives such as emulsifiers and enzymes can be used in pastry to enhance the physicochemical properties of croissant. Diacetyl tartaric acid ester of mono-diglycerides (DATEM) and transglutaminase (TGase) were used respectively on 0%, 10% and 15% RB to investigate the effect of such additives on physicochemical properties of croissant. Increased % RB and DATEM, produced a significant decrease in specific volume, together with a significant increase in colour, hardness and chewiness. With increased % RB, TGase caused significant increase in colour, hardness and chewiness but significant decrease in specific volume. The overall moisture sorption isotherm curves of the croissant belong to the Type III isotherm, also known as Flory-Huggins Isotherm (J-shaped). The critical aw obtained from the Guggenheim-Anderson-de Boer (GAB) equation showed that the shelf life of croissants were not positively impacted by the addition of DATEM and TGase and the addition of RB did not cause any significant positive effects on quality characteristics of croissants. Keywords: Rice bran(RB); DATEM; TGase; Physicochemical properties; Moisture sorption isotherm

1 Introduction gendra Prasad, Sanjay, & Khatokar M., 2011). Rice bran is an excellent source of total dietary ﬁbre ranging from 20 - 51 % (Saunders, 1990). Rice bran is a by-product obtained from the rice In spite of its excellent nutrition value, a large milling industry. Due to its high nutrient con- amount of rice bran is discarded during the pro- tent rice bran has potential to be used as food cess of rice milling and is used as animal feed ingredient. However, the use of rice bran in food and sells at considerably low prices (McKee & applications is limited mainly due to deteriora- Latner, 2000). tion that is caused by enzymatic activity (Na-

Nomenclature

DATEM diacetyl tartaric acid ester of mono - RB rice bran and diglycerides fwb ﬂour weight basis TGase Transglutaminase

Recently, the focus of interest has been on the de- DATEM functions as a dough strengthener that velopment of food products from by-products or mainly interacts with gluten proteins, as a crumb wastes and other underutilised agricultural (Is- softener or an anti-firming agent that can com- sara & Rawdkuen, 2016; Sharma, Srivastava, & plex gelatinized starch (Gaupp & Adams, 2014). Saxena, 2015). Addition of rice bran to a wide Furthermore, enzymes have been considered as range of products will contribute to the develop- good alternatives for use in baking processes. ment of new functional foods and fulfil the cus- Transglutaminase clearly has great potential as tomers’ expectations. The croissant is a layered an additive to both white and wholemeal flour bakery product, so named for its crescent shape. croissant doughs. TGase addition generally re- It is made of yeast-leavened dough, layered with sulted in stronger dough properties, but supe- butter, rolled and folded several times in succes- rior handling properties were observed especially sion, then cut into a triangle and rolled up like at lower addition levels (Basman, Koksel, & Ng, a scroll so that the centre bulges and the ends 2002). taper out. Dietary fibre is considered a critical The present work aimed to evaluate the effect ingredient in food products particularly in pas- of addition of DATEM and TGase on physic- try products. However, fibre has been known to ochemical properties and shelf life of croissant alter the texture, volume and shelf life of yeast- with added RB. RB is considered as a good leavened products. A study done by Ghufran source of dietary fiber. However, the addition Saeed, Arif, Ahmed, Ali, and Shih (2009) also in- of RB may give various detrimental effects on vestigated the impact of rice bran on the rheolog- dough handling and croissant quality characteris- ical parameters of dough. Based on the findings tics. Therefore, food additives such as emulsifiers of the study, it was clear that adding hydropho- and enzymes are used to overcome this problem. bic rice bran to wheat had a negative impact DATEM and transglutaminase were added to in- on the mixture, leading to inferior performance crease the dough strength and volume. Further- in dough development and lower water absorp- more, the optimal combination of RB, DATEM tion capacity. The physical properties of fibre and TGase on quality of croissant in terms of including water holding, and swelling capacity quality characteristics also has been studied. that may interfere the interaction between starch and gluten network, resulting in poor dough development (Collar, Santos, & Rosell, 2007). In 2 Materials and Methods high fibre bread, the addition of hydrocolloids provide various functional properties by control- 2.1 Ingredients of croissant ling the water molecule’s mobility, thus affecting the dough rheology, dough development and The ingredients used in preparation of croissants gas retention (Peressini, Pin, & Sensidoni, 2011). were wheat flour, salt, castor sugar, milk pow- Furthermore, additives with functional property der, yeast, butter, pastry margarine and eggs, (emulsifiers, antioxidants, acid or enzyme) can be which were purchased at local market. DATEM used for quality improvement in pastry products. was obtained from Zhentong Food Technology

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Co., Ltd (Henan, China). TGase was bought method (AACC, 2000); green beans were used from Ajinomoto North America Inc. (Itasca, Illi- instead of rapeseeds. The green beans displaced nois, U.S.A).The stabilized RB was purchased by the croissant were collected and their volume from Bob’s Red Mill Natural Foods (Milwaukie, measured (cm3). The weight of croissant (g) was Oregon, U.S.A). The croissant formulations were obtained by using an electronic weighing balance shown in Table1. SPO 61 (Scaltec, Germany). Specific volume was also calculated (volume divided by weight). 2.2 Baking of croissant Texture profile analysis On a wheat flour basis, the following ingredients were used to produce croissants: 45.5% of wheat Texture profile analysis was conducted by using flour, 20.4 % of pastry margarine, 20.4 % of ice a Texture Analyser TA.XT2i (Stable Micro Sys- water, 2.4 % of eggs, 1.8 % of milk powder, 0.5 tems Ltd., Surrey, UK) provided with a SMS % of salt, 4.5 % of castor sugar, 0.9 % of yeast, P/75 probe, that is a 75 mm diameter cylindrical 3.6 % of butter, 0.4 % of DATEM and 0. 4% of probe and the strain applied was 50%. The pre- TGase. Wheat flour which contained 11.5 % of test speed was 1.00 mm/s, test speed was 5.00 moisture, 0.53 % of ash, 12 % of protein, and 31.2 mm/s, post-test speed was 5.00 mm/s and the % of gluten, was mixed thoroughly with castor trigger force was 5.0 g. The hardness, springiness sugar, milk powder and yeast using cake mixer and chewiness were the main factors evaluated in (Taiwan Cake Mixer B5, Taiwan). Whole eggs, all croissant formulations (Boukid et al., 2018). ice water, butter and salt were then added and mixed. The dough was sheeted on a table to a Water activity rectangular shape and covered with wet cloth, The water activity of dough and crackers and proved in chiller for 30 mins. The dough were measured using Water Activity Analyser was then sheeted to a wider and longer shape, (AquaLab 3TE, Pullman, USA). Each sample by using a roller, and the pastry margarine was was transferred into a chamber (filled to quar- placed in the middle of the dough, given 3-folds ter capacity). The chamber was then inserted and then further proofed in chiller (7 oC) for 20 into the analyser and the measurements started. mins. The rolled pieces were put on a baking The temperature of the water activity analyser tray and left to proof for 60 mins, then baked at ranged from 24 to 25 oC and it took the device 190 oC for 15 minutes. 15 mins to get a stable value.

2.3 Physicochemical analysis Moisture content Colour measurement The moisture content of croissant was measured using Moisture Analyser MA 35 (Sartorius, Ger- Colour analysis was conducted to assess the many). Each sample was transferred onto an alu- colour of croissant the crust using a Chroma minium sample pan. The pan was then inserted meter model CR-410 (Konika Minolta Inc, Os- into the analyser and the measurements started. aka, Japan), calibrated with a white reference It took 15 mins for the device to get a constant plate which is the standard for reflectiveness. weight. The temperature used in the analyser Colour parameters were expressed as L* (light- was 95 oC. ness/darkness), a* (redness/greenness) and b* (yellowness/blueness). 2.4 Analyses of crude protein and Specific volume analysis crude fat Specific volume analysis was conducted to de- The analyses of moisture, crude protein (N termine the volume of croissant the using rape- • 6.25) were conducted using the standard seed displacement process according to the 10-05 micro-Kjeldahl method (AOAC Method 960.52).

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Figure 1: Specific volume - Effect of addition of RB on specific volume of croissant with and without addition of DATEM and TGase. a indicates significant difference in specific volume compared among the same category (with DATEM/ with TGase/ without additive). b indicates significant difference in specific volume compared among the same % RB added.

Table 1: Croissant Formulation (% by ﬂour weight basis)

Formulation (%) F1 F2 F3 F4 F5 F6 F7 F8 F9 Wheat ﬂour 45.5 45.5 45.5 45.5 35.5 35.5 30.5 30.5 30.5 Rice bran 0 0 0 10 10 10 15 15 15 DATEM 0 0.4 0 0 0.4 0 0 0.4 0 TGase 0 0 0.4 0 0 0.4 0 0 0.4 Castor sugar 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 Milk powder 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 Yeast 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 Whole egg 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 Ice water 20.4 20.4 20.4 20.4 20.4 20.4 20.4 20.4 20.4 Salt 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Butter 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 Pastry margarine 20.4 20.4 20.4 20.4 20.4 20.4 20.4 20.4 20.4

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Crude fat were conducted using a Soxhlet ex- 3 Results and Discussion traction process with petroleum ether (AOAC Method 945.16) (AOAC, 2000). 3.1 Colour measurement

The surface colour is an essential attribute of yeasted pastry, and contributes to consumer preference. A brown crust is caused by a non- 2.5 Dynamic Dewpoint Isotherm enzymatic browning reaction (Maillard type) be- Method tween amino acids and reducing sugars (Kent & Evers, 1994). Based on Table3, there were no significant difference (P>0.05) observed in light- The Dynamic Dewpoint isotherms (DDIs) were ness, redness and yellowness on crust of croissant obtained using the AquaSorp Isotherm Genera- with the addition of 10% and 15% RB respec- tor (Decagon Devices Inc., Pullman, WA, USA). tively. Addition of RB was expected to decrease Before the DDIs were obtained, the a dewpoint w the crust lightness but in this study, it showed no sensor was verified using two unsaturated aque- significant changes. When DATEM and TGase ous salt solutions purchased from the instrument were used alone respectively, they decreased the manufacturer, 13.41 molal LiCl (0.250 ± 0.003 lightness significantly. At 10% and 15 % RB ad- a ) and 6.0 molal NaCl (0.760 ± 0.003 a ). w w dition, DATEM and TGase both showed signifi- Working DDIs were obtained for each material (1 cant reduction (P<0.05) in lightness as compared to 2 g in a stainless steel sample cup half filled) to those with RB addition but without additives. using the start from current a feature (Aqua- w The lower L* crust values can be attributed to Sorp firmware version ASIG 1.19, Decagon De- Maillard browning (Breeding & Beyer, 2000). vices, Pullman, WA, USA) with a 120 ml/min This could be due to increased amino acids avail- flow rate at 25 oC. The adsorption and des- able for Maillard reaction when the DATEM and orption sections of the working isotherm were TGase were used together with increased % RB obtained in duplicate, each on a different sam- as RB, DATEM and TGase are proteins. The ple. The average DDI starting a and associated w a*value increased with the increased RB. This standard deviation for each material for both could be due to the fine bran presence that pro- adsorption and desorption parts of the working duce darker yellowish color of the crumb (Popov- isotherm are given in Table2. Weight change at Raljic, Mastilovic, Lalicic-Petronijevic, & Popov, each a was converted to moisture content (% w 2009). db) using the SorpTrac software (version 1.03.3, Decagon Devices, Pullman, WA, USA) using the initial measured moisture content value (Table 3.2 Specific volume 2). Based on Figure1, there were significant reduction (P<0.05) in specific volume with increase % of RB. The reduction may be attributed to 2.6 Statistical Analysis the higher level of gluten present in wheat flour compared to composite blends which could not be properly stretched by carbon dioxide (CO2) Statistical analyses were conducted using gas during fermentation and proofing (Oladun- Minitab statistical software (Minitab 16, moye, Akinoso, & Olapade, 2010). The decrease Minitab Inc, Pennsylvania). One-way analysis of in croissant specific volume might be due to the variance (ANOVA) was performed to compare high water absorption by the rice bran and the the data. The Tukey test was used to compare reduced air entrapment, resulting in heavier but the average mean values obtained. The confi- smaller dough (higher weight but lower volume). dence limits used in this study were based on a When DATEM was used solely, the specific vol- level of 95% (P<0.05). ume was significantly higher (P<0.05) compared

IJFS February 2021 Volume 10 pages SI26–SI40 Quality characteristics of rice bran croissant SI31 to that where DATEM worked with 10% and 15% 3.3 Textural properties RB respectively. Rogers and Hoseney (1983) reported that addition of DATEM improved the Based on Table4, there were no significant loaf volume of wheat bread. The positive effect changes in hardness of croissant with increased on bread volume of emulsifiers is caused by their % of RB (P>0.05). Besides, increased % RB also dough strengthening properties by forming liq- did not show any significant changes in springi- uid films with a lamellar structure in the inter- ness of croissant. Other than that, there was sig- phase between the gluten strands and the starch nificant increase (P<0.05) in chewiness with ad- (Stampfli & Nersten, 1995). Emulsifiers such as dition of 15% RB. This can explain by higher pro- DATEM, with amphiphilic structure and proper- portion of RB added contributed to the chewi- ties, will increase the strength of the dough by in- ness of croissant. As rice bran is an excellent teracting with hydrophobic regions of the gluten source of fibre (Saunders, 1990), due to its wa- proteins, forming hydrogen bonds with the amino ter binding capacities, fibre inhibits water loss groups of glutamine (Azizi & Rao, 2004; Gomez during storage and prevents staling that affects et al., 2004; Stampfli & Nersten, 1995). Such crumb firmness (Walter, 2014). The fact that factors would contribute to improved gas re- firmness was impaired with increased fibre con- tention, better texture and increased volume of tent may be associated with dietary fibre exhibit- baked product (Chin, Goh, Rahman, & Hashim, ing increased water capacity, so water does not 2007). When TGase was used alone, it showed become available for gluten that is formed while significant higher (P<0.05) specific volume com- kneading (Ranasalva & Visvanathan, 2014) and pared to those which TGase used together with cause increased croissant hardness and chewi- RB. The higher specific volume might due to ness. the effect of TGase on the rheological behaviour When DATEM was used alone, the hardness of whereby the TGase transforming weak gluten croissant decreased significantly (P<0.05) com- into a stronger one (Basman et al., 2002). Similar pared to that where DATEM was used with 10 % results also reported by Koksel, Ozay, K. W. Ng, and 15 % RB. According to Stampfli and Nersten and F. Steffe (2001). Gerrard et al. (1998) (1995) and Sawa, Inoue, Lysenko, Edwards, and suggested that TGase alters the structure of Preston (2009), the higher the amylose complex- the gluten proteins by crosslinking in dough de- forming power of the emulsifier, the lower the velopment. Those could be either disulphide initial bread crumb firmness. DATEM, which linkages, formed through oxidative processes or are monoacylglycerols with one stearic acid in other covalent crosslinks. Based on Table3, it the structure, might be expected to have a sim- can be observed that the specific volume signif- ilar effect on bread firmness. Ribotta, Perez, icantly decreased (P<0.05) when TGase worked Leon, and Anon (2004) have reported the re- with increased % RB. Basman et al. (2002) reducing effect of DATEM on firmness for wheat ported that TGase catalyse the formation of non- breads and this can be explained by its capac- disulphide covalent crosslinks between peptide- ity to aggregate gluten proteins, which create a bound glutaminyl residues and ε-amino groups gluten network that can improve the entrapment of lysine residues in protein, thereby decreas- of air and result in better crumb texture. Ac- ing the protein aggregates in the gluten network. cording to Gaupp and Adams (2014), in order Moreover, TGase yield intra and intermolecular for a given emulsifier to work properly in a dough ε-N-(γ-glutamyl)-lysine crosslinks between pro- system, it is necessary to consider the balancing teins reinforcing gluten network, thereby increas- of ingredients used to assist emulsifiers. There- ing dough strength and tenacity (Joye, Lagrain, fore, when DATEM worked together with RB, & Delcour, 2009) and decreasing the specific vol- it is assumed that the emulsifier failed to inter- ume. act with the protein in dough system due to disruption of ingredients after addition of fibre into the system. When TGase was used alone, the hardness of croissant was reduced significantly (P<0.05). This is in accordance to the study of

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Figure 2: Crude protein and crude fat - Effect of addition of RB on crude protein and crude fat of croissant with and without addition of DATEM and TGase. a indicates significant difference in crude protein and crude fat compared among the same category (with DATEM/ with TGase/ without additive). b indicates significant difference in crude protein and crude fat compared among the same % RB added.

Figure 3: Moisture sorption isotherm without additives - Eﬀect of addition of RB on moisture sorption isotherm without addition of additives

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Table 2: Moisture Content and Water Activity of Croissant Formulations

Formulation Moisture content (% wb) Water activity (aw) 1 11.23 0.899 2 15.85 0.823 3 17.63 0.844 4 9.1 0.867 5 15.89 0.886 6 13.18 0.818 7 17.25 0.851 8 14.72 0.815 9 12.34 0.862

Gerrard et al. (2001) who stated that in addition thus serving as a tool to overcome the prevailing to the increased size, transglutaminase-treated malnutrition problem. croissants had a desirable flakiness and crumb When DATEM was added alone, it showed sig- texture. However, when TGase worked with in- nificant reduced (P<0.05) crude protein and increased % RB, the hardness was significantly increased crude fat. Decreased crude protein may creased. This may be due to the quantity of fibre due to the dough improving effect of emulsifiers in dough increased but the water given into the seems to be related to their effect in reducing dough system still remain constant, therefore re- the repulsing charges between gluten proteins sulting in hardening of final product. Besides, and thereby causing them to aggregate. This ef- the quantity of TGase used might be not suffi- fect appears to be of particular importance in cient to catalyse the formation of non-disulphide composite flours, as the wheat gluten has been covalent crosslinks between peptide-bound glu- diluted (Eduardo, Svanberg, & Ahrne, 2016). taminyl residues and ε-amino groups of lysine When DATEM worked with 10% and 15% RB, residues in protein in order to give softer texture. there was a significant increase (P<0.05) in crude However, higher concentration of TGase like 1.0 fat. This was also in agreement with study of % and 1.5 % increased firmness of bread due to O. Ameh et al. (2013) reported that there was formation of a tough dough caused by excessive increase of crude fat with increased level of RB crosslinking (Basman et al., 2002). supplementation. When TGase was used solely, it showed significant lower (P<0.05) crude protein value. The 3.4 Crude protein and crude fat results are in accordance with those found by Marco and Rosell (2008) who reported that free Based on Figure2, crude protein increased sig- amino group of protein in samples significantly nificantly with 15% RB addition but 10% RB decreased with the addition of TGase. The ex- addition did not showed similar result. On the tent of TGase catalyzed reaction was also con- other hand, the addition of 10% and 15% RB firmed by Huang et al. (2010) who observed a both showed significant increase in crude fat of progressive decrease in the amount of free amino croissant respectively. Increase in crude protein groups. According to Gujral and Rosell (2004), content and crude fat is in agreement with study this could be because of involvement of amino of O. Ameh, Gernah, and D. Igbabul (2013) and acids groups of protein in the cross-linking reac- Farrell (1994) These authors had reported that tion, and this change from free to total amino rice bran is a good source of proteins, lipids, di- groups in terms of their concentrations. Higher etary fibre and minerals and could be an effective crude protein value obtained when TGase was tool in supplementing lysine and methionine de- used with increased % RB could be explained by ficient foods such as wheat, maize and sorghum;

IJFS February 2021 Volume 10 pages SI26–SI40 SI34 Wan-Ibadullah et al. BWtotadtvsWt AE ihTGase With DATEM With additives Without 3946.93 b 3801.77 a 3596.16 15 10 0 RB % niae infiatdffrnei ades pigns n hwns oprdaogtesm Badded. RB % same the among compared chewiness and springiness hardness, in difference significant indicates niae infiatdffrnei ades pigns n hwns oprdaogtesm aeoy(ihDTM ihTae ihu additive). without TGase/ with DATEM/ (with category same the among compared chewiness and springiness hardness, in difference significant indicates ades()Srnies()Ceies(m ades()Srnies()Ceies(m ades()Srnies()Ceies(Nm) Chewiness (m) Springiness (N) Hardness (Nm) Chewiness (m) Springiness (N) Hardness (Nm) Chewiness (m) Springiness (N) Hardness al :Eeto diino Bo oormaueeto risn ihadwtotadto fDTMadTGase and DATEM of addition without and with croissant of measurement colour on RB of addition of Effect 3: Table 560.24 60.31 15 10 BWtotadtvsWt AE ihTGase With DATEM With additives b Without a 60.69 0 RB % niae infiatdffrnei * *adb oprdaogtesm Badded RB % same the among compared b* and a* L*, in difference significant indicates niae infiatdffrnei * *adb oprdaogtesm aeoy(ihDTMwt Gs/wtotadditive). without TGase/ DATEM/with (with category same the among compared b* and a* L*, in difference significant indicates ± ± ± al :Eeto diino Bo etr fcosatwt n ihu diino AE n TGase and DATEM of addition without and with croissant of texture on RB of addition of Effect 4: Table 123.82 177.96 145.00 *a *L *L *a b* a* L* L* a* L* b* a* L* aB aB aA ± ± ± 0.08 0.04 1.88 0.76 0.77 0.83 aA aA aA ± ± ± 0.09 0.07 0.02 9.40 9.22 9.23 aA aA aA ± ± ± 0.02 0.14 0.40 1894.32 1530.11 1407.79 aA aA aA 22.40 23.23 23.30 ± ± ± 57.30 81.38 177.74 ± ± ± 0.40 0.53 0.58 aB bB bA aA aA aA 4023.32 3741.99 2570.14 55.34 44.49 54.25 ± ± ± ± ± ± 1.62 1.87 0.37 64.95 97.36 175.97 aB bB aB aB aB bB 9.59 9.67 9.27 0.81 0.81 0.90 ± ± ± 0.58 0.34 0.05 ± ± ± 0.05 0.06 0.01 aA aA aA aA aA aA 21.22 21.37 22.13 ± ± ± 1966.87 1382.27 1168.75 0.36 0.31 0.35 bB abB aA ± ± ± 96.83 103.97 59.56 52.83 48.84 52.77 aB bA bB ± ± ± 0.83 1.84 0.45 6468.41 4549.12 2640.05 aB bC aB 9.62 9.85 9.37 ± ± ± 122.58 114.85 184.80 ± ± ± 0.21 0.34 0.30 aA bA cB aA aA aA 0.78 0.78 0.85 21.48 21.70 22.04 ± ± ± 0.06 0.07 0.11 ± ± ± 0.51 0.12 0.91 aA aA aA aAB aB aA 2595.51 1776.60 1279.66 ± ± ± 76.99 107.25 141.17 aA bA cA IJFS February 2021 Volume 10 pages SI26–SI40 Quality characteristics of rice bran croissant SI35 the protein reducing effect of TGase cancelled tent, which eventually affect textural properties off with the addition of RB as RB is a good and the shelf life of the product. source of protein. There was a significant in- The addition of TGase into the formulations crease (P<0.05) in crude fat when TGase worked showed similar isotherm curves which were also with 10% and 15% RB. This was due to the ad- categorized as a Type III (J-shaped) isotherm dition of RB into the croissant as processed RB (Figure5). According to Barbosa-Cánovas et al. is a good source of fat (Bhosale & Vijayalakshmi, (2008), proteins have plasticizing nature, result- 2015). ing in increased availability of all polar groups. Heating at high temperatures generally increases adsorption. Therefore, with the addition of 3.5 Moisture sorption isotherm 15% RB and enzyme TGase, the adsorption curve was the steepest. When the % of RB in- It was found that the isotherm curves for the creased, TGase shifted the curve downwards to a formulations with increased % RB without any higher moisture content. Bread staling increased additives were all belong to Type III (J-shaped) by TGase addition, and affected specially to isotherm (Figure3), which also known as the hardness, chewiness and gumminess (Caballero, Flory-Huggins isotherm. Moisture gain is very Gomez, & Rosell, 2007). The affinity to water low up to the point where the crystals begin to promoted by TG in gluten (Gerrard et al., 1998) dissolve in the absorbed water at the surface of could also limit the water availability for starch crystal (Barbosa-Cánovas, Fontana, Schmidt, & and accelerate its retrogradation. The isotherm Labuza, 2008). As compared to 0% RB addi- curves indicated that TGase did not help to sta- tion, 10 % RB addition showed a moisture migra- bilize the croissant. tion towards lower moisture content while 15 % RB shifted the isotherm curve to a higher moisture content. Plasticization is not easy for car- 3.6 Critical water activity bohydrates due to their internal bonding structure that consists lots of hydrogen bonding. High According to Carter, Galloway, Campbell, and temperature and moisture are required for gela- Carter (2015), the water activity at which the tinization to open up structure to adsorption. inflection point in the DDI adsorption curve oc- Carbohydrates that have more hydrogen bonds curred was identified as the critical water activ- and more open chains resulting in higher water ity (RHc) at the temperature of the isotherm. holding capacity (Barbosa-Cánovas et al., 2008) Due to its association with glass transition, this can be observed in 15 % RB addition as higher RHc has been identified as the critical water % RB contains higher fibre. activity level for the loss of stability in amor- Based on Figure4, the addition of DATEM phous powders. The RHc gives direct informa- into the formulations showed similar isotherm tion about the maximum allowed relative vapour curves which were also categorized as a Type pressure for storage of amorphous or partially III (J-shaped) isotherm (Brunauer, 1945). When amorphous food systems (Barbosa-Cánovas et DATEM was used with the RB increased to 10 al., 2008). They also indicate that at a con- %, the isotherm shifted upwards to a higher mois- stant storage temperature, the glass transition ture content. However, when RB increased to 15 may occur because of water sorption if the stor- %, the isotherm shifted downwards to a lower age relative vapour pressure exceed the critical moisture content. DATEM as an emulsifier con- values. Based on the results obtained (Figure6), tains both hydrophilic and hydrophobic parts. the RHc values decreased as the % RB increased According to Barbosa-Cánovas et al. (2008), the without addition of any additives. Besides, when -OH groups on the glycerol will absorb moisture % RB increased, both DATEM and TGase also but the hydrophobic interactions between indi- showed a trend of decreasing RHc values. This vidual molecules block the moisture absorption. indicated that with increased % RB and addition Hence, DATEM caused inconsistent moisture mi- of additives, the croissant can enter glass transi- gration and unable to stabilize the moisture con- tion state at a lower water activity. The product

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Figure 4: Moisture sorption isotherm DATEM - Eﬀect of addition of RB on moisture sorption isotherm with addition of DATEM

Figure 5: Moisture sorption isotherm TGase - Eﬀect of addition of RB on moisture sorption isotherm with addition of TGase

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Figure 6: Critical water activity - Eﬀect of addition of RB on critical water activity with and without addition of DATEM and TGase

can be transformed into a highly viscous, solid- of croissant were not positively impacted by the like glass, ‘frozen’ over a temperature range and addition of DATEM and TGase. It can be con- may exhibit only rotational motion and vibra- cluded that DATEM and TGase did not provide tions (Barbosa-Cánovas et al., 2008). Therefore, any significant positive effect on quality charac- it can observed that the addition of DATEM and teristics of croissant with the addition of RB. TGase both cannot help in stability of croissant. Acknowledgements 4 Conclusions This work was supported by Geran Putra (Grant It can be concluded that with increased % No. 9550600), which was awarded by the Univer- RB without any additives, there was signifi- siti Putra Malaysia. cant decreased specific volume, increased chewiness (only with 15 % RB), and increased crude References protein and crude fat content. When DATEM and TGase were used alone, they showed signifi- AACC. (2000). American association of cereal cant decreased lightness, increased specific vol- chemists, aacc. Approved Methods of the ume and decreased hardness. They also de- AACC. AACC International, Minnesota. creased crude protein and increased crude fat AOAC. (2000). Official methods of analysis (17th significantly. When DATEM and TGase worked ed., method nos: 979.09, 920.39). washing- with increased % RB, there was significant de- ton, dc. Association of Official Analytical creased lightness and specific volume, increased Chemists. hardness, and increased chewiness. The mois- Azizi, M. H., & Rao, G. V. (2004). Effect of sur- ture sorption isotherm curves of the croissant factant gels on dough rheological character- belong to the Type III isotherm, also known as istics and quality of bread. Critical Reviews Flory-Huggins Isotherm (J-shaped). The critical in Food Science and Nutrition, 44 (7-8), aw obtained from the Guggenheim-Anderson-de 545–552. doi:10.1080/10408690490489288 Boer (GAB) equation showed that the shelf life

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Barbosa-Cánovas, G. V., Fontana, A., Schmidt, Collar, C., Santos, E., & Rosell, C. M. (2007). As- S. J., & Labuza, T. (2008). Water activity sessment of the rheological profile of fibre- in foods: Fundamentals and applications. enriched bread doughs by response surface doi:10.1002/9780470376454 methodology. Journal of Food Engineering, Basman, A., Koksel, H., & Ng, P. K. W. (2002). 78 (3), 820–826. doi:10 . 1016 / j . jfoodeng . Effects of increasing levels of transglutam- 2005.11.026 inase on the rheological properties and Eduardo, M., Svanberg, U., & Ahrne, L. (2016). bread quality characteristics of two wheat Effect of hydrocolloids and emulsifiers on flours. European Food Research and Tech- the shelf-life of composite cassava-maize- nology, 215 (5), 419–424. doi:10 . 1007 / wheat bread after storage. Food Science & s00217-002-0573-3 Nutrition, 4 (4), 636–644. doi:10.1002/fsn3. Bhosale, S., & Vijayalakshmi, D. (2015). Pro- 326 cessing and nutritional composition of rice Farrell, D. J. (1994). Utilization of rice bran in di- bran. Current Research in Nutrition and ets for domestic-fowl and ducklings. Worlds Food Science, 3 (1), 74–80. doi:10.12944/ Poultry Science Journal, 50 (2), 115–131. CRNFSJ.3.1.08 doi:10.1079/WPS19940012 Boukid, F., Carini, E., Curti, E., Bardini, G., Gaupp, R., & Adams, W. (2014). Diacetyl Tar- Pizzigalli, E., & Vittadini, E. (2018). Ef- taric Esters of Monoglycerides (DATEM) fectiveness of vital gluten and transglu- and Associated Emulsifiers in Bread Mak- taminase in the improvement of physico- ing. In Emulsifiers in food technology chemical properties of fresh bread. LWT- (Chap. 6, pp. 121–146). doi:10 . 1002 / Food Science and Technology, 92. doi:10 . 9781118921265 . ch6. eprint: https : / / 1016/j.lwt.2018.02.059 onlinelibrary.wiley.com/doi/pdf/10.1002/ Breeding, C. J., & Beyer, R. S. (2000). Food 9781118921265.ch6 chemistry: Principles and applications. . Gerrard, J. A., Fayle, S. E., Wilson, A. J., New- In G. L. Christen & J. S. Smith (Eds.), berry, M. P., Ross, M., & Kavale, S. (1998). (Chap. Eggs, pp. 421–431). West Sacra- Dough properties and crumb strength of mento: Science Technology System. white pan bread as affected by microbial Brunauer, S. (1945). The adsorption of gases and transglutaminase. Journal of Food Science, vapors. Physical adsorption, 1. 63 (3), 472–475. doi:10.1111/j.1365-2621. Caballero, P. A., Gomez, M., & Rosell, C. M. 1998.tb15766.x (2007). Improvement of dough rheology, Gerrard, J. A., Newberry, M. P., Ross, M., Wil- bread quality and bread shelf-life by en- son, A. J., Fayle, S. E., & Kavale, S. (2001). zymes combination. Journal of Food En- Pastry lift and croissant volume as affected gineering, 81 (1), 42–53. doi:10 . 1016 / j . by microbial transglutaminase. Journal of jfoodeng.2006.10.007 Food Science, 65 (2), 312–314. doi:10.1111/ Carter, B. P., Galloway, M. T., Campbell, G. S., j.1365-2621.2000.tb15999.x & Carter, A. H. (2015). The critical water Ghufran Saeed, S. M., Arif, S., Ahmed, M., Ali, activity from dynamic dewpoint isotherms R., & Shih, F. (2009). Influence of rice bran as an indicator of crispness in low moisture on rheological properties of dough and in cookies. Journal of Food Measurement and the new product development. Journal of Characterization, 9 (3), 463–470. doi:10 . food science and technology, 46 (1), 62. 1007/s11694-015-9254-3 Gomez, M., del Real, S., Rosell, C. M., Ronda, F., Chin, N. L., Goh, S. K., Rahman, R. A., & Blanco, C. A., & Caballero, P. A. (2004). Hashim, D. M. (2007). Functional effect of Functionality of different emulsifiers on the fully hydrogenated palm oil-based emulsi- performance of breadmaking and wheat fiers on baking performance of white bread. bread quality. European Food Research and International Journal of Food Engineering, Technology, 219 (2), 145–150. doi:10.1007/ 3 (3). doi:10.2202/1556-3758.1196 s00217-004-0937-y

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Gujral, H. S., & Rosell, C. M. (2004). Func- Nutrition Sciences, 4, 43–48. doi:10.4236/ tionality of rice flour modified with a mi- fns.2013.49A2007 crobial transglutaminase. Journal of Cereal Oladunmoye, O., Akinoso, R., & Olapade, Science, 39 (2), 225–230. doi:10.1016/j.jcs. A. (2010). Evaluation of some physical- 2003.10.004 chemical properties of wheat, cassava, Huang, W., Li, L., Wang, F., Wan, J., Tilley, M., maize and cowpea flours for bread mak- Ren, C., & Wu, S. (2010). Effects of trans- ing. Journal of Food Quality, 33, 693–708. glutaminase on the rheological and mixo- doi:10.1111/j.1745-4557.2010.00351.x lab thermomechanical characteristics of oat Peressini, D., Pin, M., & Sensidoni, A. (2011). dough. Food Chemistry, 121 (4), 934–939. Rheology and breadmaking performance of doi:10.1016/j.foodchem.2010.01.008 rice-buckwheat batters supplemented with Issara, U., & Rawdkuen, S. (2016). Rice bran: A hydrocolloids. Food Hydrocolloids, 25 (3), potential of main ingredient in healthy bev- 340–349. doi:10.1016/j.foodhyd.2010.06. erage. International Food Research Jour- 012 nal, 23 (6). Popov-Raljic, J. V., Mastilovic, J. S., Lalicic- Joye, I. J., Lagrain, B., & Delcour, J. A. (2009). Petronijevic, J. G., & Popov, V. S. (2009). Use of chemical redox agents and exoge- Investigations of bread production with nous enzymes to modify the protein net- postponed staling applying instrumental work during breadmaking - a review. Jour- measurements of bread crumb color. Sen- nal of Cereal Science, 50 (1), 11–21. doi:10. sors, 9 (11), 8613–8623. doi:10 . 3390 / 1016/j.jcs.2009.04.001 s91108613 Kent, N. L., & Evers, A. D. (1994). Technol- Ranasalva, N., & Visvanathan, R. (2014). Devel- ogy of cereals: An introduction for students opment of bread from fermented pearl mil- of food science and agriculture. Woodhead let flour. Journal of Food Processing and Publishing. Technology, 5 (5). Koksel, H., Ozay, D., K. W. Ng, P., & F. Steffe, Ribotta, P. D., Perez, G. T., Leon, A. E., & J. (2001). Effects of transglutaminase en- Anon, M. C. (2004). Effect of emulsifier and zyme on fundamental rheological proper- guar gum on micro structural, rheological ties of sound and bug-damaged wheat flour and baking performance of frozen bread doughs. Cereal Chemistry - Cereal Chem, dough. Food Hydrocolloids, 18 (2), 305–313. 78, 26–30. doi:10.1094/CCHEM.2001.78.1. doi:10.1016/S0268-005X(03)00086-9 26 Rogers, D. E., & Hoseney, R. C. (1983). Bread- Marco, C., & Rosell, C. M. (2008). Effect of making properties of datem. Bakers Digest, different protein isolates and transglutam- 12–&. inase on rice flour properties. Journal of Saunders, R. M. (1990). The properties of rice Food Engineering, 84 (1), 132–139. doi:10. bran as a food stuff. Cereal Foods World, 1016/j.jfoodeng.2007.05.003 35, 632–662. McKee, L. H., & Latner, T. A. (2000). Under- Sawa, K., Inoue, S., Lysenko, E., Edwards, N. M., utilized sources of dietary fiber: A review. & Preston, K. R. (2009). Effects of purified Plant Foods for Human Nutrition, 55 (4), monoglycerides on canadian short process 285–304. doi:10.1023/A:1008144310986 and sponge and dough mixing properties, Nagendra Prasad, M. N., Sanjay, K., & Khatokar bread quality and crumb firmness during M., S. (2011). Health benefits of rice bran - storage. Food Chemistry, 115 (3), 884–890. a review. Journal of Nutrition & Food Sci- doi:10.1016/j.foodchem.2009.01.010 ences, 01. doi:10.4172/2155-9600.1000108 Sharma, R., Srivastava, T., & Saxena, D. C. O. Ameh, M., Gernah, D., & D. Igbabul, B. (2015). Studies on rice bran and its (2013). Physico-chemical and sensory eval- benefits-a review. Int. J. Eng. Res. Appl, uation of wheat bread supplemented with 5 (5), 107–112. stabilized undefatted rice bran. Food and

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IJFS February 2021 Volume 10 pages SI26–SI40 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI41–SI56

Optimization of a Process for a Microgreen and Fruit Based Ready to Serve Beverage

Anjali Sharmaa, Prasad Rasanea, b*, Anirban Deyc, Jyoti Singha, Sawinder Kaura, Kajal Dhawana, Ashwani Kumara, and Hari Shankar Joshid

a Department of Food Technology and Nutrition, Lovely Professional University, Jalandhar Punjab-144411 b Centre of Food Science and Technology, Banaras Hindu University, Varanasi 221005, India c Pimpernel Food Products Pvt. Ltd. Hooghly, West Bengal 712136, India d College of Applied Food and Dairy Technology, Minbhawan, New Baneshwor, Kathmandu, Nepal, G.P.O. Box: 11898 *Corresponding author [email protected] Tel: +918968976119

Received: 6 August 2018; Published online: 24 February 2021

Abstract

A process for preparation of a microgreen and fruit based beverage was optimized using spinach (Spinacia oleracea) microgreen, pomegranate (Punicagranatum), pineapple (Ananascomosus) and sugar. The blended juice in diﬀerent ratios was analysed for total soluble solids, viscosity, sedimentation, acidity, metal chelation activity, free radical scavenging activity and reducing power. The optimized beverage had 17.26 mL 100 mL−1 spinach microgreen juice, 57.07 mL 100 mL−1 pomegranate juice, 1.01 g 100 g−1 sugar and 24.66 mL 100 mL−1 pineapple juice. The product was high in nutrients, particularly protein, minerals (sodium, potassium and iron) and vitamins (vitamin C), and bioactive compounds (total phenols and total carotenoids), and had high antioxidant activity (metal chelation activity, free radical scavenging activity and reducing power). The antioxidants and bioactive compounds present in juice were designed to help reduce oxidative stress during inﬂammatory cases such as arthritis. Keywords: Antioxidants; Microgreen blend; Response Surface Methodology; Physicochemical properties

1 Introduction as edible garnishing and to provide vivid colour, intense ﬂavour and tender texture to food such Microgreens are young and tender edible greens as salads, soups and sandwiches (Di Gioia et al., produced from diﬀerent species of vegetables, 2017). Microgreens are considered as highly per- aromatic herbs and herbaceous plants. Micro- ishable products because they are very delicate greens are usually harvested after 7-14 days of and have short shelf life i.e. 1-2 days at ambi- germination when they grow to 2.5-7.6 cm (1-3 ent temperature. Microgreens are highly rich in inch) in height and the cotyledonary leaves are bioactive compounds, vitamins, minerals, antiox- fully developed with a small pair of true leaves idants and phytonutrients such as α-tocopherol (Di Gioia, Bellis, Mininni, Santamaria & Serio, and β-carotene as compared to mature leaves 2017; Kou et al., 2013; Xiao, Lester, Luo & (Kou et al., 2013; Xiao et al., 2014). Due to Wang, 2012). They are increasingly used by chefs their high nutritive value, health promoting and

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a4 SI42 Sharma et al. disease preventing properties thay are considered is popular amongst athletes for the treatment of as a “functional food”. Over the last few years, injuries and other physical aches (Debnath, Dey, they have begun to appear in farmer’s market, Chanda & Bhakta, 2012). It also has an anti- upscale markets and restaurants as the demand inflammatory effect on soft tissue injury and re- for microgreens increases. Therefore, it is be- duces swelling and pain (Kumar, Chandra, Ku- coming important to study their utility in vari- mar & Prince, 2016). Pomegranate is obtained ous commercial preparations that could improve from arils which are rich sources of bioactive their shelf life and delivery of their valuable nu- compounds with abilities to reduce inflammation trients to the consumers (Mir, Shah & Mir, 2017; and fight the enzyme that destroys the cartilage Xiao et al., 2014). (Anahita, Asmah & Fauziah, 2015; Bhowmik, Spinaciaoleraceais belongs to Amaranthaceae Gopinath, Kumar & Kumar, 2013). The com- family and is commonly known as spinach (Me- bination of fruits makes a healthy diet to provide hta & Belemkar, 2014). It is considered as a all the basic nutrients that are present in differ- “poor man’s vegetable” because of its low pro- ent juices (Akusu, Kiin-Kabari & Ebere, 2016; duction cost and high yield (Yadav, Kalia, Ku- Landon, 2007). The consumption of fruit juice mar & Jain, 2013). It is one of the most may be influenced by sensory quality and nu- nutritious leafy vegetable, ranking second be- tritive value. Recently herbal formulations have hind kale in total folate and carotenoid con- been given due attention owing to their thera- tent. In India, the plant is known as palak peutic potential (Clark, 1995). and has long been used as a diuretic, laxat- The present work focuses on optimizing a process ive and anti-inflammatory agent. Spinach mi- for preparation of a microgreen and fruit based crogreen is harvested 7-14 days after germina- beverage with acceptable sensory and physico- tion and 1-3 inches in height. They are sown chemical properties. Spinach microgreen was at closer density than regular spinach and thus blended with fruits such as pomegranate and the leaves are smaller and hence the name (Ber- pineapple to increase its acceptability and nu- gquist, 2006). The spinach microgreen is a trient potential. rich source of carotenoids (such as lutein, vi- olaxanthin, zeaxanthin and β-carotene), phenolic compounds (para-coumaric acid, ferulic acid, ortho-coumaric acid) and flavonoids which act as powerful antioxidants to fight against free radical damage and protect the body from many diseases (Mehta & Belemkar, 2014; Rao, Tabassum, 2 Materials and Methods Babu, Raja & Banji, 2015). Quercetin is a bio- flavonoid present in spinach which exhibits antioxidant, anti-proliferative, anti-inflammatory 2.1 Cultivation of microgreens and hepatoprotective properties. Spinach is also associated with easing pain of arthritis due to its anti-inflammatory property (Gaikwad, Shete & Otari, 2010). Other vegetarian sources like The spinach (Spinaciaoleraceais) microgreens fruits, particularly pineapple contains nutrients were cultivated in the experimental farm un- such as carbohydrates, vitamin C, β-carotene, der low pressure controlled polyhouse condi- protein, fat, ash and fibre, and bioactive com- tions in Lovely Professional University, Pun- pounds such as flavonoids and phenols which acts jab, India. Indirect and low air circulation as antioxidant, anti-carcinogenic, antimicrobial, was provided. The temperature was regulated anti-mutagenic and anti-inflammatory agents (da between 18 and 26 oC and the relative humidity Silva, Nogueira, Duzzioni & Barrozo, 2013; Goh, was controlled to 50%. Pineapple (Queen Vari- Mohd Adzahan, Leong, Sew & Sobhi, 2012). ety) and pomegranate (Mridula Variety) were Pineapple contains bromelain, a proteolytic en- purchased from the farmers’ market of Jaland- zyme which digests the proteins in the food. It har, India.

IJFS February 2021 Volume 10 pages SI41–SI56 Microgreen and fruit based RTS SI43

2.2 Optimization by response Protein content surface methodology (RSM) The protein content was analysed by the Kjeld- hal method suggested by Rangana (1997). A The product was optimized using response sur- weighed sample (0.5 g) was digested along with face methodology (RSM). Product variables concentrated sulphuric acid (20 mL) and 5 g di- included spinach microgreen juice (10-20 mL gestion mixture (10 g potassium sulphate and 1 −1 100 mL ), pomegranate juice (40-60 mL 100 g copper sulphate) in a Kjeldhal digestion flask. −1 −1 mL ), sugar (0-2 g 100 g ) and pineapple juice The contents were cooled, diluted with a small which were added to make up a total formulation amount of distilled water and transferred into a of 100 mL in required experiments. Twenty tri- 50 mL volumetric flask. The volume was made als were carried out to optimize the microgreen up to the mark with addition of distilled water. based functional juice. A measured aliquot (5 mL) was taken in a dis- tillation flask followed by 10 mL of 40% NaOH. Ammonia liberated was collected through a con- 2.3 Proximate composition denser in a flask containing 10 mL of 0.1 N HCl, prior to which methyl red indicator was added. Moisture content The methyl red indicator solution containing liberated NH3 was titrated against 0.1 N NaOH. The moisture content was determined by AOAC The amount of NaOH used to neutralize the (2000) method. The sample (5 g) was weighed indicator was recorded. One blank containing accurately and then weighed again in a petri concentrated sulphuric acid and digestion mix- ± o plate. It was then heated at 110 2 C for 4 h in ture was also run along with the experimental a hot air oven. The plate was removed. This was samples. followed by cooling in a desiccator and weighed By this method, the percentage of nitrogen at one hour intervals until constant weight was present in the sample was calculated and then attained. The moisture content was determined the crude protein content was calculated as fol- by the following equation: lows: W [A − B] × 0.0014 × vol. of digest Moisture content(%) = W − 1 × 100 (1) N(%) = × 100 2 W Aliquot taken × S (3) Where W1 and W2= weight of petri plate along P rotein(%) = N(%) × 6.25 (4) with sample before and after drying respectively Where A = Sample titre (mL), B = Blank titre and W= weight of sample. (mL), S = Weight of sample taken

Ash content Fat content The Fat content was determined by the Soxh- The ash content was determined by the method let extraction method (AOAC, 2000). Dried of Romelle, Rani and Manohar (2016). The samples (2 g) were extracted with Petroleum sample (2 g) was taken in a silica dish and ig- ether (100%) in the Soxhlet extraction apparatus nited over a low flame to char the organic matter. for 6-8 h in a pre-weighed round bottom flask. After complete charring, the dish was placed in a The extract containing fat and petroleum ether muffle furnace and heated at 550 oC for 3-4 h, till was evaporated over a boiling water bath and greyish to off white ash was obtained. The dish dried in an oven at low temperature and weighed. containing ash was cooled in a desiccator and The differences in the weight of the round bottom weighted. The percentage ash was calculated as flask represented the ether extract (fat content) follows: present in the sample and were calculated as: wt. of ash W Ash content(%) = × 100 (2) Fat content(%) = (W − 2 ) × 100 (5) wt. of sample 1 W

IJFS February 2021 Volume 10 pages SI41–SI56 SI44 Sharma et al.

Where, W (g) = Weight of sample, W1 (g) = 2.4 Antioxidant properties Weight of empty round bottom flask, W2 (g) = Weight of empty round bottom flask + Fat con- Reducing power tent The microgreen juice blend was evaluated for reducing power using the Ferricyanide-ferric chlor- Titratable Acidity ide method. Microgreen based juice blend (0.5 Titratable acidity was estimated by the method mL) was added to test tubes along with 2.5 mL according to Lugwisha (2014). A known volume potassium phosphate buffer (0.2 M, pH 6.6) and of sample was titrated against 0.1 N NaOH freshly prepared 1% potassium ferricyanide (2.5 standard solutions by using phenolphthalein as mL). The mixture was incubated in a water bath o an indicator up to the end point (Pink color). at 50 C for 20 minutes. After adding 2.5 mL of The titratable acidity was calculated as: 10% trichloroacetic acid to the mixture, it was then subject to centrifugation at 5000 rpm for 5 minutes. The upper layer of the mixture (2.5 T × NA × V ol × A × 100 mL) was taken and mixed with 2.5 mL of dis- TA = w m eq (6) Vs × Val × 1000 tilled water. Freshly prepared 0.1% FeCl3 (0.5 mL) was added and the absorbance was meas- Where TA is the Titratable acidity (%), Tw is ured at 700 nm against a blank Shiban, Al-Otaibi the Titre weight, NA is the Normality of alkali, and Al-Zoreky (2012). V olm is the Vol. make up,Aeq is the Equivalent of acid, Vs is the Volume of sample taken and Val is the Volume of aliquot taken. FRSA (Free radical scavenging activity) Minerals and Vitamin (Ascorbic acid) FRSA (Free radical scavenging activity) was assessed using the 2,2-diphenyl-1-picrylhydrazyl Sodium and potassium contents were determ- method, with certain modifications. The micro- ined by the flamephotometery process suggested green based juice blend (0.1 mL) was dissolved by Rangana (1997). Iron content of the micro- with 2.9 mL of DPPH solution. The mixture was green blend was estimated by using the method shaken vigorously and left to stand in the dark at of Achikanu, Eze-Steven, Ude and Ugwuokolie room temperature for 30 minutes. The amount (2013). The sample solution (2.5 mL) was pipet- of FRSA was determined specrtophotometrically ted in 0.4 mL of NaOH (5 M) to maintain the pH at 517 nm (Jenitha & Anusuya, 2016) The free between 4.0-4.5. Acetate buffer (0.7 mL, pH 4.5) radical scavenging activity was calculated as % was added along with 0.5 mL of Hydroquinone inhibition from the following equation: (25%). α1α1 Dipridyl 0.1% of 0.5 mL was added. Distilled water (0.35 mL) was used to make the OD sample FRSA(%Inhibition) = (OD control − ) × 100 volume up to 5 mL. The absorbance was taken OD control against a blank at 520 nm. Ascorbic acid content (7) was measured by using dye, 2,6-dichlorophenol- OD - Optical density (Absorbance) indophenol titration method according to Rekha et al. (2012). Metal chelating activity

Total soluble solids (TSS) FeSO4 solution (0.25 mL) and 0.5mL of sample was taken in a test tube. Tris-HCL buﬀer (1 mL, TSS was determined using a hand-held refracto- pH 7.4) and 2,2 –bipyridyl solution was taken meter of range 0-30 oBrix. The TSS was recorded along with 2.5 mL of ethanol and made up the by placing 1-2 drops of juice sample on the prism volume up to 5 mL with distilled water. The mix- of the hand-held refractometer. The results were ture was incubated at room temperature for 10 expressed as oBrix (Fawole & Opara, 2013). minutes and the absorbance was taken at 522 nm

IJFS February 2021 Volume 10 pages SI41–SI56 Microgreen and fruit based RTS SI45 against a blank (Mohan, Balamurugan, Elaya- determined by using the following equation: raja & Prabakaran, 2012). The metal chelation ρ1t1 activity scavenging eﬀect (M ) was calculated η1 = × η2 (9) chel ρ t by the following formula: 2 2 Where, ρ1=density of unknown liquid, (A0 − A1) ρ2=density of liquids (water), t1=time of other Mchel = × 100 (8) A0 liquids, t2=time of known liquid, η2=viscosity of known liquid. Where A0= absorbance of control, A1= absorbance of sample 2.6 Sensory evaluation

Total Phenolic Content (TPC) The microgreen based functional juice formulations were subjected to sensory evaluation using Total phenolic content was determined spectro- a 10-point hedonic scale which comprises colour photometrically at 765 nm by using folin- ciocal- and appearance, aroma, taste, mouthfeel, con- teu’s reagent (Kamtekar, Keer & Patil, 2014). sistency and overall acceptability. The sensory Juice extract was mixed with 0.2 mL of folin- evaluation was performed by a panel of 50 semi- ciocalteu’s reagent and the volume made up to trained judges (25 males and 25 females; age: 20- 3 mL with distilled water. The mixture was in- 40 years) from the campus of Lovely Professional cubated for 30 minutes at room temperature. 1 University, Punjab (India). mL of 20% Sodium carbonate was added and the mixture boiled for 1 minute in a boiling bath and the absorbance was measured at 765 nm by using 3 Results and Discussion a UV-Vis spectrophotometer. 3.1 Nutritional composition and antioxidant activity of mature Total carotenoid content spinach and spinach Total carotenoid content of the microgreen based microgreen juice blend was determined spectrophotometric- ally. The sample (3 mL) was mixed with 10 mL of The nutritional composition of spinach micro- distilled water. Cold acetone (20 mL) was added green and mature spinach are compared in Table and it was allowed to stand for 15 minutes. The 1. There is a significant difference (p<0.05) filtered extract (1/3) was mixed with petroleum between them in the amount of nutrients such ether (20 mL). Distilled water was added for the as protein, minerals and vitamin C. Moreover, separation phase. The total carotenoid was de- the antioxidant activity in the form of redu- termined at 450 nm wavelength using a UV-Vis cing power, metal chelation and free radical spectrophotometer (Ding & Syazwani, 2016). scavenging activity was found to be significantly (p<0.05) higher in the spinach microgreen as compared to their mature counterpart. It 2.5 Viscosity (Ostwald was reported by Janovská,Stockováand Stehno viscometer) (2010); Xiao et al. (2012); Sun et al. (2013) and Weber (2017) that microgreens have high levels Viscosity of the microgreen based functional of vitamins and bioactive compounds and are juice was measured using an Ostwald viscometer dense sources of mineral nutrition as compared (Borosil, BT1UR3502007, India). The liquid was to mature plants. Pinto, Almeida, Aguiar and added to the viscometer, pulled into the upper Ferreira (2015) showed that microgreens have a reservoir by suction, and then allowed to drain higher content of minerals and lower nitrate con- by gravity into the lower reservoir, based on the tent than the mature leaves, and thus makes time of flow through a volumetric capillary. The them an excellent source of minerals whilst redu- viscosity of different concentrations of juice was cing exposure to harmful nitrates. Microgreens

IJFS February 2021 Volume 10 pages SI41–SI56 SI46 Sharma et al. are a good source of both the microelements and samples and also sugar is an essential factor for macroelements with no traces of toxic elements the flavour. Sugar provides sweetness which is as shown by Xiao et al. (2016). the most significant attribute for the acceptance of juice (Endrizzi, Gasperi, Rodbotten & Naes, 2014). 3.2 Optimization of process for microgreen and fruit based beverage 3.4 Effect of juice blend on physicochemical properties of The experimental setup for optimization and val- the beverage ues for sensory and physico-chemical parameters for the experiment are as shown in the Tables2 Total soluble solids and3, respectively. As evident from Table5, spinach and pomegranate had significant (p<0.01) pos- 3.3 Effects of juice blends on itive effects on TSS. This might be due to the sensory properties of the high sugar content present in pomegranate. beverage Added sugar also had a significant effect on TSS but was less as the quantity of added sugar Table4 shows the coefficient estimates for the was lower. Fawole and Opara (2013) reported sensory properties as a response. As evidenced that TSS depends on the maturity of fruit. As from the Table, pomegranate had a significant the fruit is going towards maturity, starch is (p<0.01) positive effect on colour and appear- converted into sugars by hydrolysis and this ance, aroma, taste, consistency, mouthfeel and enhances the sweetness and flavour. It was overall acceptability scores, while spinach negat- shown by Reboucas, Rodrigues, de Freitas and ively (p<0.01) affected the sensory properties ex- Ferreira (2016) that as the concentration of juice cept consistency. As the amount of pomegranate and sugar increases, the TSS of the juice blend juice in the blend increased the overall accept- also increases. The increase in TSS may also ability of the juice blend also increased, owing to be due to the conversion of polysaccharides into the bright red colour it imparts and also provides oligosaccharides and monosaccharides as stated good mouthfeel. Lawless, Threlfall, Howard and by Wisal et al. (2013), Oyeleke (2013) and Jan Meullenet (2012) and Mohideen et al. (2015) and Dorcus Masih (2012). It was also recorded showed that red colour is due to the anthocy- by Lawless et al. (2012) that the increase in TSS anins and it positively affects consumer accept- increases the overall acceptance of the product. ance. However, sp inach provides a dark brown colour and gives a bitter mouthfeel. It was ob- Viscosity served by Lawless et al. (2012) that the reaction of anthocyanins and tannins forms brown Pomegranate and sugar also had an interactive coloured complexes and thus imparts dark col- significant (p<0.01) positive effect on the vis- our to the juice and a slight sour taste (Pimen- cosity of the juice blend (Fig.1B). This might tel, Madrona & Prudencio, 2015) decreasing con- be due to the presence of a greater amount of sumer acceptance. Consistency of the blend was pulp. It is reported by Kar and Kaya (2014) also positively affected by the amount of spin- that as the concentration of juice increases, the ach and pomegranate juice. On the other hand, viscosity also increases. Pomegranate and spin- sugar had no significant effect on colour, appear- ach had a significant (p<0.01) positive effect on ance and aroma, while it positively affected the sedimentation. As the concentration of pine- taste, consistency, mouthfeel and overall accept- apple and spinach increases the sedimentation ability of the product (Fig.1A). It was stated increases (Table3). It was claimed by Abedi, by Wisal, Ullah, Zeb and Khan (2013) that ad- Sani and Karazhiyan (2014) that viscosity and dition of sugar increases the consistency of juice sedimentation increases with an increase in pec-

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Table 1: Nutritional composition of mature spinach and spinach microgreen

Nutrients Spinach Mature Spinach Microgreen Protein (%) 1.19±0.11b 2.45±0.06a Moisture (%) 90.04±0.11b 94.2±0.20a Ash (%) 1.05±0.04b 1.23±0.03a Potassium (mg) 132.0±0.04b 161.0±0.03a Sodium (mg) 70.02±0.09b 78.02±0.03a Total phenols (GAE g−1) 0.59±0.08b 0.95±0.06a Total carotenoids (µg 100g−1) 10.80±0.05b 15.08±0.05a Vitamin C (mg) 9.06±0.28b 11.8±0.008a Reducing Power (%) 39.16±0.45b 48.86±0.15a Metal chelation activity (% inhibition) 33.41±0.05b 39.59±0.36a Free radical scavenging activity (% inhibition) 36.95±0.70b 43.07±0.14a Iron (mg) 3.59±.32b 4.03±0.02a The values are presented as Mean ± Standard deviation The values represented with different superscripts differ significantly at p < 0.05

Table 2: Sensory attributes of the blended juice as aﬀected by diﬀerent constituents

Run Spinach Pomegranate Sugar Pineapple Color and Aroma Taste Consistency Mouth Overall juice (%) juice (%) (%) (%) Appearance (%) (%) (%) (%) feel (%) acceptability (%) 1 15 50 1 34 8.4 8.4 7.0 7.7 7.2 7.4 2 6.59 50 1 42.41 8.7 8.8 7.7 7.5 7.5 7.3 3 20 40 0 40 7.6 7.6 6.1 7.4 6.1 6.3 4 10 60 0 30 8.9 8.9 7.5 7.8 7.4 7.3 5 23.41 50 1 25.59 7.9 8.1 6.4 7.9 6.5 6.6 6 15 50 1 34 8.2 8.4 7.1 7.7 7.2 7.2 7 15 50 2.68 32.32 8.3 8.5 7.3 7.8 7.4 7.6 8 20 60 0 20 8.6 8.7 6.9 7.9 7.0 7.1 9 10 40 0 50 8.4 8.6 6.8 7.1 6.9 6.4 10 20 60 2 18 8.4 8.5 7.0 8.1 7.1 7.2 11 15 50 0 30 8.3 8.6 6.8 7.6 6.8 6.5 12 10 40 2 43 8.4 8.4 7.0 7.2 7.1 7.0 13 15 33.18 1 55.82 7.4 7.5 6.2 6.9 6.4 6.4 14 15 50 1 34 8.3 8.5 6.8 7.7 6.7 6.7 15 20 40 2 38 7.6 7.7 6.4 7.5 6.5 6.5 16 15 66.82 1 16.18 9.0 9.0 7.8 8.1 7.9 7.7 17 15 50 1 34 8.3 8.4 7.0 7.7 7.2 7.1 18 10 60 2 28 8.7 8.9 7.6 8.2 7.8 7.6 19 15 50 1 34 8.4 8.4 7.0 7.6 7.1 7.2 20 15 50 1 34 8.3 8.3 7.1 7.7 7.2 7.4

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Figure 1: Response surface plots for: (A) Overall acceptability, (B) Viscosity, (C) Acidity, (D) Metal chelation, (E) FRSA, (F) Reducing power

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Table 3: Physicochemical attributes of the blended juice as aﬀected by diﬀerent constituents

Run Spinach Pomegranate Sugar Pineapple TSS Acidity Metal FRSA Reducing Viscosity Sedimentation juice (%) juice (%) (%) juice (%) (o Brix) (%) chelation (%) (%) power (%) (Pa sec) (%) 1 15 50 1 34 10.03 0.33 66.12 75.12 74.12 76.13 17.05 2 6.59 50 1 42.41 9.04 0.26 46.23 56.23 54.36 72.34 15.08 3 20 40 0 40 11.00 0.31 64.08 74.08 71.03 74.73 17.05 4 10 60 0 30 12.00 0.33 59.36 69.36 66.46 73.53 18.03 5 23.41 50 1 25.59 12.08 0.28 69.12 79.11 73.98 78.25 18.05 6 15 50 1 34 10.02 0.32 65.36 76.36 73.68 76.03 17.04 7 15 50 2.68 32.32 11.03 0.33 65.02 75.02 75.59 77.34 17.05 8 20 60 0 20 13.03 0.34 69.55 79.55 83.59 78.61 19.02 9 10 40 0 50 8.02 0.28 50.47 60.47 57.39 69.43 16.03 10 20 60 2 18 13.08 0.32 72.02 82.23 80.32 80.91 19.02 11 15 50 0 30 10.01 0.33 70.98 80.98 75.41 73.64 17.04 12 10 40 2 43 8.07 0.27 42.01 52.01 60.88 71.47 16.02 13 15 33.18 1 55.82 9.06 0.31 44.59 54.59 60.27 69.25 16.04 14 15 50 1 34 10.03 0.33 66.26 76.26 73.39 76.03 17.02 15 20 40 2 38 11.07 0.31 57.22 67.22 70.27 73.42 17.05 16 15 66.82 1 16.18 14.07 0.38 70.12 80.12 78.02 79.32 19.06 17 15 50 1 34 10.03 0.32 64.15 74.15 73.39 76.03 17.03 18 10 60 2 28 11.07 0.34 64.15 70.91 67.55 78.25 18.04 19 15 50 1 34 10.02 0.33 67.26 76.53 73.39 76.45 17.01 20 15 50 1 34 10.05 0.33 66.75 77.32 74.25 77.82 17.05

Table 4: Coeﬃcient estimates of sensory properties for fruit based microgreen juice

Factors Appearance (%) Aroma (%) Taste (%) Mouth feel (%) Consistency (%) Overall acceptability (%) Intercepts 8.31 8.40 7.00 7.10 7.68 7.17 A -0.26* -0.25* -0.34* -0.31* 0.093* -0.17* B 0.39* 0.38* 0.39* 0.38* 0.35* 0.38* C -0.29 -0.034 0.11* 0.15* 0.083* 0.22* A2 7.057*10−3 0.017 -8.248*10−3 -0.055 0.010 -0.094 B2 -0.028 -0.054 -0.026 -2.332*10−3 -0.061* -0.059 C2 7.057*10−3 0.052 8.248*10−3 -0.020 0.010* -0.059 AB 0.13* 0.14* 0.013 0.038 -0.075 0.000 AC 0.00 0.013 0.013 -0.012 -0.025 -0.075 BC -0.050 -0.012 0.037 -0.012 0.050* -0.050 Model Significant Significant Significant Significant Significant Significant A Spinach juice, B Pomegranate juice, C Sugar, A2 Quadratic terms of spinach juice, B2 Quadratic terms of pomegranate juice, C2 Quadratic terms of sugar, AB Interactive term of spinach juice and pomegranate juice, AC Interactive term of spinach juice and sugar, BC Interactive term of pomegranate juice and sugar The values within the column of each attribute denoted with * are significant at p<0.01

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Table 5: Coeﬃcient estimates of textural and antioxidant properties in fruit based microgreen juice

Factors TSS Acidity Metal FRSA Viscosity Reducing Sedimentation (o Brix) (%) chelation (%) (%) (Pa sec) power (%) (%) Intercepts 10.30 0.33 66.01 76.00 76.41 73.69 17.32 A 1.09* 6.856*10−3* 6.25* 6.50* 1.83* 6.29* 0.64* B 1.45* 0.020* 6.90* 6.68* 2.87* 4.99* 0.95* C 0.25* -1.464*10−3 -1.32* -1.55* 1.02* 0.062 0.012 A2 0.27* -0.020* -3.12* -3.19* -0.37* -3.30* -5.830*10−3* B2 0.64* 6.104*10−3* -3.23* -3.30* -0.73* -1.54* 0.29 C2 0.12* 8.003*10−3 0.53 0.46 -0.30 0.70* 0.10 AB -0.30* -1.000*10−2* -1.34* -0.91 0.061 0.86* -0.10 AC 0.12 -2.500*10−3 -0.090 0.34 -0.72* -1.08* 0.000 BC -0.13 0.000 2.82* 2.44* 0.79* -0.61* 0.025 Model Significant Significant Significant Significant Significant Significant Significant A Spinach juice, B Pomegranate juice, C Sugar, A2 Quadratic terms of spinach juice, B2 Quadratic terms of pomegranate juice, C2 Quadratic terms of sugar, AB Interactive term of spinach juice and pomegranate juice, AC Interactive term of spinach juice and sugar, BC Interactive term of pomegranate juice and sugar The values within the column of each attribute denoted with * are significant at p<0.01

tin concentration. The viscosity is determined conversion of pectic acid into pectinic acid, which by the pectin colloidal content. As the fruit ma- decreases the pH of the juice blend (Wisal et tures, the proto-pectin in the central lamina is al., 2013). It was shown by Rodbotten et al. converted to hydrocolloids which are soluble and (2009) that an increase in acidity decreases the easily disperse through the juice thus increas- consumer acceptance of juices. ing the viscosity as shown by Wojdylo, Teleszko and Oszmianski (2014). Gabsi, Trigui, Barring- Antioxidant activity: reducing power, ton, Helal and Taherian (2013) reported that the FRSA and metal chelation viscosity also increases with an increase in the amount of sugar. Table3 depicts the different antioxidant activities of the juice blend. Pomegranate had a posit- Acidity ive significant (p<0.01) effect on metal chelation (Fig1D), FRSA (Fig.1E) and reducing power As shown in Fig.1C, the acidity of the juice (Fig.1F) of the juice blend (Table5). In the blend increases as the pomegranate increases case of spinach, as it increases, the metal chela- and had a significant (p<0.01) positive effect. tion, FRSA and reducing power also increases. This might be due to the acidic nature of Sugar had no significant effect on these proper- pomegranate. On the other hand, spinach is ties of the juice blend. The increase in antiox- basic in nature and thus the acidity decreases idant activity might be due to the presence of a as the concentration of spinach microgreen in- high amount of antioxidants in the microgreen creases (Table3). Sugar has no significant effect based juice blend. It is reported by Raghav- on acidity. Therefore, the interactive effect of endra, Araveeti, Raghuveer Yadav, Sudharshan pomegranate and spinach microgreen is negative Raju and Siva Kumar (2013) that the reductones in the case of acidity. It is reported by Kamol, present in vegetables exert antioxidant action by Howlader, Dhar and Aklimuzzaman (2014) that breaking the free radical chain or by donating a the acidity is directly dependent on the matur- hydrogen atom. It is reported by Viuda-Martos ity of fruit. The premature fruit has maximum et al. (2011) that pomegranate contains certain acidity while mature fruit has minimum acidity. types of compounds such as punicalagin isomers, It was claimed that acidity increases due to the ellegic acid derivatives and anthocyanin which

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Table 6: Optimization of microgreen based blend

Experiments Predicted values Experimental values Color and Appearance (%) 8.49a 7.7±0.20b Aroma (%) 8.57a 7.6±0.15b Taste (%) 7.01a 6.9±0.25b Consistency (%) 7.92a 8.0±0.08a Mouth feel (%) 7.23b 7.6±0.25a Over all acceptability (%) 7.31b 7.6±0.28a TSS (o Brix) 12.10a 11.6±0.57a Acidity (%) 0.33a 0.35±0.02a Metal chelation (%) 71.44a 72.0±0.57a FRSA (%) 81.07b 82.0±0.28a Reducing power (%) 78.88a 79.0±0.33a Viscosity (Pa sec) 78.86a 76.7±0.41b Sedimentation (%) 18.40a 18.3±0.57a The experimental values are presented as Mean ± Standard deviation The values represented with different superscripts differ significantly at p < 0.05

Table 7: Nutritional composition of fruits and spinach microgreen juice

Nutrients Spinach Microgreen Pineapple Pomegranate Microgreen blend Protein (%) 2.45±0.06b 0.45±0.04d 1.86±0.16c 3.62±0.18a Moisture (%) 94.2±0.20a 83.6±0.05c 86.1±0.05b 80.05±0.09d Ash (%) 1.13±0.03a 0.86±0.09c 0.46±0.07d 1.02±0.05b Potassium (mg) 161.0±0.03b 99.2±0.32d 180±0.08a 156.03±3.0c Sodium (mg) 78.02±0.03a 5.06±0.12c 4.0±0.32d 37.0±1.0b Total phenols (GAE g−1) 0.95±0.06d 6.45±0.35a 3.34±0.11c 5.07±0.01b Total carotenoids (µg 100g−1) 15.08±0.05a 13.04±0.16b 8.5±0.22d 11.38±0.53c Vitamin C (mg) 11.8±0.0080c 25.0±0.23a 11.3±0.14d 21.30±0.79b Reducing Power (%) 48.86±0.15c 39.0±0.25d 59.4±0.26b 76.33±4.40a Metal chelation activity (% inhibition) 39.59±0.36c 22.5±0.42d 47.1±0.52b 72.07±0.57a Free radical scavenging activity (% inhibition) 43.07±0.14c 40.17±0.09d 62.3±0.45b 82.01±0.28a Iron (mg) 4.03±0.02a 0.22±0.05d 0.60±0.13c 3.10±0.04b The values are presented as Mean ± Standard deviation The values represented with different superscripts differ significantly at p < 0.05

IJFS February 2021 Volume 10 pages SI41–SI56 SI52 Sharma et al. have the potential to scavenge free radicals and The moisture content of the microgreen blend inhibit lipid oxidation. Baby spinach contains was found to be less than the individual values of most of the flavonoids, carotenoids, vitamin C, spinach microgreen, pineapple and pomegranate. vitamin E and β-carotene which possess anti- At the same time, the values of different prop- oxidant benefits and acts as anti-inflammatory erties such as reducing power, metal chelation agents (Nemadodzi, 2015). It has been reported activity and free radical scavenging activity were by Arfan et al. (2013) that the spinach leaves found to be higher in the blend as compared to have para-coumaric acid derivatives which pos- the ones found for spinach microgreen, pineapple sess strong antioxidant activity. and pomegranate. The values obtained for total phenols, carotenoids and vitamin C were also 3.5 Optimization of microgreen higher which could be beneficial for reducing in- flammations and have a potent role in dealing based blend with oxidative stress due to free radical damage (Janovskáet al., 2010). According to the study Based on the sensory scores of the juice blend, conducted by Xiao et al. (2014), microgreens con- rheological properties and antioxidant proper- tain high concentrations of phytonutrients like ties, the optimum formulation for the develop- carotenoids and vitamin C. It was reported by ment of the microgreen based blend was selec- Leahu, Damian, Carpiuc, Oroian and Avramiuc ted using RSM. The optimized formulation was (2013) that vitamin C content decreases with the selected as 17.26 mL 100 mL−1 spinach micro- maturation of fruit thus it is highest at the be- green juice, 57.07 mL 100 mL−1 pomegranate ginning of ripening and minimum at full matur- juice, 1.01 g 100 g−1 sugar and 24.66 mL 100 ity. The polyphenol content increases with fruit mL−1 pineapple juice. The predicted and ex- maturation. It was also shown by Lawless et al. perimental values for the optimized product are (2012) that blending three juices enhances nut- shown in Table6. The obtained experimental raceutical properties and also consumer accept- values showed little difference with the predic- ance of the blend. tions. The use of spinach microgreen and pineapple that contains the anti-inflammatory potential was kept as the highest priority, with highest weights. The desirability of the selected formulation is 0.821. 4 Conclusions 3.6 Nutritional composition of microgreen based beverage Response surface methodology was used to op- The nutritional composition of the optimized mi- timize a microgreen and fruit based beverage. crogreen based beverage is shown in Table7. The Spinach microgreen was compared to its ma- nutritional composition of spinach microgreen is ture counterpart and found to have a better higher than the nutritional value of mature spin- nutritional and phytochemical profile, and thus ach. This is because the microgreens contain was selected to prepare the beverage. The op- high amounts of bioactive compounds like vit- timum conditions for preparation of the micro- amins, minerals and antioxidants as compared to green and fruit based beverage on a sensory mature greens Kou et al. (2013). The amount of and physicochemical basis were 17.26 mL 100 total carotenoids and ascorbic acid found in the mL−1 spinach microgreen juice, 57.07 mL 100 microgreens were in accordance with the values mL−1 pomegranate juice, 1.01 g 100 g−1 sugar obtained by Bergquist (2006). The values of all and 24.66 mL 100 mL−1 pineapple juice. The the nutrients present in pomegranate and pine- optimized beverage had overall better physico- apple were found to be in accordance with the chemical properties as compared to fruits (pine- values obtained by Viuda-Martos et al. (2011), apple and pomegranate) and spinach microgreen Kumar et al. (2016) and Debnath et al. (2012). juice alone.

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pacted by storage temperature, packaging ﬁlm, and chlorine wash treatment. LWT- Food Science and Technology, 55 (2), 551– 558. doi:10.1016/j.lwt.2013.09.009 Yadav, R. K., Kalia, P., Kumar, R. & Jain, V. (2013). Antioxidant and nutritional activity studies of green leafy vegetables. Int J Agric Food Sci Tech, 4, 707–12.

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Chickpea Protein Isolation, Characterization and Application in Muﬃn Enrichment

Sobhy A. El Sohaimya, c*, Marageta A. Brennanb, Amira M. G. Darwisha, and Charles S. Brennanb

a Food Technology Department, Arid Lands Cultivation Research Institute, City of Scientiﬁc Research and Technological Applications, 21934 Alexandria, Egypt. b Wine, Food and Molecular Biosciences Department, Faculty of Agriculture and Life Sciences, Canterbury, New Zealand c Department of Technology and Organization of Public Catering, Institute of Sport, Tourism and Service, South Ural State University, 454080 Chelyabinsk, Russian Federation *Corresponding author [email protected] Tel.: +2034593420 Fax: +2034593423

Received: 31 August 2018; Published online: 24 February 2021

Abstract

The aim of this study was to enhance the nutritional value and the functional characteristics of muffins by enriching with chickpea protein isolate, while keeping their rheological characteristics. Chickpea Protein isolate (CPI) was prepared by alkaline solubilization (pH 11), followed by isoelectric precipitation at pH 4.5. SDS-PAGE revealed three subunits with molecular weights of 47, 30 and 85 kDa; representing the globulin fractions, legumin-like and vicilin-like proteins. Maximum protein solubility (83.32%) was obtained at pH 11. CPI exhibited an emulsifying activity index of 25.17 m2 g−1, emulsion stability index of 14.09 min. The foaming capacity and stability were 62% and 94.49%, respectively. Water and oil absorption were 3.65 and 2.30 mL g−1, respectively. CPI was added to muffin batter at 0, 2.5, 5, 7.5 and 10%. CPI fortified muffins showed reduction in moisture content, which influenced texture profile analysis through increasing hardness, gumminess and chewiness values. Additionally, both protein content and protein digestibility of muffins increased to 22.2 and 94.08%, respectively. CPI-enriched muffins were darker (lower L) with yellowish crumbs (higher b). Finally, preliminary sensory evaluation showed high consumer acceptance for CPI-enriched muffins. Keywords: Chickpea proteins isolate; Enriched muffins; In-Vitro Protein digestibility; Colour analysis; Texture Profile Analysis; Sensory evaluation

1 Introduction appreciate improvements in product flavor but they do not neglect their health benefits (Valmor- ida & Castillo-Israel, 2018; Wardy et al., 2018). Consumers throughout the world enjoy baked Chickpeas (Cicer arietinum L.) are an oldworld food products, especially muffins, due to their or- pulse and were first grown in the Levant and ganoleptic characteristics (Gao, Brennan, Mason ancient Egypt. They have a nutlike flavor and & Brennan, 2016, 2017). Their high-level con- are used to complement grains (such as whole sumption makes them useful as potential carri- grains); to form a complete protein. The protein ers of bioactive compounds. Recently, consumers

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a5 SI58 El-Sohaimy et al. quality of legumes such as chickpeas are signific- ingredients were obtained from local markets loc- antly improved by heat treatment since heat des- ated in Christchurch, New Zealand. troys and/or inactivates anti-nutritional factors. Therefore, this might be important for vegans, individuals adhering to variations of plant based diets or low socio-economic individuals (O’Neil, Nicklas & Fulgoni III, 2014; Wallace, Murray & 2.2 Preparation of defatted Zelman, 2016). Like other legumes, chickpea’s albumins and globulins represent the two major chickpea flour protein fractions. The albumin fraction consti- tutes up to 15-25% whereas the globulins, rep- Defatting of chickpea flour was carried out ac- resented mainly by vicinin and legumin, reach up cording to Folch, Lees and Stanley (1957) with to 60-80% of the extractable proteins. Albumins some modifications. The chickpea samples were display a higher nutritive value due to their high homogenized in chloroform/methanol (v/v; 2/1); content of lysine and sulfur amino acids. Chick- the final volume was 20 fold the sample volume pea proteins are appreciated due to their high (1 g in 20 ml of solvent mixture). After disper- biological value, well balanced amino acid con- sion, the whole mixture was agitated for 60 min tent and low content of anti-nutritional factors. in an orbital shaker at room temperature. Then, However, there have been concerns about chick- the homogenate was filtered with a folded filter pea protein isolates due to their low fat con- paper to recover the liquid phase. Finally, the tent among other reasons (Aloweidat, 2014; Car- chickpea flour was dried at 43oC/ 36 h in a hot bonaro, Cappelloni, Nicoli, Lucarini & Carno- air flow. vale, 1997). Earlier researchers have investigated the physicochemical properties of chickpea protein isolates and their use in food enrichment as a dietetic alternative for individuals with special caloric or metabolic requirements (Aguilar & Vélez-Ruiz, 2016). Herein, this study aims to en- 2.3 Preparation of chickpea rich muffins with chickpea isolate proteins to po- protein isolate tentially increase their nutritional and functional qualities while preserving rheological characteristics. The chickpea protein isolate was prepared according to Chang, Alli, Konishi and Ziomek (2011), El-Sohaimy, Sitohy and El-Masry (2007). 2 Materials and Methods Fifty grams of defatted chickpea flour were sus- pended in 1000 mL deionized water (1:20, w/v), 2.1 Raw materials the pH values varied in the range of 3.0 to 12.0 using 0.1 N NaOH and 0.1 N HCl. The suspen- Organic chickpea flour (Cicer arietinum L.) (aka sions at different pH values were stirred for 1 h Besan flour, ACO, Australia); 25% protein, 4.5% to assess optimum solubility. The soluble isolate fat and 9.7% moisture. The ingredients used for fractions (at the desired pH) were centrifuged at muffin preparation are: wheat flour (Chantal, 6,000 x g for 30 min 20oC. The supernatant was NZ); 10.5% protein, 1.4% fat and 70.1% carbo- collected and acidified to pH values ranging from hydrates, skimmed milk powder (Go Milk, NZ); 1 to 6 to facilitate protein precipitation and de- 8.3% protein, 0.3 % fat, 13.8% carbohydrates, termine the isoelectric point. Precipitates were 125mg sodium and 300 mg calcium, margar- then centrifuged at 10,000 x g for 45 min at 4oC. ine from (Anchor, NZ); protein <1.0 g, fat 2g, The precipitated protein fractions were collec- carbohydrates <1g and sodium 24 mg, sugar ted, neutralized and freeze dried. The total pro- (Chelsea White sugar NZ), baking powder (Ed- tein content in the isolates was determined by monds, NZ), and salt (Essentials, Australia). All Kjeldahl method.

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2.4 Characterization of chickpea A protein solubility curve was constructed by us- protein isolate ing the average of soluble protein percentage values calculated at each pH value. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Functional properties of protein (SDS-PAGE) isolate The protein isolate was dissolved in aqueous solu- Emulsifying activity index (EAI) and tion at six different concentrations (0.015, 0.03, emulsion stability index (ESI) 0.062, 0.125, 0.5 and 1 mg mL−1) and applied EAI and ESI were measured using the method to the gel for better resolution of the bands. of Pearce and Kinsella (1978) with some modi- SDS-PAGE was carried out by the technique re- fications. Fifteen mL of 1% neutralized pro- ported by Laemmli (1970); using 4% stacking tein solution was mixed with 5 ml of commercial gel and 12% separating gel. Sample solutions sunflower oil. The mixture was homogenized at (20 µL) were prepared by dissolving 10 mg of 7,500 rpm for 1 min, using homogenizer (MZIP freeze dried protein extract in 1 mL sample buf- Model 114, China). Then, 50 µL aliquots were fer [distilled water, 0.5 M Tri HCl (pH 6.8), gly- taken from emulsions at 0 and 10 min from the cerol, SDS (10%), bromophenol blue (1%) and bottom of the tube and mixed with 10 mL of β-mercaptoethanol]. The samples were heated 0.1% sodium dodecyl sulphate (SDS) (1:200 di- at 98 oC for 10 min, then applied to the sample lution). The absorbance of the diluted solutions wells. The standard protein marker (260, 160, was measured at 500 nm immediately after emul- 110, 80, 60, 50, 40, 30, 20, 15, 10 and 3.5 KDa) sion formation (A ) and at 10 min (A ). EAI (Bio-Rad Hercules, USA) was used for molecu- 0 10 and ESI were calculated using the following equa- lar weight estimation. Electrophoretic migration tions: was monitored at constant current (14 mA/gel) m2 2T × F × A EAI = 0 (2) for 1.5 to 2 h. Gels were fixed with a fixing solu- g C × θ × 10, 000 tion [water/methanol/acetic acid, 700: 200: 100 ∆t mL] for 30 min and then stained with commassie ESI = A0 × (3) brilliant blue R-250 for 1 h. The stained gels ∆A were destained by frequent change of the fixing ∆A = A0 − A10 and ∆t = 10min (4) solution. Where: T= 2.303; F: dilution factor (200); A0: absorbance measured at 500 nm immediately Protein solubility after emulsion form ation; At: absorbance measured at 500 nm after 10 min of emulsion forma- Chickpea protein isolate solubility (5% suspen- tion c: protein concentration (0.01 g/mL) and θ: sion) was determined at pH values ranging from dispersed phase (oil) volume fraction (15). 1.0 to 12.0 according to Klompong, Benjakul, Kantachote and Shahidi (2007). For better solu- Foaming capacity (FC) and foaming bilization, the suspensions were stirred at room stability (FS) temperature for 1 h, using a magnetic stirrer. The pH values were adjusted using HCl (0.1 N) FC and FS were assessed according to the and NaOH (0.1 N). The suspensions at different method described by Tsutsui (1988) with some pH values were centrifuged at 6,000 x g for 30 modifications. The protein solution was agitated min. The total protein was determined in the in a blender at high speed (Breville, platinum, supernatants by Kjeldahl method. Protein sol- China) for 5 min and then transferred into gradu- ubility (PS) was calculated using the following ated cylinders. Foam capacity was calculated ac- equation. Samples were tested in triplicate. cording to following equation: Protein content in supernatant Vafter agitation − Vprior agitation PS(%) = × 100 FC(%) = × 100 Total protein content in sample Vprior agitation (1) (5)

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Similarly, FS value was determined, however 2.6 Muffin characteristics samples were allowed to stand at room temperature for 30 min and the residual foam volume Muffin properties (moisture content, height, (VResidual foam) was calculated according to the specific volume and colour) were assessed fol- following equation: lowing (Rahman, Hiregoudar, Veeranagouda, Ramachandra et al., 2015) procedures. Moisture V FS(%) = Residual foam × 100 (6) content of different muffin recipes was determ- VTotal foam ined according to AOAC (1990). Height was measured with a digital caliper, from the bottom to the highest point of the muffin. Spe- Water and oil absorption cific volume was determined by milletseed displacement method and was expressed as spe- The water/oil absorption capacity of chickpea cific volume (cm3 g−1). Colour parameters were protein isolate was determined by the method determined for muffin crust and crumb via Ul- of Chandra and Samsher (2013). One gram of trascan VIS Hunter Lab (MiniScan XE Plus, the isolate was mixed with 10 mL of distilled Model 45/0-L, Hunter Associates Inc, Reston, water/sunflower oil (specific gravity: 0.88) and VA, USA). Values were expressed by Hunter (L, allowed to stand at ambient temperature (30± o a, and b) values which correspond, respectively, 2 C) for 30 min, then was centrifuged at 3,500 to: value of lightness (0-100 representing dark x g for 30 min. Water/oil absorption (WOA) in to light), value of redness and greenness degree mg/l was calculated according to the equation: (higher positive indicating more red) and value of yellowness and blueness degree (higher value WOA = V − V (7) water (oil) Initial supernatant indicating more yellow).

2.5 Batter and muffin preparation 2.7 Protein content and In-Vitro Four muffin batter formulations were prepared protein digestibility of muffins by replacing a percent of the wheat flour with chickpea protein isolate (CPI) according to Rah- Total protein content of enriched muffins was de- man, Hiregoudar, Veeranagouda, Ramachandra termined by Kjeldahl method as described in et al. (2015) with some modifications. The AOAC (1990). One g of muffin sample was samples were identified as [control, M1 (CPI 2.5), placed into a digestion flask, along with 15 mL M2 (CPI 5), M3 (CPI 7.5) and M4 (CPI 10)]. of concentrated sulfuric acid (H2SO4). Addition- The recipes used for different muffin preparations ally, seven grams of potassium sulfate and a cata- were exhibited in (w/w %) as shown in table1. lyst, usually copper, were added to the flask. The The ingredients were weighed using a kern 572 mixture was transferred to a digestion tube and balance (Scout Pro SP602, OHAUS Corpora- boiled at 400oC using a heating block until form- tions, USA). Egg and margarine were mixed in ation of white fumes, then heating was continued a laboratory scale kitchen mixer (Kitchen aid, for about 60-90 min. The tube was cooled then St. Joseph, USA) at speed 4 then speed 8 for 10 water (250 mL) was cautiously added. The pH and 50 s, respectively. Flour, sugar, salt, milk of the mixture was raised using sodium hydrox- powder and water were mixed at speed 2 for 10 ide (45% NaOH solution); this converts the am- + s, then speed 8 for 50 s. Forty-five grams of bat- monium (NH4 ) ions, which are dissolved in the ter was filled into paper cups in a muffin pan and liquid, to the ammonia gas (NH3). The nitrogen baked in the oven (MIWE condo) at 180oC for has been separating away from the digestion mix- 20 min. Baked muffins were left to cool at room ture by distilling the ammonia (converting it to a temperature for 1 h in order to avoid moisture volatile gas, by raising the temperature to boiling condensation on their undersurface, and finally point). The distilled vapor has been trapped in packed in polypropylene bags and stored in a dry a special trapping solution of about 15 mL HCl environment prior to analysis. in 70 mL of water, and then the trapping flask

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Table 1: Chickpea protein enriched muﬃn batter recipes (CPI, Chickpea Protein Isolate)

Ingredients (w/w)% Control M1 (CPI 2.5) M2 (CPI 5) M3 (CPI 7) M4 (CPI 10) Wheat ﬂour 32.94 32.12 31.29 30.47 29.65 Chickpea protein isolate 0.00 0.82 1.65 2.47 3.29 White sugar 19.70 19.70 19.70 19.70 19.70 Salt 0.40 0.40 0.40 0.40 0.40 Baking powder 1.66 1.66 1.66 1.66 1.66 Fat (margarine) 16.46 16.46 16.46 16.46 16.46 Skimmed milk powder 2.49 2.49 2.49 2.49 2.49 Liquid whole eggs 9.89 9.89 9.89 9.89 9.89 Water 16.46 16.46 16.46 16.46 16.46

was removed. As the ammonia dissolves in the 2.8 Texture Profile Analysis acid trapping solution, it neutralizes some of the (TPA) of muffins present HCl. The excess HCl was then back titrated with a standard NaOH. The indicator dye The texture profile analysis was carried out using was added to the acid/ammonia trapping solu- texture analyzer (TA/TX-plus texture analyzer; tion. In this way the amount of ammonia dis- Texture analyzer, Stable Micro system, Surrey, tilled off from the digestive solution could be cal- UK) equipped with a 5-kg load cell. Exponent culated; this amount corresponds to the nitrogen software was used for testing procedures, present- content of the protein. The volume of sodium ation formats and data analysis to provide the hydroxide solution was noted, and the nitrogen most powerful and flexible testing analysis solu- was calculated by the following equation: tion available. The muffin samples were placed at the center of a heavy-duty platform (HD P/90), (ml standard acid - ml blank) × N of acid × 1.4007 %N = (8) and subjected to compression (50%) using a 75 weight of sample in grams mm diameter flat aluminum probe (P/75) at test speed of 1 mm/s. Firmness is the maximum peak %P = %NX5.7 (9) force during the first compression cycle. Springi- ness (the height that the muffin sample recovered In-vitro protein digestibility was carried out during the time elapsed between the end of the for CPI-enriched muffins by the multienzyme first compression and the start of the second one) method of Bodwell, Satterlee and Hackler (1980), and cohesiveness (the ratio of the peak area dur- Carbonaro et al. (1997). Porcine pancreatic tryp- ing the second compression to the one during the sin (type IX, 15 310 units/mg protein), bovine first compression) were calculated from the force pancreatic chymotrypsin (type II, 48 units/mg time curve (Bourne, 2002). of solid), porcine intestinal peptidase (P-7500, 115 units/g of solid) and bacterial protease (type XIV, 4.4 units /mg of solid) (Sigma-Aldrich, Ger- 2.9 Sensory evaluation many) were employed for the enzymatic digestion. In-vitro protein digestibility was calculated Ten well trained panelists from staff members of according to the equation: Food Technology Department, City of Scientific Research, and Technological Applications, Alex- Y = 234.84 − 22.56X (10) andria, Egypt carried out preliminary sensory evaluation of enriched muffins. Samples were Where, Y is the In-vitro digestibility of protein randomly assigned to each panelist. The pan- (%) and X is the pH of the suspension after 20 elists were asked to evaluate each sample: shape, min of digestion. mouth feel, flavour, crumb texture, crumb col-

IJFS February 2021 Volume 10 pages SI57–SI71 SI62 El-Sohaimy et al. our, crust colour and crust texture, through a concentrations aid the confirmation of band pos- nine point hedonic scale according to (Ihekoronye itions with increased intensity at higher concen- & Ngoddy, 1985). The ratings are: Dislike ex- trations. tremely (1), Dislike very much (2), Dislike moderately (3), Dislike slightly (4), Neither like nor Proteins solubility dislike (5) Like slightly (6) Like moderately (7), Like very much (8) and Like extremely (9). The solubility of isolated proteins at different pH values is presented in figure2. A sharp minimum 2.10 Statistical analysis solubility (24.92 and 28.65 %) was observed at acidic pH values (4 and 5). On the other hand, Statistical factorial analysis was performed us- the protein isolate showed the highest protein sol- ing analytical software SPSS® 13.0 (Statistical ubility at pH 11 (83.32 %). This observation il- Package for the Social Sciences, 2005). Differ- lustrates that the major isolated chickpea pro- ences were considered significant at P < 0.05. teins are acidic and are soluble in alkaline medium. The protein solubility profile is similar to those reported for several legume proteins in 3 Results and Discussion earlier studies (Carbonaro et al., 1997; Liu, Hung & Bennett, 2008). These results suggest that al- 3.1 Chickpea protein isolate kaline medium, pH 11, is the optimum pH for average yield solubilization of most chickpea proteins.

Protein isolates from chickpea defatted flour were prepared in two steps: the first was the solubil- 3.3 Functional properties of ization and extraction of protein from chickpea protein isolate flour at alkaline pH. The optimum pH for the extraction of maximum amount of protein was 11; Functionality is any property of a food ingredi- maximum chickpea protein extraction rate was ent, except its nutritional values, that has a great 80 %. The protein was precipitated at isoelectric impact on its utilization. Chickpea protein isol- point (pH 4.5), which recovered maximum sol- ate functional properties are presented in table uble protein (78%), as described by El-Sohaimy 2. et al. (2007). Extraction of chickpea protein reached a recovery percent of 82.94% yield. Emulsifying activity index (EAI) and emulsion stability index (ESI) 3.2 Characterization of chickpea protein isolate EAI and ESI were determined for the chickpea protein isolate to support its applications Protein profile in food industry. Table2 shows the EAI and ESI of chickpea protein isolate. Chickpea pro- Figure1 illustrates the SDS-PAGE profiles of tein isolate exhibited EAI and ESI values of six different concentrations of chickpea protein 25.17±0.07 m2/ g−1 and 14.09±0.40 min, re- isolate (0.015, 0.03, 0.062, 0.125, 0.5 and 1 mg spectively. Several studies have reported the ml−1). SDS-PAGE gel analysis revealed that the emulsifying properties of chickpea protein isol- chickpea protein profiles were composed mainly ate (Alvarez, Cuesta, Herranz & Canet, 2017; of three bands; the major protein subunit (MW Ladjal-Ettoumi, Boudries, Chibane & Romero, 47 kDa), followed by a 30 KDa protein and fi- 2016). To form emulsions, proteins migrate nally an 85 kDa protein. Similar observation to the oil-water interface and re-align to al- was reported by Papalamprou, Doxastakis and low positioning of hydrophobic groups towards Kiosseoglou (2010), who stated that these pro- the oil phase and hydrophilic groups towards tein constituents belong to the globulin fractions, the aqueous phase. Reducing interfacial tension legumin like and vicilin like proteins. Different between oil and water phases enables emulsion

IJFS February 2021 Volume 10 pages SI57–SI71 Characterization of Chickpea Protein Enriched Muﬃn SI63

Figure 1: SDS-PAGE for Chickpea Protein Isolate Lane M: protein marker; Lane 1: 0.015 mg ml−1; lane 2: 0.03 mg mL−1; lane 3: 0.062 mg mL−1; lane 4: 0.125 mg mL−1; lane 5: 0.5 mg mL−1and lane 6: 1mg mL−1.

Table 2: Chickpea protein isolate functional properties

Parameter Unit Value Emulsifying activity index (EAI) m2g−1 25.17±0.07 Emulsion stability index (ESI) min 14.09±0.40 Foaming capacity (FC) % 62.00±2.83 Foaming stability (FS) % 94.49±1.67 Water absorption mlg−1 3.65±0.07 Oil absorption mlg−1 2.30±0.14 Data presented as mean±SD (samples were run in duplicate)

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Figure 2: Chickpea protein isolate solubility proﬁle. Data presented as the mean±SD value (samples were run in triplicate)

droplets to form, which subsequently leads to Water and oil absorption higher emulsion stability (Johnston, Nickerson & Low, 2015). The water/oil absorption properties of chickpea protein isolate are shown in table2. The isolated protein showed water and oil absorption of 3.65±0.07 and 2.30±0.14 mL/g respectively. These values are similar to those previously reported by Aloweidat (2014), who also reported Foaming properties (Foaming capacity that oil absorption capacity of protein is par- and foaming stability) tially related to the physical confinement of oil by means of the protein matrix. Therefore, the source of the protein might be important. Foam capacity and foam stability of chickpea protein isolate are shown in table2. The protein isolate showed foam capacity of 62.00±2.83 3.4 Characteristics of muffins %, which might be related to the presence of enriched with chickpea globulin fractions, which can encapsulate and re- protein tain air. Therefore, rapid migration, unfolding and rearranging of the air-water interface are ne- The moisture content, colour, height and volume cessary to exhibit good foam capacity (Alleoni, properties of baked CPI-enriched muffins are il- 2006). On the other hand, chickpea showed re- lustrated in tables3 and4. Enrichment of latively high foaming stability (94.49±1.67 %), muffins with chickpea protein caused a reduction based on air retaining, which could support in moisture content of fortified muffins com- its recommendation in food industries, such as pared to control, and this reduction correlates bakery products and ice creams. Similar foaming with the increase in CPI concentration. How- properties of chickpea proteins have been repor- ever, this decrease was not significant in the first ted earlier by Boye et al. (2010). two blends (CPI 2.5 and CPI 5.0%). Rahman,

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Table 3: Properties of chickpea protein enriched muﬃns

Sample Moisture Height SP. Volume (%) (mm) (cm3g−1) Control 23.05±2.05a 42.11±1.80c 92.00±4.00a M1 (PI 2.5) 22.95±1.03a 43.42±1.69a 88.00±2.00c M2 (PI 5) 22.75±0.62a 42.75±0.51b 90.00±3.06b M3 (PI 7.5) 21.83±1.28b 43.08±0.25ab 92.00±2.00a M4 (PI 10) 21.41±1.02c 40.11±0.64d 33.00±1.16a Data presented as mean±SD (samples were run in triplicate), Mean in the same column followed by different superscript letters are significantly different (p>0.05)

Table 4: Colour properties of chickpea protein enriched muﬃns

L A b Sample Crust Crumb Crust Crumb Crust Crumb Control 64.03±0.85a 80.24±1.59b 16.92±0.75b 3.58±0.17c 45.24±0.87a 29.90±0.56e M1 (CPI 2.5) 62.23±2.13c 80.15±1.41b 17.65±0.81a 3.94±0.50bc 44.51±1.02b 31.06±0.94d M2 (CPI 5) 63.12±1.76b 80.74±2.24a 16.07±2.65c 4.11±0.08b 43.88±1.31c 32.83±0.42c M3 (CPI 7.5) 55.03±3.15e 78.51±1.09c 16.41±0.49c 4.85±0.06bc 41.07±2.09e 33.68±0.70b M4 (CPI 10) 56.95±2.42d 77.84±0.76d 16.79±0.63b 4.87±0.20a 42.54±2.12d 34.82±0.43a Data presented as mean±SD (samples were run in duplicate), Means in the same column followed by different superscript letters are significantly different (p>0.05) CPI= Chickpea protein isolate Colour parameters by Ultrascan VIS Hunter Lab (MiniScan XE Plus, Model 45/0-L, Hunter Associates Inc, Reston, VA, USA). L, a, and b represent: value of the lightness (0-100 representing dark to light), value of redness and greenness degree (higher positive indicating more red) and value of yellowness and bluenessdegree (higher value indicating more yellow) respectively.

Hiregoudar, Veeranagouda, C T et al. (2015) combinations. Crusts tended to be darker (lower reported similar behavior with increasing levels L values; 56.95±2.4), more reddish (higher val- of enrichment in muffin batter. There was no ues; 16.79±0.63) and yellowish (lower b values; significant variation in either the height or the 42.54±2.12) than crumbs. L (Lightness) results specific volumes of the four enrichment treat- illustrate a significant impact of protein enrich- ments in comparison with the control, except ment on the muffins. This is because the interac- for M1 (CPI 2.5%) which showed a decrease in tion between the ingredients during baking, pos- specific volume with a value close to control. sibly due to increasing of Millard browning reac- These results could help to improve muffin qual- tions concurrent with higher protein enrichment ity (height and volume) like control muffins that percent, subsequently resulted in darker muffins consumers are familiar with. This emphasizes in both crust and crumb. Similar observations that the enrichment of muffins with chickpea pro- have been recorded by Bhaduri (2013). Compar- tein didn’t negatively affect the physical proper- ing the redness degree to control, the CPI en- ties of the product. Colour characteristics of for- richment did not cause high variations in crumb tified muffins are presented in table4. The col- and crust despite their significance. The yellow our properties were more pronounced in the crust colour of CPI affected the crumb colour, b val- compared to the crumb for all parameters and all ues in the muffins increased with higher substi-

IJFS February 2021 Volume 10 pages SI57–SI71 SI66 El-Sohaimy et al. tutions of CPI, however, b values decreased for that starts crosslinking during batter prepara- the crust. This could be attributed to Millard tion. The reason for that is mixed networks reactions that might cause consumption of the which form with proteins during baking based on protein amino acids and reducing sugars to pro- hydrophobic interactions and S-S bonds, which duce a brown colour that diverts the tendency determine volume and texture (Deleu, Wilder- for lightness (to become darker) rather than yel- jans, Van Haesendonck, Brijs & Delcour, 2016; lowish. Generally, similar patterns were reported Deleu, Wilderjans, Vanhaesendonck, Brijs & Del- by Bhaduri (2013), (Rahman, Hiregoudar, Veer- cour, 2017). Moreover, diﬀerent types of pro- anagouda, C T et al., 2015) and (Wardy et al., teins can impact each other’s network forma- 2018). tion (Lambrecht, Rombouts, Nivelle & Delcour, 2017). 3.5 Protein content and In-Vitro Protein Digestibility 3.7 Sensory evaluation

Figure3 and4 exhibits the protein content The mean sensory score of chickpea protein for- and in vitro protein digestibility of CPI-enriched tified muffins is illustrated in table6. Muffin muffins. Figure (3) shows a significant increase in shape had a high acceptability score with up to protein content of enriched muffins compared to 10% of protein isolate (similar to the shape of control, as a function of CPI concentration. The control, 100% wheat flour). The mouth feel of increasing of protein content in enriched muffin the product showed significant differences among results, consequently, in increasing its nutritional control and all blends, with sensory score gradu- quality, due to the quality of chickpea protein, ally decreasing with higher CPI inclusion; the compared to the control muffins (89.47%). score in control was (like very much) while in The protein digestibility (%) of chickpea pro- 10 % CPI was (like moderately). The low score tein in muffins is represented in figure (4). It is at high level of protein isolate in muffins might directly proportional to the enrichment percent- be due to the higher value of gumminess and age of protein isolate with scores of 91.44, 92.97, chewiness of protein isolate compared with wheat 93.40 and 94.08% for M1, M2, M3 and M4 re- flour. The muffin samples (2.50, 7.50 and 10 spectively. Unlike other legume proteins, chick- % of protein isolate) showed a higher score in pea protein has been shown to have improved flavour, which resulted in good acceptance (like digestibility upon heating, which can be mainly very much) for panelists. All blends of protein ascribed to protein denaturation and inactivation isolate had a score of 8.50 to 7.50, in accordance of protease inhibitors (Carbonaro et al., 1997). with sensorial acceptance (like moderately). The supplemented muffins had a yellow reddish crust colour. It is clear from data, that crust colour of 3.6 Texture Profile Analysis fortified muffins in the ratio range (2.5% - 10% (TPA) of muffins CPI) had almost the same score (like very much), meaning that fortification of muffins with chick- Table5 illustrates the Texture Profile Analysis pea protein isolate didn’t negatively affect the (TPA) of CPI-enriched muffins showing: hard- crust colour. The same trend in crust colour was ness, cohesiveness, springiness, gumminess and obtained for crumb colour of fortified muffins. chewiness parameters. Hardness, springiness and No significant differences among control and all cohesiveness are crucial textural parameters for blends; sensory score was the same (Like very consumers (Shevkani & Singh, 2014). Chickpea much). The results presented in table6 show the protein isolate enrichment resulted in a signific- texture of crust and crumb, there are no significant increase in hardness, gumminess and chew- ant differences among control and all blends from iness of produced muffins as a function of increas- 2.5% to 10% CPI, which means that wheat flour ing levels of protein isolate substitution. These can be substituted with chickpea protein isolates results could be referred to increased protein with up to 10 % without any negative effect on

IJFS February 2021 Volume 10 pages SI57–SI71 Characterization of Chickpea Protein Enriched Muﬃn SI67

Figure 3: Protein content of enriched muffins. Data presented as mean±SD (samples were run in triplicate), a,bMeans followed by different superscript letters differ significantly (p>0.05)

Figure 4: In Vitro protein digestibility of enriched muffins. Data presented as mean±SD (samples were run in triplicate), i,jMeans followed by different superscript letters differ significantly (p>0.05)

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Table 5: Texture Proﬁle Analysis (TPA) of chickpea protein enriched muﬃns

Sample Hardness Cohesiveness Springiness Gumminess Chewiness (g) (cm) (N) (g cm) Control 176.67±19.66e 1.05±0.14a 1.02±0.08a 183.11±15.99e 187.05±20.44e M1 (CPI 2.5%) 180.00±10.00d 1.07±0.06a 1.05±0.06a 187.73±3.21d 196.16±8.71d M2 (CPI 5%) 200.00±10.00c 1.05±0.01a 1.00±0.00a 205.00±7.07c 205.00±7.07c M3 (CPI 7.5%) 213.33±11.55b 0.95±0.06a 1.00±0.00a 210.00±14.14b 210.00±14.14b M4 (CPI 10%) 296.67±32.15a 1.11±0.11a 1.00±0.00a 315.00±7.07a 315.00±7.07a Data presented as mean±SD (samples were run in triplicate), Means in the same column followed by different superscript letters are significantly different (p>0.05) CPI= Chickpea protein isolate

Table 6: Sensory evaluation of chickpea protein enriched muﬃns

Organoleptic M1 M2 M3 M4 propertiesControl (2.5% CPI) (5% CPI) (7.5% CPI) (10% CPI) Shape 8.15a±0.63 8.08ab±0.73 8.00b±0.66 7.80ab±0.84 7.60ab±0.84 Mouth feel 8.40a±0.70 8.20ab±0.63 7.80abc±1.03 7.80bc±0.79 7.50bc±0.52 Flavor 8.50a±0.70 8.60a±0.70 8.30ab±0.73 8.00ab±0.67 7.50b±0.85 Crust colour 7.90ab±0.99 7.70ab±1.16 8.20a±0.73 7.70ab±0.74 7.45ab±0.95 Crust texture 7.80a±0.92 7.70a±0.82 7.90a±0.92 8.30a±0.73 8.07a±0.96 Crumb colour 8.70a±0.53 8.20a±0.79 8.10a±0.87 7.80a±0.63 8.04a±0.66 Crumb texture 8.90a±0.71 8.10a±0.74 7.70a±1.33 8.00a±0.47 8.36a±1.05 Overall acceptance 8.55a±0.49 8.15ab±0.69 7.73abc±0.97 7.79abc±0.76 7.47bc±0.33 Means in the same column followed by different superscript letters are significantly different (p>0.05) CPI= Chickpea protein isolate

the consumer perception of texture characterist- 4 Conclusion ics of muffins. The overall acceptance in control was 8.90±0.49 while in 10% protein isolate was The present study was designed to increase the 8.36±0.57. There is no reduction in overall ac- nutritional and rheological properties of muffins ceptance in all blends with chickpea protein isol- by adding chickpea protein isolate in different ate and a high organoleptic score (from like mod- blends (up to 10 %). Chickpea protein profile on erately to like very much). The fortification of SDS-PAGE revealed protein subunits with mo- muffins with up to 10% chickpea protein isolate lecular weights of (47, 30 and 85 kDa) in des- shows no negative effect on overall acceptance of cending order, which could belong to globulin the final product. These results agree with Her- fractions, legumin-like and vicilin-like protein. ranz, Canet, Jose Jimenez, Fuentes and Dolores Chickpea protein isolate (CPI) showed relatively Alvarez (2016) who reveled that fortification of high emulsifying activity index of 25.17 m2 g−1, muffins with chickpea flour resulted in a chickpea emulsion stability index 14.09 min, foaming ca- like taste which was not a driver of disliking for pacity 62% and foaming stability 94.49%. Wa- the panelists. ter and oil absorption scored 3.65 and 2.30 mL g−1, respectively. Chickpea protein isolate incorporation resulted in increases in hardness, gumminess and chewiness of baked muffins due to

IJFS February 2021 Volume 10 pages SI57–SI71 Characterization of Chickpea Protein Enriched Muffin SI69 increasing protein concentration. Sensory eval- Bhaduri, S. (2013). A comprehensive study on uation showed consumer acceptance of enriched physical properties of two gluten-free flour muffins where they achieved high scores. Since fortified muffins. Journal of Food Pro- consumers enjoy baked food products; the forti- cessing and Technology, 04. doi:10 . 4172 / fication of muffins with up to 10% chickpea pro- 2157-7110.1000251 tein isolate has no negative effect on the over- Bodwell, C., Satterlee, L. & Hackler, L. (1980). all acceptance of the final product. Our results Protein digestibility of the same protein encourage the employment of chickpea protein preparations by human and rat assays and isolate as a vehicle to produce higher nutritional by in vitro enzymic digestion methods1 2. value products. More research is going on to The American journal of clinical nutrition, achieve the maximum replacement of wheat flour 33, 677–86. doi:10.1093/ajcn/33.3.677 with protein isolate and chickpea flour in muffins Bourne, M. (2002). Food texture and viscosity : to develop a high nutritionally valued and glu- Concept and measurement / m.c. bourne. tenfree muffin that does not affect the rheological Boye, J., Aksay, S., Roufik, S., Ribéreau, S., properties Mondor, M., Farnworth, E. & Rajamo- hamed, S. (2010). Comparison of the func- Acknowledgements tional properties of pea, chickpea and lentil protein concentrates processed us- Authors appreciate the support provided by, ing ultrafiltration and isoelectric precipit- Wine, Food and Molecular Biosciences Depart- ation techniques. Food Research Interna- ment, Faculty of Agriculture and Life Sciences, tional, 43 (2), 537–546. Molecular, Func- Canterbury, New Zealand, to achieve this work. tional and Processing Characteristics of Whole Pulses and Pulse Fractions and their Emerging Food and Nutraceutical Applic- References ations. doi:https : / / doi . org / 10 . 1016 / j . foodres.2009.07.021 Aguilar, V. & Vélez-Ruiz,J. (2016). Character- Carbonaro, M., Cappelloni, M., Nicoli, S., Lucar- ization of two chickpea varieties and the ini, M. & Carnovale, E. (1997). Solubility- effect of cooking on their physico-chemical digestibility relationship of legume pro- and functional properties of flours. Journal teins. Journal of Agricultural and Food of Food Research, 5, 67. doi:10.5539/jfr. Chemistry, 45 (9), 3387–3394. doi:10.1021/ v5n5p67 jf970070y. eprint: https://doi.org/10.1021/ Alleoni, A. C. C. (2006). Albumen protein and jf970070y functional properties of gelation and foam- Chandra, S. & Samsher, a. (2013). Assessment ing. Scientia Agricola, 63, 291–298. doi:10. of functional properties of different flours. 1590/S0103-90162006000300013 African journal of agricultural research, 8, Aloweidat, M. Y. (2014). Growth performance 4849–4852. doi:10.5897/AJAR2013.6905 and yield components of five legume crops Chang, Y.-W., Alli, I., Konishi, Y. & Ziomek, under rain-fed conditions. E. (2011). Characterization of protein frac- Alvarez, M., Cuesta, F., Herranz, B. & Canet, tions from chickpea (cicer arietinum l.) W. (2017). Rheometric non-isothermal ge- and oat (avena sativa l.) seeds using pro- latinization kinetics of chickpea flour-based teomic techniques. Food Research Interna- gluten-free muffin batters with added bi- tional, 44 (9), 3094–3104. doi:10 . 1016 / j . opolymers. Foods, 6, 3. doi:10 . 3390 / foodres.2011.08.001 foods6010003 Deleu, L. J., Wilderjans, E., Van Haesendonck, AOAC. (1990). Official Methods of Analysis, I., Brijs, K. & Delcour, J. A. (2016). Pro- 15th ed. Association of Official Analytical tein network formation during pound cake Chemists. INC., Arlig-ton, Virginia, USA. making: The role of egg white proteins and wheat flour gliadins. Food Hydrocolloids,

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61, 409–414. doi:10.1016/j.foodhyd.2016. lysate of yellow stripe trevally (selaroides 05.001 leptolepis) as influenced by the degree of Deleu, L., Wilderjans, E., Vanhaesendonck, I., hydrolysis and enzyme type. Food Chem- Brijs, K. & Delcour, J. (2017). 15 n-labeling istry, 102 (4), 1317–1327. doi:10 . 1016 / j . of egg proteins for studying protein net- foodchem.2006.07.016 work formation during pound cake making. Ladjal-Ettoumi, Y., Boudries, H., Chibane, M. & Cereal Chemistry Journal, 94. doi:10.1094/ Romero, A. (2016). Pea, chickpea and len- CCHEM-07-16-0183-R til protein isolates: Physicochemical char- Folch, J., Lees, M. & Stanley, G. (1957). A simple acterization and emulsifying propertiesu. method for the isolation and purification Food Biophysics, 11 (1), 43–51. doi:10 . of total lipides from animal tissues. The 1007/s11483-015-9411-6 Journal of biological chemistry, 226, 497– Laemmli, U. K. (1970). Cleavage of struc- 509. tural proteins during assembly of head Gao, J., Brennan, M. A., Mason, S. L. & Bren- of bacteriophage-t4. Nature, 227 (5259), nan, C. S. (2016). Effect of sugar replace- 680+. doi:10.1038/227680a0 ment with stevianna and inulin on the Lambrecht, M. A., Rombouts, I., Nivelle, M. A. texture and predictive glycaemic response & Delcour, J. A. (2017). The role of wheat of muffins. International Journal of Food and egg constituents in the formation of a Science and Technology, 51 (9), 1979–1987. covalent and non-covalent protein network doi:10.1111/ijfs.13143 in fresh and cooked egg noodles. Journal of Gao, J., Brennan, M. A., Mason, S. L. & Bren- Food Science, 82 (1), 24–35. doi:10.1111/ nan, C. S. (2017). Effects of sugar substitu- 1750-3841.13558 tion with “stevianna” on the sensory char- Liu, L. H., Hung, T. V. & Bennett, L. (2008). acteristics of muffins. Journal of Food Qual- Extraction and characterization of chick- ity. doi:10.1155/2017/8636043 pea (cicer arietinum) albumin and globulin. Herranz, B., Canet, W., Jose Jimenez, M., Journal of Food Science, 73 (5), C299– Fuentes, R. & Dolores Alvarez, M. (2016). C305. doi:10.1111/j.1750-3841.2008.00773. Characterisation of chickpea flour-based x gluten-free batters and muffins with ad- O’Neil, E., Nicklas, A. & Fulgoni III, V. L. ded biopolymers: Rheological, physical and (2014). Chickpeas and hummus are associ- sensory properties. International Journal ated with better nutrient intake, diet qual- of Food Science and Technology, 51 (5), ity, and levels of some cardiovascular risk 1087–1098. doi:10.1111/ijfs.13092 factors: National health and nutrition ex- Ihekoronye, A. I. & Ngoddy, P. O. (1985). amination survey 2003-2010. Nutrition & Integrated food science and technology Food Sciences. for the tropics. (pp. 296–301). Macmil- Papalamprou, E. M., Doxastakis, G. I. & lan. Retrieved from http : / / www . Kiosseoglou, V. (2010). Chickpea protein gproxx . com / http : / / blogxd . info / isolates obtained by wet extraction as dspace / uk / integrated food science and emulsifying agents. Journal of the Science technology for the tropics.pdf of Food and Agriculture, 90 (2), 304–313. Johnston, S. P., Nickerson, M. T. & Low, N. H. doi:10.1002/jsfa.3816 (2015). The physicochemical properties of Pearce, K. N. & Kinsella, J. E. (1978). Emul- legume protein isolates and their ability sifying properties of proteins-evaluation of to stabilize oil-in-water emulsions with and a turbidimetric technique. Journal of Ag- without genipin. Journal of Food Science ricultural and Food Chemistry, 26 (3), 716– and Technology - MYSORE, 52 (7), 4135– 723. doi:10.1021/jf60217a041 4145. doi:10.1007/s13197-014-1523-3 Rahman, R., Hiregoudar, S., Veeranagouda, M., Klompong, V., Benjakul, S., Kantachote, D. & Ramachandra, C. T., Nidoni, U., Roopa, Shahidi, F. (2007). Antioxidative activity R. S., . . . Ganjyal, G. M. (2015). Effects and functional properties of protein hydro- of wheat grass powder incorporation on

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physiochemical properties of muffins. Inter- Science and Technology, 53 (1), 262–269. national Journal of Food Properties, 18 (4), doi:10.1111/ijfs.13582 785–795. doi:10 . 1080 / 10942912 . 2014 . 908389 Rahman, R., Hiregoudar, S., Veeranagouda, M., C T, R., Kammar, M., Nidoni, U. & R S, R. (2015). Physico-chemical, textural and sensory properties of muffins fortified with wheat grass powder. Karnataka J. Agric. Sci. 28, 79–82. Shevkani, K. & Singh, N. (2014). Influence of kidney bean, field pea and amaranth protein isolates on the characteristics of starch-based gluten-free muffins. Interna- tional Journal of Food Science and Techno- logy, 49 (10), 2237–2244. doi:10.1111/ijfs. 12537 El-Sohaimy, S., Sitohy, M. & El-Masry, R. (2007). Isolation and partial characterization of chickpea, lupine and lentil seed proteins. World Journal of Agricultural Sci- ences, 3, 123–129. Tsutsui, T. (1988). Functional-properties of heat-treated egg-yolk low-density lipopro- tein. Journal of Food Science, 53 (4), 1103– 1106. doi:10 . 1111 / j . 1365 - 2621 . 1988 . tb13539.x Valmorida, J. S. & Castillo-Israel, K. A. T. (2018). Application of plackett-burman experimental design in the development of muffin using adlay flour. In A. Al- baarri (Ed.), International symposium on food and agro-biodiversity (isfa) 2017 (Vol. 102). IOP Conference Series-Earth and Environmental Science. 2nd Interna- tional Symposium on Food and Agro- biodiversity (ISFA), Semarang, INDONE- SIA, SEP 26-27, 2017. doi:10.1088/1755- 1315/102/1/012081 Wallace, T. C., Murray, R. & Zelman, K. M. (2016). The nutritional value and health benefits of chickpeas and hummus. Nutri- ents, 8 (12). doi:10.3390/nu8120766 Wardy, W., Jack, A. R., Chonpracha, P., Alonso, J. R., King, J. M. & Prinyawiwatkul, W. (2018). Gluten-free muffins: Effects of sugar reduction and health benefit information on consumer liking, emotion, and pur- chase intent. International Journal of Food

IJFS February 2021 Volume 10 pages SI57–SI71 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI72–SI81

Nutritional Evaluation of Unripe Plantain, Moringa Seed and Defatted Sesame Seed Cookies

Mopelola A. Sodipoa*, Matthew O. Oluwamukomia, Zianab A. Oderindea, and Olugbenga O. Awolua

a Federal University of Technology, Akure, School of Agriculture and Agricultural Technology, Department of Food Science and Technology, Ondo State, Nigeria *Corresponding author [email protected] Tel.: +23480600326123

Received: 14 December 2018; Published online: 24 February 2021

Abstract

Cookies are widely consumed all over the world and can bring important nutrients, especially to children. Composite flours consisting of plantain, moringa seed and sesame seeds were used to produce cookies in this study. Response surface methodology was employed in setting up the cookie’s formulation. The samples with the best protein and fibre contents were sample F (71.34% plantain, 6.66% moringa and 22.00% sesame seed, with 14.08% protein and 3.02% fibre) and sample I (68.00% plantain, 10.00% moringa and 22.00% sesame seed, with 14.35% protein and 2.29% fibre). Cookies from 100% wheat flour were prepared as control. The protein, fat, ash and crude fibre contents of formulated cookies were significantly (p≤0.05) higher than control, whereas carbohydrate content was lower. There was no significant difference between the formulated cookies and the control in terms of overall acceptability, aroma and crunchiness. The formulated cookies showed higher biological value, net protein utilization, true digestibility, protein efficiency ratio, net protein ratio and protein retention efficiency than control, resulting in a significant increase in rat growth and development. The composite cookies containing unripe plantain, moringa seed and defatted sesame, at the blending ratio obtained in this study showed better nutritional quality than control cookies. These composite cookies, therefore, would be suitable for mitigating protein-energy malnutrition in children. Keywords: Unripe plantain; Cookies; Protein quality

1 Introduction is often referred to as cooking banana and is a chief staple food in many parts of Africa (Muller, Cookies have become one of the most desir- 1988). When plantain is matured but unripe, able snacks for both youth and elderly people it has a high amount of dietary fiber (8.82%) due to their low manufacturing cost, conveni- and resistant starch (16.2%), micronutrients and ence to eat, long shelf-life and ability to serve helps to reduce blood sugar level, besides it as a vehicle for important nutrients (Akubor is low in protein and fat (Ayodele & Godwin, & Ukwuru, 2003; Giarnetti, Paradiso, Caponio, 2010). Defatted sesame flour contains protein Summo & Pasqualone, 2015; Hooda & Jood, (55.70%), ash (9.83%), crude fiber (1.64%), total 2005; Pasqualone, Bianco & Paradiso, 2013).The carbohydrate (29.40%) and is high in sulphur- fruit of plantain is very similar to that of ba- containing amino acids (ElAdawy, 1997). Ses- nana except that it is less sweet and larger. It ame increases plasma gamma-tocopherol and en-

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a6 Cookies produced from unripe plantain, moringa seed and defatted sesame seed SI73 hances vitamin E activity which is known to pre- were the independent variables, while the de- vent cancer and heart disease (Bailey & Hui, pendent variable was the proximate composition 1996). Moringa pods, flowers, fruits, leaves, roots (Table2). The blends with the best protein and and seeds are all useful in food and medicine, as crude fibre contents were selected for subsequent they contain various valuable nutrients. Moringa analyses. seed contains essential amino acid and has a good fatty acid composition (Ogunsina, Radha & In- 2.4 Proximate Analysis of Flour drani, 2011). Considering the health benefits of unripe plantain, moringa and sesame flour, their Blends incorporation as composite blends in the prepar- Proximate analysis for moisture, protein (N x ation of cookies may enhance the nutritional and 6.25), fat, ash, and crude fiber of samples were health status of the consumers and reduce total determined according to AOAC (2005) proced- dependence on wheat flour. This research aimed ures. Carbohydrate content was calculated by at evaluating the nutritional benefits and sens- difference. ory quality of cookies produced from non-wheat- composite flour. 2.5 Preparation of cookies

2 Materials and Methods Cookies were prepared according to the method of AACC (2000) with some modifications in the 2.1 Materials recipe: flour 250 g, sugar 75 g, vegetable short- ening 100 g, baking powder 2.5 g, one egg, milk Matured unripe plantain (Musa AAB), moringa 30 g, flavour 5 g and 5 ml of water was added. seed (Moringa olifera), sesame seeds (Sesamum The dry ingredients (flour, sugar, flavour, milk indicum) and baking ingredients including but- and baking powder) were thoroughly manually ter, salt, sugar, flavour, baking powder and eggs mixed in a bowl for 3 min. Vegetable shorten- were purchased at Oja-Oba, Akure. Four-week- ing was added and mixed until uniform. Egg old weanling albino rats (Rattus norvegicus) were was then added, and the mixture kneaded. The obtained from the Department of Health Sciences dough was rolled and cut with a 50 mm diameter Animal House, Obafemi Awolowo University, Ile cookie cutter. The cookies were placed on baking Ife, Nigeria. trays, leaving 25 mm spaces in between and were baked in an oven at 150oC for 30 min. Following 2.2 Preparation of unripe baking, the cookies were cooled at ambient temperature, packed in polyethylene bags and stored plantain, moringa and for subsequent analyses. defatted sesame seed flours

The unripe plantain and moringa seed flours were 2.6 Nutritional experiments prepared by the method of Akubor and Ukwuru Experimental isonitrogenous rodent diets con- (2003) with little modification while defatted ses- taining 10% protein were prepared by incorpor- ame seed flour was prepared by the method of ating the complementary foods and casein (con- ElAdawy (1997). trol) into a basal diet prepared as described by Osundahunsi and Aworh (2003). The study 2.3 Formulation of flour blends was approved by the ethical review committee of the Federal University of Technology, Ak- Composite flour formulation was designed us- ure, Ondo State, Nigeria. Sixteen four-week- ing optimum mixture model of response sur- old weanling albino rats (Rattus norvegicus) ob- face methodology (Design expert 8.0.3.1, trial tained from the Department of Health Sciences version) and is presented in Table1. Unripe Animal House, Obafemi Awolowo University, Ile plantain, moringa and defatted sesame flours Ife were weighed and divided into four groups

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Table 1: Mixture Design of Flour Blends from Response Surface Methodology (RSM)

Run Unripe Plantain (g) Moringa seed (g) Sesame seed (g) 1 75.92 5.50 18.57 2 78.00 5.00 17.00 3 75.20 7.75 17.04 4 72.85 5.15 22.00 5 74.30 5.00 20.70 6 71.34 6.65 22.00 7 75.20 7.75 17.04 8 70.11 8.81 21.06 9 68.00 10.00 22.00 10 72.85 5.15 22.00 11 73.06 7.80 19.13 12 78.00 5.00 17.00 13 73.00 10.00 17.00 14 73.00 10.00 17.00 15 68.00 10.00 22.00 16 71.08 10.00 18.91

Table 2: Proximate Composition of composite ﬂours

Run Moisture (%) Ash (%) Fibre (%) Protein (%) Fat (%) CHO (%) A 5.01 4.34 2.20 12.51 9.81 66.11 B 5.56 3.63 1.97 14.26 10.55 64.01 C 4.48 3.84 4.00 12.96 10.45 64.24 D 5.62 3.17 2.08 12.20 10.91 65.98 E 6.32 2.98 2.36 14.00 11.07 66.05 F 5.27 2.29 3.02 14.08 10.65 64.24 G 4.48 3.84 4.00 12.96 10.45 64.24 H 5.05 3.24 4.71 13.69 10.65 62.64 I 4.23 4.13 2.28 14.35 11.26 63.72 J 5.62 3.17 2.08 12.20 10.91 65.98 K 6.12 3.37 2.25 14.17 10.89 63.19 L 5.56 3.63 1.97 14.26 10.55 64.01 M 6.42 2.96 2.24 14.00 10.92 63.43 N 6.42 2.96 2.24 14.00 10.92 63.43 O 4.23 4.13 2.28 14.35 11..26 63.72 P 6.02 2.94 2.24 13.65 9.87 65.26

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(experimental, control and basal) and were ac- presented in Table2. The formulations with climatized for a period of 7 days to the labor- high protein and crude fibre contents selected atory conditions prior to the experiment with were sample F (71.34% plantain, 6.66% moringa free access to drinking water and pellet diets. and 22.00% sesame) consisting of 14.08% protein The experimental animals were reweighed and and 3.02% fibre and sample I (68.00% plantain, regrouped to 4 groups so that the average weights 10.00% moringa and 22.00% sesame) consisting were equal. Two groups were fed with different 14.35% protein and 2.28% fibre. The protein con- formulation of the diet (cookies) while the re- tent of the flour blends was higher than that of maining groups were fed with casein as control the wheat flour; this may be due to the pres- and nitrogen free diet (basal diet) as presented ence of the defatted sesame and moringa seed in Table3. The rats were housed in individual flours (Chinma, Igbabul, Omotayo et al., 2012). metabolic cages while water and food were sup- Also, the fibre content in the composite mixture plied ad libitum for 21 days. of the flour blends was higher than wheat flour. The weights of the animals and feed intake were It might be a result of incorporation of matured recorded daily for 21 days. Faeces and urine unripe plantain which contains more resistant samples were collected, and physical appearance starch, and hence, high fibre content (Ayodele of the animals was recorded. At the end of the & Godwin, 2010). test period, the animals were anaesthetized and sacrificed; the liver, kidney and heart were removed and weighed. The Protein Efficiency Ra- tio (PER), Net Protein Retention (NPR), Bio- 3.2 The proximate composition of logical Value (BV), True Digestibility (TD), Net cookies Protein Utilization (NPU) and mean weight gain were calculated. The proximate composition of cookies and control (100% wheat flour) are presented in Table 4. It was observed that cookies from composite 2.7 Sensory properties of cookies flour had higher protein, moisture, fat and ash contents than that of the control. The addition of The organoleptic assessment of the cookies was defatted sesame and moringa seed flour could be carried out using twenty untrained panelists. A responsible for the higher protein content of the 9-point hedonic scale ranging from 9 (liked ex- composite flour (Ayo, Ayo, Popoola, Omosebi tremely) to 1 (disliked extremely) was used. & Joseph, 2014). The control had higher carbohydrate (69.79%) compared with 64.23% and 2.8 Statistical analysis 61.41% of cookies from composite flour. The percentage fat content of the composite flour cook- All analyses were conducted in triplicates. Mean ies increased from 10.66% to 16.65% in sample F scores of the results and their standard error of and 11.27% to 17.82% in sample I. The increase mean were reported using SPSS. Data was sub- may be due to the addition of vegetable shorten- jected to analysis of variances, and Duncan mul- ing and some other ingredients such as milk used tiple range (Duncan, 1955) test was used to point in the production of the composite flour cook- out significant differences. ies. The low values of moisture content obtained were in accordance with the report of Olagunju and Ifesan (2013) of low moisture in cookies pro- 3 Results and Discussion duced from wheat and germinated sesame seed. This is desirable for long shelf life and it was be- 3.1 Proximate composition of low the maximum level of (6%) recommended re- composite flour blends quirement of Nigerian Industrial Standard (NIS) for cookies by Standard Organization of Nigeria The proximate compositions of unripe plantain, (SON). The value of fibre was higher in sample moringa seed and defatted sesame flour are F compared with control.

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Table 3: The basic composition of the basal diet

Materials Amount (%) Vegetable oil 10 Vitamin premix 1 Mineral premix 2 Cellulose non-nutritive 5 Test protein casein (not included in nitrogen free diet) 10 Glucose 5 Sodium chloride 0.25 Bone meal 2 Oyster shell 1 Sugar 10 Corn starch(was added to make up to 100%) Osundahunsi and Aworh (2003)

Table 4: Proximate composition of cookies (%)

Sample Moisture Fat Crude Fibre Protein Ash Carbohydrate F 2.49±0.20ab 16.65±0.02b 2.25±0.03a 13.83±0.19a 2.56±0.21b 64.23±0.11b I 3.14±0.16a 17.82±0.12a 1.93±0.24a 14.53±0.35a 3.49±0.04a 61.41±0.23b Control 1.87±0.07b 14.95±0.11c 2.05±0.01a 9.54±0.22b 1.80±0.14c 69.79±0.32a Values on column with the same superscript are not significantly different. Key: Sample F; cookies produced from unripe plantain (71.341%), moringa (6.659%) and sesame flour (22%). Sample I; cookies produced from unripe plantain (68%), moringa (10%) and sesame flour (22%). Control sample; cookies produced from 100% wheat flour.

3.3 Sensory evaluation of cookies ies from formulated and control were accepted for sensory evaluation. This is in accordance The sensory evaluation of the cookies produced with the report of Olaoye and Oladoye (2007) on from the selected formulations was carried out breadfruit flour biscuit. This cookie was gluten using a 9 point hedonic scale. The subjective free so can serve as alternative for children and result of 20 panelists on the cookies produced adults that are allergic to gluten in wheat flour from unripe plantain, moringa seed and defat- that can cause celiac diseases (Caponio, Summo, ted sesame flour compared with cookies produced Clodoveo & Pasqualone, 2008). from 100% wheat is presented in Table5. The result shows that there was no significant different (p≤ 0.05) in the overall acceptability between 3.4 Nutritional evaluation of the composite flour cookies 649 (71.34% plantain, cookies 6.66% moringa and 22.00% sesame flour) and cookies from 100% wheat flour 352. There were Figure1 shows the average weight of the anim- no significant differences between the crunchiness als. Group 1 fed with the basal diet experienced a and aroma of the three samples, which agreed significant decrease in weight while those groups with the result of Chinma et al. (2011) who used fed with experimental diets group 2 (cookies tigernut and pigeon pea for biscuits, while sample :71.43% unripe plantain + 6.66% moringa seed + 352 (control) was preferable in colour and taste 22.00% defatted sesame seed) and group 3 (cook- to the composite flour samples. Both the cookies: 68.00% unripe plantain + 10.00% moringa

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Table 5: Sensory evaluation of cookies

Sample Appearance Aroma Taste Crunchiness Overall acceptability 842 7.07 ± 0.43b 7.87 ± 0.27a 6.93 ± 0.46b 8.00 ± 0.35a 7.13 ± 0.43b 649 7.20 ± 0.35b 7.40 ± 0.41a 7.33 ± 0.30b 8.13 ± 0.27a 7.60 ± 0.34ab 352 8.40 ± 0.16a 7.53 ± 0.22a 8.53 ± 0.19a 8.20 ± 0.30a 8.40 ± 0.24a Values on column with the same superscript are not significantly different. Keys: Sample 842: cookies from flour of unripe plantain 71.431%+moringa seed 6.659%+ defatted sesame seed 22%). Sample 649: cookies from flour of unripe plantain 68% moringa seed 10%+ defatted sesame seed 22%). Sample 352: cookies from 100% wheat flour.

Figure 1: Average weight of the animals (g)

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Figure 2: Biological value, true digestibility and net protein utilization of test diets and casein diet

Table 6: Protein quality indices of experimental animals

Indices Group 1 Group 2 Group 3 Group 4 Feed intake (g) 262.90 260.80 235.80 330.60 Feacal nitrogen 5.6 6.48 5.84 6.30 Urine nitrogen 1.47 2.66 2.38 1.54 Protein eﬃciency ratio (PER) 2.05 2.06 2.73 Net protein ratio (NPR) 3.05 3.08 3.86 Protein retention eﬃciency (PRE) 48.80 49.28 61.76 Keys: Group 1: animal fed with basal diet (nitrogen free) Group 2: animal fed with cookies (71.43% unripe plantain + 6.66% moringa seed + 22.00% defatted sesame seed) Group 3: animal fed with cookies (68.00% unripe plantain + 10.00% moringa seed + 22.00% defatted sesame seed) Group 4: animal fed with 10% casein

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Table 7: Average weight of animal organs

Groups Liver Kidney Heart Group 1 3.47±0.40a 0.58±0.07c 0.32±0.08a Group 2 4.24±0.25a 0.79±0.01a 0.39±0.03a Group 3 3.43±1.75a 0.69±0.03b 0.35±0.10a Group 4 4.03±1.20a 0.82±0.08a 0.36±0.03a Values on column with the same superscript are not signiﬁcantly diﬀerent. Keys: Group 1: animal fed with basal diet (nitrogen free) Group 2: animal fed with cookies (71.43% unripe plantain + 6.66% moringa seed + 22.00% defatted sesame seed) Group 3: animal fed with cookies (68.00% unripe plantain + 10.00% moringa seed + 22.00% defatted sesame seed) Group 4: animal fed with 10% casein

seed + 22.00% defatted sesame seed), and with children, and also may serve as a means to reduce casein diets experienced an increase in weight. Protein-Energy Malnutrition (PEM) in children. This agreed with Sodipo and Fashakin (2011); The Biological Value (BV) of animals fed with Ikujenlola and Fashakin (2005) in the prepara- casein diets (group 4) was higher than the BV tion of complementary diets. The weight of rats of animals fed with experimental diets (group 2 fed with experimental diets was significantly dif- and 3) as presented in fig2. The True Digestib- ferent (p≤0.05) from rats fed with casein. There ility of protein of animals fed with experimental was increase in growth of the animals fed with diets, group 3, was the highest, followed by anim- experimental diets and casein. It was observed als fed with casein diets (group 4), while animals that there was an increase in weight of anim- fed with experimental diets, group 2, recorded als fed with experimental diets and a decrease the least true digestibility. Group 4 recorded the in weight of animals fed with basal diets (nitro- highest Net Protein Utilization (NPU) followed gen free diet), which was also in accordance with by group 3 and group 2, respectively. The higher the report of Sodipo and Fashakin (2011). This the BV, TD, and NPU the better the protein indication shows that daily protein intake is es- quality, the standard for protein quality measure- sential for growth and development in humans, ment ranged from 70 to 90% (Ali, El Tinay, Mal- especially children. Table5 shows the protein lasy & Yagoub, 2010). The result obtained for quality indices of the experimental animals. The protein quality of animals fed with experimental animals fed with casein diets had highest PER, diets indicated that the protein content present PRE and NPR. This agreed with the report of in the experimental cookies was well utilized and Abiose, Ikujenlola and Aboderin (2015) that ca- appeared to be significant in animal growth and sein had the highest value in PER. The PER and development. The True Digestibility (TD) is a NPR obtained for the composite cookies agreed measure of total essential amino acid profile in with the report of Adepeju, Abiodun, Dauda and the diet. The values obtained from animals fed Fatiregun (2016) that used soybean sesame ogi. with both experimental and casein diets ranged Since, the protein efficiency ratio (PER), net pro- from 78.90 to 93.63, which showed that the ex- tein ratio (NPR) and protein retention efficiency perimental and casein diets contain high numbers (PRE) are very important parameters for the de- of essential amino acids. Table6 shows the av- termination of effective and efficient growth rate, erage weight of animal organs that include liver, cookies produced from the composite flour blends kidney and heart. It was observed there were could be essential for growth and development of no significant differences in the weights of an-

IJFS February 2021 Volume 10 pages SI72–SI81 SI80 Sodipo et al. imal liver and heart within groups. The weight Ali, M. A. M., El Tinay, A. H., Mallasy, L. O. & of group 2 and group 4 kidneys was not signi- Yagoub, A. E. A. (2010). Supplementation ficantly different while group1 and group 3 kid- of pearl millet flour with soybean protein: neys was significantly different from others. This Effect of cooking on in vitro protein digest- could be as a result of the amount of food taken ibility and essential amino acids composi- by the animal and components of the food. tion. International Journal of Food Science and Technology, 45 (4), 740–744. doi:10 . 4 Conclusion 1111/j.1365-2621.2010.02187.x AOAC. (2005). Official Methods of Analysis. It was observed that cookies produced from com- 16th Edn., Association of Official Analyt- posite flour containing unripe plantain, moringa ical Chemists, Washington, DC., USA. and sesame, had high protein content and were Ayo, J. A., Ayo, V. A., Popoola, C., Omosebi, M. considered acceptable by the panelists in com- & Joseph, L. (2014). Production and eval- parison with cookies made from wheat flour. The uation of malted soybean-acha composite composite cookies also promoted growth of rats, flour bread and biscuit. African journal of due to high protein efficiency ratio and biolo- Food science and Technology, 5 (1), 21–28. gical value. Therefore, cookies prepared from Ayodele, O. H. & Godwin, E. V. (2010). Gly- this composite flour may be used to ameliorate cemic indices of processed unripe plantain protein-energy malnutrition in children and can (musa paradisiaca) meals. African Journal be found acceptable also by adults. of Food Science, 4 (8), 514–521. Bailey, A. E. & Hui, Y. H. (1996). Edible oil and fat products: General applications. John References Wiley & Sons, Limited. Caponio, F., Summo, C., Clodoveo, M. L. & AACC. (2000). Approved Methods of the Amer- Pasqualone, A. (2008). Evaluation of the ican Association of Cereal Chemists. 10th nutritional quality of the lipid fraction Edn., American Association of Cereal of gluten-free biscuits. European Food Re- Chemists Press, St. Paul, MN., USA. search and Technology, 227 (1), 135–139. Abiose, S. H., Ikujenlola, A. V. & Aboderin, F. I. doi:10.1007/s00217-007-0702-0 (2015). Nutritional quality assessment of Chinma, C. E., Igbabul, B. D., Omotayo, O. O. complementary foods produced from fer- et al. (2012). Quality characteristics of mented and malted quality protein maize cookies prepared from unripe plantain and fortified with soy bean flour. Polish Journal defatted sesame flour blends. American of Food and Nutrition Sciences, 65 (1), 49– Journal of Food Technology, 7 (7), 398–408. 56. doi:10.1515/pjfns-2015-0004 Chinma, C. E., James, S., Imam, H., Ocheme, Adepeju, A. B., Abiodun, A., Dauda, O. & O. B., Anuonye, J. C. & Yakubu, C. M. Fatiregun, A. (2016). Nutritional evalu- (2011). Physicochemical and sensory prop- ation of weaning food prepared from fer- erties, and in-vitro digestibility of biscuits mented sorghum, germinated soybean and made from blends of tigernut (cyperus es- defatted sesame seed. School of Sciences, culentus) and pigeon pea (cajanus cajan). Federal University of Technology, Akure, Nigerian Journal of Nutritional Sciences, Nigeria. 32 (1), 55–62. Akubor, P. I. & Ukwuru, M. U. (2003). Func- Duncan, D. B. (1955). Multiple range and mul- tional properties and biscuit making poten- tiple F tests. Biometrics, 11 (1), 1–42. tial of soybean and cassava flour blends. ElAdawy, T. A. (1997). Effect of sesame seed Plant Foods for Human Nutrition, 58 (3), protein supplementation on the nutritional, 1–12. physical, chemical and sensory properties of wheat flour bread. Food Chemistry, 59 (1), 7–14. doi:10 . 1016 / 0308 - 8146(95 ) 00197-2

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Giarnetti, M., Paradiso, V. M., Caponio, F., mentary diet prepared from germinated Summo, C. & Pasqualone, A. (2015). Fat maize, cowpea and pigeon pea. Journal of replacement in shortbread cookies using an Food Agriculture & Environment, 9 (3-4, 1), emulsion filled gel based on inulin and ex- 23–25. tra virgin olive oil. LWT - Food Science and Technology, 63 (1), 339–345. doi:10.1016/j. lwt.2015.03.063 Hooda, S. & Jood, S. (2005). Organoleptic and nutritional evaluation of wheat biscuits supplemented with untreated and treated fenugreek flour. Food Chemistry, 90 (3), 427–435. doi:10.1016/j.foodchem.2004.05. 006 Ikujenlola, V. A. & Fashakin, J. B. (2005). Bioassay assessment of a complementary diet prepared from vegetable proteins. Journal of Food Agriculture and Environ- ment, 3 (3/4), 20. Muller, H. G. (1988). An introduction to tropical food science. Cambridge University Press. Ogunsina, B. S., Radha, C. & Indrani, D. (2011). Quality characteristics of bread and cookies enriched with debittered moringa oleifera seed flour. International Journal of Food Sciences and Nutrition, 62 (2), 185–194. doi:10.3109/09637486.2010.526928 Olagunju, A. I. & Ifesan, B. O. T. (2013). Nu- tritional composition and acceptability of cookies made from wheat flour and germinated sesame (sesamum indicum) flour blends. Current Journal of Applied Science and Technology, 702–713. Olaoye, O. & Oladoye, O. (2007). Breadfruit flour in biscuit making: Effects on product quality. African Journal of Food Science, 1. Osundahunsi, O. F. & Aworh, O. C. (2003). Nu- tritional evaluation, with emphasis on protein quality, of maize-based complementary foods enriched with soya bean and cowpea tempe. International Journal of Food Science and Technology, 38 (7), 809– 813. doi:10.1046/j.1365-2621.2003.00736.x Pasqualone, A., Bianco, A. M. & Paradiso, V. M. (2013). Production trials to improve the nutritional quality of biscuits and to enrich them with natural anthocyanins. CYTA - Journal of Food, 11 (4), 301–308. doi:10 . 1080/19476337.2012.753113 Sodipo, M. A. & Fashakin, J. B. (2011). Physico-chemical properties of a comple-

IJFS February 2021 Volume 10 pages SI72–SI81 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI82–SI94

Chemical Characteristics and Sensory Properties of Novel Snacks Produced with Okara Fortiﬁed with Omega-3 from Fish Oil

Trinidad Sandra Alvarez´ a and Daniela Lorena Lamasb*

a Facultad de Ciencias Exactas y Naturales, FACEN - Universidad Nacional de Asunción,UNA, Asunción, Paraguay - Consejo Nacional de Ciencia y Tecnolog´ıa(CONACYT). Mar del Plata, Buenos Aires, Argentina b Instituto de Investigaciones Marinas y Costeras (IIMyC), CONICET- Consejo Nacional de Investigaciones Cient´ıficasy TécnicasUNMdP, Instituto Nacional de Investigacióny Desarrollo Pesquero. Mar del Plata, Buenos Aires, Argentina *Corresponding author [email protected] Tel.: +54-223-420-9100

Received: 4 February 2019; Published online: 24 February 2021

Abstract

The aim of the present work was to develop a new soy-based food product and evaluate its chemical and sensory properties. A soy-based snack was mixed with rice (Oryza sativa), fortified with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) Omega-3 fatty acids from fish by-products in encapsulated and emulsion form. Soy beans were subjected to grinding processes, maceration in pure distilled water, filtration and pasteurization, to obtain vegetable drinks and generate solid residue. Finally, a sensory analysis of the product obtained was carried out. The snacks protein content was around 17%. From the sensory evaluation, it could be concluded that the snacks with 50% of okara had great acceptability and the addition of Omega-3 was also acceptable. So, okara represents an excellent raw material that can be utilized for dietary protein fortification. On the other hand, it proposes based on soy and fish by-products allow sustainable development and contribute to the economy of each sector. Keywords: Okara, soybean by-product; Sensory analysis; Protein; Omega-3

1 Introduction Rhee, Kim, Kim, Jung & Rhee, 2004). As a result, demand for a snack with enhanced nutrition exists (Navam, Tajudini, Srinivas, Sivarooban & Currently, consumers are becoming aware of the Kristofor, 2014). Many researchers have stud- importance of diet in relation to health problems. ied health-promoting snacks that use buckwheat Snacks foods as a cereal-based product, are the (Dapˇcević Hadnadev, Torbica & Hadnadev, most commonly consumed and are an important 2013), barley (Frost, Adhikari & Lewis, 2011) source of starch and other dietary carbohydrates wheat flour, and millet (Verma & Patel, 2013). (dietary fibre), which play an important role in The idea of developing products using a mix- the energy requirement and nutrient intake of hu- ture of cereals and legumes to combat hunger man. However, these cereal-based products tend and malnutrition problems in developing coun- to be lower in certain nutrients including pro- tries is a long-standing one. Soybean cultivars tein (Meng, Threinen, Hansen & Driedger, 2010;

Nomenclature

PUFAs polyunsaturated fatty acids CMC carboxymethylcellulose EPA eicosapentaenoic acid Fish oil C encapsulated ﬁsh oil DHA docosahexaenoic acid Fish oil E Emulsion of ﬁsh oil

vary in their crude protein and lipid contents, Friedman and Brandon (2001). The fortification lipoxygenase activities and fatty acid composi- of foods with PUFAs of the Omega-3 series is a tions (Vong & Liu, 2016). Previous works repor- current issue of interest. The daily intake of these ted that protein content of soybean range from compounds in populations of developed countries 42 to 48% (Guimaraes et al., 2018; Kanojia, is below the recommended dose (Ferguson, Smith Singh, M. Khandelwal & Azam, 2016). Due & James, 2010; Iafelice et al., 2008). Among the to its high protein content, polyunsaturated li- health benefits associated with the intake of these noleic acid and isoflavones, soybean is qualified fatty acids are the reduction of cardiovascular as a functional food (Guimaraes et al., 2018). diseases, anti-inflammatory, anti-allergic effects, This fact has generated great interest from food good development and function of the brain, the industry to incorporate ingredients from soybean retina and the nervous system, and the poten- into products that provide beneficial properties tial protection against certain types of cancer to the organism and which are called “functional (Iafelice et al., 2008; Kolanowski & Weissbrodt, foods” (Genovese & Lajolo, 2002). 2008). As well, there are studies that suggest On the other hand, soymilk and tofu manufac- that the consumption of Omega-3 during preg- turing produce great amounts of residue called nancy can have a good influence on the foetus okara. This is considered a waste by-product (Yupa & Claribel, 2015). In recent years, world- and it is disposal, due to the high water, is an wide, the number of available foods fortified with important environmental concern (Waliszewski, PUFAs has increased rapidly in an attempt to Pardio & Carreon, 2002). Dry okara, which con- address this deficiency. However, there are no tains about 50% dietary fiber, 25% protein and studies on fortification of soy snacks. Thus, the 10% lipid (Li et al., 2013), is an excellent altern- incorporation of fatty acids of the Omega-3 series ative for incorporation into food products, not could be an extra factor to the benefits of them. only as a form of food enrichment, but also as Hence, the overall objective of this work was a way of using and valuing this residue (Vong & to develop a snack with soybean (Glycine max) Liu, 2016). okara to achieved a high protein content, along Also, several clinical studies showed the asso- with rice (Oryza sativa) and improved it nu- ciation of soy foods with reduction in blood tritional value by using Omega-3 from fish by- serum cholesterol levels, risk of cardiovascular products in encapsulated and emulsion form. In diseases in humans, mammary and prostate can- addition to that, the chemical characteristics and cers in women and men, respectively, osteo- the effect of adding Omega-3 to the product sens- porosis among menopausal women and increased ory preference were determined. Sensory proper- bone density (Messina & Loprinzi, 2001). Be- ties were evaluated in terms of overall visual and neficial effects, such as prevention of diabetes taste acceptability of the products. and obesity, protection against intestinal and renal diseases, related to the promotion of human health and nutrition, were also reported by

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2 Materials and Methods lished in the formulations. The determination of fats was carried out by Randall (1974) method 2.1 Materials and lipids were extracted and quantified by the method of Bligh and Dyer (1959). Crude fibre The oilseeds were obtained commercially from was estimated by gravimetric treatment (AOAC, Vivo and Sano, (Ciudad del Este, Paraguay). 1990). Carbohydrate contents were calculated by They were received in polyethylene bags and difference. stored free of oxygen, light and moisture at room To determine the fatty acid profile of the lipid temperature, until their use. fraction, it was subject to methylation and All materials and reagents for making capsules, subsequent gas chromatography (ISO 12966-2, beverages and snacks were food grade. Cit- 2011). Briefly, 60 mg of lipid sample was mixed ric acid, calcium chloride and guar gum were with 2 ml of hexane and 0.3 ml of KOH/MeOH commercially purchased from KUBO Company reagent in a glass tube in order to convert fatty and sodium alginate was obtained from Qu´ımica acids to methyl esters (FAMEs). The sample Bol´ıvar. The orange flavouring, stevia, carboxy- was mixed vigorously; then, 2 ml of NaCl and 2 methylcellulose and a mix of whole seeds with ml of hexane were added and mixed again. The sunflower, black and white sesame, chia and sample was allowed to stand for 5 minutes, and squash were purchased in commercial stores of the upper hexane layer was separated and trans- Mar del Plata city (Argentina). Ray liver oil was ferred to a clean tube. The FAMEs were determ- provided by OmegaSur S.A. (Mar del Plata, Ar- ined with a Shimadzu GC2010 (Kyoto, Japan) gentina) and the average Omega-3 PUFA content equipped with a flame-ionization detector (260 was 158mg g-1, being the EPA and DHA 43 and oC) and capillary column (30 m x 0.32 mm; 0.25 115mg respectively. In addition, soybean oil was µm film thickness; Omegawax 320, Darmstadt, provided by Cargil S.A. (Bah´ıaBlanca, Argen- Germany). A split injector (50:1) at 250 oC was tina). used. The column temperature was programmed All the reagents used for the determinations were starting at a constant temperature of 120 oC dur- of analytical grade and high purity. ing 20 min, heated to 200 oC at 1 oC minute−1, held at 200 oC during 1 minute, heated again to 220 oC at 5 oC/min, and finally held at 220 oC 2.2 Physicochemical for 20 minutes. The separation was carried out characterization with nitrogen as carrier gas. The oven temperature was increased to 240 oC at a rate of 5 oC To characterize the seeds, 3 sub-lots of them con- minute−1 and held for 5 minutes. A volume of 1 taining 10 seeds each were weighed and their µL of sample was manually injected (in duplic- average weight was calculated. The transversal ate), and FA peaks were identified by comparison length was calculated in the same way. of their retention times with those of external ref- The proximate composition on raw and snacks erence standards (Supelco FAME Mix C4-C24, products was determined according the methods Pennsylvania, USA). Retention times and peak of the Association of Official Analytical Chem- areas were processed by Shimadzu GC Solution ists (AOAC, 1990). software. Moisture was quantified by drying in a furnace The calories of snacks were determined consid- at a temperature of 105 oC to constant weight ering all the ingredients used in the formula- (AOAC, 1990). The ashes were determined by tion (expressed in grams) and their correspond- calcination in muffle at 550 oC of temperature, ing quantities. Finally, the conversion of grams until obtaining white ash and constant weight to calories was performed. For nutritional la- (AOAC, 1990). The proteins were determined by belling, the US Food and Drug Administration the Kjeldahl method using the conversion factor (FDA) suggests caloric content be calculated by 5.70 AOAC (1984). For the snacks the conver- a procedure using 4, 9, and 4 calories gram−1 of sion factor used was 5.70 and 5.95 to soy and rice protein, fat, and carbohydrate, respectively. respectively, according to the proportions estab-

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2.3 Formulations of soy snacks for 35 minutes. Finally, the capsulates were obtained by dripping the mix in 0.1 M CaCl2 solu- Soybeans were sorted, and once the broken and tion and dried at 30 oC for 24 hours in stove and immature ones were removed, the rest were 7 days in desiccator. washed. Next, wet okara was obtained according to the protocol described by Turhan, Temiz and Sagir (2007) with modifications. Soybeans (100 2.5 Sensory analysis of formulated g) were soaked for 12 hours at room temperature foods in 600 ml of distilled water. Then, the hydrated soybean was drained by rinsing hot water (90- The sensory attributes of okara were analysed by 95 oC), and cooking was done to inactivate the 16 untrained panellists (men and women, 25 to lipoxygenase enzyme and the antitrypsin. The 65 years old). Colour, aroma, flavour and texture bitter water was removed. The milling was done of the 3 snacks formulated with equal propor- in a domestic mill (Philips) and the process time tion of okara and rice flour were evaluated. The was standardized to 3 minutes. After washing, samples were served in polystyrene dishes coded the slurry was filtered through cheesecloth and with letters. They were presented in individual pressed obtaining the residue called okara. Fig. sectors, with good natural light and a temper- 1 illustrated the sequence of soymilk processing ature of 23 oC. The analysis was performed on steps and the obtaining fresh okara to manufac- portions of 10 g each. The participants were in- ture the snacks. structed to eat a snackand drink water between The following ingredients were used for the pro- the samples in order to cleanse the palate if it was duction of snacks: okara rice flour, carboxy- necessary. The panellists evaluated the accept- methylcellulose, soybean oil, stevia, fish oil in ance of the samples using a hybrid hedonic scale capsule and emulsion form and a mix of whole of 9 points (Drake, 2007), where 1 = I was very seeds, sunflower, sesame. Six formulations were upset, and 9 = I liked it extremely. There was developed and each one was identified as G code a space for observations and comments. Then a with the numbers from 1 to 6. Table1 sum- descriptive sensory analysis was carried out using marizes the percentage distribution of the com- different descriptors. The panellists were previ- ponents of each one and Fig.2 shows the mix- ously instructed in the descriptors used in this tures prepared. The mixtures were kneaded and study like flavour as (soybean, to rice, to fish, brought to 4 mm in thickness, cut into a circular sweet or salty), odour (to bean soy, to rice, fish shape of 2.5 cm in diameter and baked at 170 oC smell, without define odour) colour (brownish, for 20 minutes. Once baked, they were cooled to green, whitened) and oral texture (hard, crispy, room temperature. humid, soft, dry). Then, two discriminatory and preference test were made between these three snacks and then one among the snacks enriched 2.4 Preparation of Omega-3 with capsules and emulsions. The panellists eval- capsules uated each treatment individually on the individual score sheets. For a preference test, the For the fish oil encapsulation process, sodium al- panellists were also provided a “your preference” ginate was dispersed in distilled water and al- sheet to indicate which snack they preferred and lowed to stand overnight at room temperature to briefly discuss why. During the instructed, for complete hydration of the biopolymer. Then the difference between a descriptive and a pref- the discontinuous emulsion (10 g) was prepared erence test was clearly explained. The purpose which was formulated with a 1: 2 ratio of oil of including a preference test was also discussed phase dispersed to continuous phase. The pre- with the panellists. Finally, a general product ac- emulsion was obtained by homogenization of me- ceptance questionnaire was developed in order to dium speed during 2 minutes using an Ultra- achieve a sensory profile of the product. The ana- turrax (Omni Mixer, Germany). The emulsifica- lysis was performed 48 hours after the product tion was carried out by centrifuging at 3500 rpm was prepared.

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Figure 1: Generation of okara from the manufacture of soy milk

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Table 1: Formulation of okara snacks Sample Ingredient g% G1 G2 G3 G4 G5 G6 Okara 40 36 24 5 24 24 Rice ﬂour 0 4 24 44 24 24 CMC 18.2 18 15 15 15 14 Soy oil 19.2 19 17 17 17 17 Stevia 1 1 1 1 1 1 Distilled water 18.2 18.4 15 15 14 15 Seed mix 3 3 4 3 4 4 Fish oil C 0 0 0 0 1 0 Fish oil E 0 0 0 0 0 1

Figure 2: Mixture of ingredients with diﬀerent okara levels for snacks. G1 40% okara; G236% okara; G3 24% okara; G4 5% okara; G5 24% okara, G6 24% okara

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2.6 Statistic analysis One of the reasons for the wide use of soy protein in foods is its high quality (measured accord- All analyses were performed in duplicate, and ing to PDCAAS Protein Digestibility-Corrected were expressed as mean value ± standard de- Amino Acid score and defined by FAO/WHO viation. The difference between the mean val- (2011). It has been demonstrated that the amino ues was evaluated by the Duncan test, and it acid content added to its high bioavailability was considered significant with a P value of 5%. qualifies it with the highest value (1 or 100%) For the statistical analysis we used the Infostat equalling in quality the whey, casein and albu- (2017) software, of general application developed min proteins (Vanegas Perez, Restrepo Molina under the Windows platform. & Lopez Vargas, 2009). On the other hand, some beneficial health effects have been attributed to soy protein as hypocholesterolemic and 3 Results and Discussion hypotriglyceridemic actions (Tovar et al., 2002). Also, some specific clinical conditions require 3.1 Proximate composition of the nutrition based on soy, as immunoglobulin E- raw material mediated milk allergy; post infectious diarrhoea due to lactose intolerance; galactosaemia; and Soybeans were accepted according the maturity other cases as the use as a vegan human milk sub- requirements and health conditions established stitute; and treatment of common feeding prob- by (Guzman Tituaña, 2018). The total lipid con- lems such as fussiness, gas, and spit-up, mainly tent was 65.1 %, with 7.1 % protein, 4.7 % ash in infants (Merritt & Jenks, 2004). However, the and moisture 2.12 %. Table2 shows the fatty food industry in many cases prefers to replace acid profile of the lipid fraction of soybean oil. whey, casein and albumin proteins by soy pro- It can be seen that the content of linoleic acid is tein, not only for health reasons but also for costs the highest, followed by oleic and palmitic. These (Hoogenkamp, 2007; USDA, 2007). results are consistent with bibliography data for soybean oils (O’Brien & Timms, 2004). Fresh okara was obtained as by-product of soy- 3.2 Formulation and making the bean milling and aqueous fraction extraction snack. Physicochemical and for producing soydrink. Immediately it was caloric value weighted and dried at 60 oC during overnight. The moisture content was 79.11 % and it The early recipes in this study involved ingredi- was corroborate with the ones reported by ents and processing modifications to produce the Wachiraphansakul and Devahastin (2007) (75-80 proper flavour and texture. When the dough was %), He and Chen (2013) (80 %) and by Tie Su, constituted by okara ratio more than 50 %, the Pedroso Yoshida, Contreras-Castillo, Quinones products broke apart into crumbs. When the and Venturini (2013) (80 %). By this way, okara and rice were added in the same ratio, okara spoilage during storage and processing was products formulated resulted with less ligation avoided maintaining characteristic flavour and problems. Major rice content produced a cake colour. Mean proximate analysis of the dried snack without a pleasant airy texture. samples were as follows: protein 38.37 g %, All the snacks formulated were analysed in terms fat 15.13 g %, fiber 35.74 g %, total carbo- of their chemical and caloric properties. How- hydrates 7.65 g % and ash 3.11 %. These data ever, only the snacks formulated with the same are in accordance with values reported by Wal- content of rice and okara were submitted for sens- iszewski et al. (2002) for different okara samples. ory analysis. Data on the chemical composition Similar results of protein content were previ- of snacks are presented in Table3. The val- ously reported by Ostermann-Porcel, Quiroga- ues obtained are within the ranges expected for Panelo, Rinaldoni and Campderros (2017). Also, this type of products. Moisture values are in ac- Katayama and Wilson (2008) reported similar cordance with the water content added in each values for the commercial dried okara powder. case. In addition, these values are in agreement

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Figure 3: Sensory assessment. a) The score distribution of the three snacks studied. b) The descriptive proﬁle of the attributes of each sample

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Table 2: Fatty acid composition of soybean oil to reduce or regulate plasma cholesterol and tri- using Gas Chromatography acylglycerol levels and to promote health. Table 3 also shows the results obtained for caloric value Fatty acid Soy oil (%) in each sample. A wide range of variation for en- 16:00 10.21 ± 0.35 ergy levels for cereal bars (from 72.8 to 321.70 18:00 4.05 ± 0.04 kcal.100 g−1) has been reported in the literature 20:00 0.19 ± 0.01 (Dutcosky, Grossmann, Silva & Welsch, 2006; 22:00 0.19 ± 0.02 Gutkoski, Maria de Almeida Bonamigo, Marli de Freitas Teixeira & Pedó, 2007). Also, energy P SFA 14.64 level of 385.95 kcal.100 g−1 of bars with high con- 18:1n9 21.19 ± 0.45 tent of rice bran has been reported by Garcia, Lobato, Benassi and Soares Junior (2012). The P MSFA 21.19 energy level of the snack developed in the present 18:2n6 55.49 ± 1.66 work is in accordance with the levels reported 18:3n3 8.67 ± 0.22 in literature. On the other hand, the ingredi- P PUFA 64.16 ents used in the formulation allowed obtaining products with low caloric value, since a 20g for Total: 100 portion was calculated. P SFA, sum of unsaturated; P MUFA, Regarding the fatty acid profile all the products sum of monounsaturated; P PUFA, sum of fortified showed values of EPA and DHA accord- polyunsaturated. ing to the contents of fish oil used for enrich- Values are expressed by mean value ± ment. The predominant fatty acid was and DHA standard error (n=2). Fatty acids that were (C22:6), followed by oleic acid (C18:1), palmitic less than 1% were omitted acid (C16:0) and EPA (C20:5). Recommend- ations concerning the daily intake of n-3 fatty acids fluctuate about 500mg of EPA + DHA in the form of food or supplements (Kris-Etherton, with data reported by Navam et al. (2014) on Grieger & D Etherton, 2009; Simopoulos, 2002). snacks formulated with fortified cereals with dif- EPA is the precursor of prostaglandins, throm- ferent percentages of soybeans. Ash content is boxanes and leukotrienes, and DHA is a com- also in accordance with the okara content ad- ponent of the phospholipid membrane of the ded in each case. Veronica, Olusola and Ade- brain and cells of the retina, consequently, both bowale (2006), reported similar values of ash in fatty acids are essential for human health, show- maize-based snacks with defatted soybean. The ing significant effect in the prevention of cardi- protein content is in the range of 15 to 19%, ovascular diseases and the development of cog- higher than the 11% reported in okara and wheat nition respectively (Fournier et al., 2007; Zhong, snacks by Katayama and Wilson (2008) and the Madhujith, Mahfouz & Shahidi, 2007). Its bene- 12 % reported by Ostermann-Porcel et al. (2017) fits on human health have motivated, in recent in gluten-free cookies developed using okara and decades, the development of products with its in- commercial manioc flour. On the other hand, corporation as additives, emulsions or capsules this value was in the range of fortified corn tor- (Bermudez-Aguirre & Barbosa-Canovas, 2011; tilla preparations with 20-25% okara reported Kolanowski & Laufenberg, 2006; Ye, Cui, Taneja, by Waliszewski et al. (2002). The snacks with Zhu & Singh, 2009). According to the results greatest content of okara in the formulation (G1 obtained, it can be expected that snacks forti- and G2), showed the lowest total carbohydrates fied with emulsion and capsulated might provide and the highest fibre, protein, and fat content. 126 and 51 mg of EPA and DHA, which is ap- Dietary fibre plays an important role in many proximately the 25% and 10% respectively of physiological processes and in the prevention of the omega-3 recommended minimal daily intake diseases of different origin. Therefore, daily in- (Kris-Etherton et al., 2009; Simopoulos, 2002). take of food rich in dietary fibre is important

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Table 3: Nutritional value of okara snacks Sample G1 G2 G3 G4 G5 G6 % Moisturea 18.55 18.63 15.25 14.25 13.49 12.91 % Asha 2.58 2.77 2.58 0.71 2.42 2.50 % Proteina 19.36 18.98 17.34 15.49 17.64 17.31 % Fata 26.47 25.29 21.83 21.18 22.03 21.93 %Carbohydratesa 12.76 13.76 19.92 23.71 20.06 19.81 % Fibre 20.27 20.55 23.08 24.66 24.36 25.54 % Kcala 366.72 358.58 345.51 347.42 349.07 345.85 Kcala(20g) 73.34 71.71 69.10 69.48 69.81 69.17 a Indicate that in the line, mean values of each okara are not diﬀerent by the Duncan test (p < 0.05).

3.3 Sensory analysis capsulated fish oil. Results showed that, in consumer preference Results of acceptance sensory studies are shown tests, all samples presented a good acceptability. in Figure3. Only the snacks formulated with the However, the order of preference by the panellists same rate of rice and okara were submitted for was G5 > G6. sensory analysis (G3, G5 and G6). According to the total sensory scores, all the samples exhibited great acceptable sensory characteristics. As re- gards to the flavour, odour and colour there were 4 Conclusion no statistical differences among the formulations. According to the descriptive sensory scores, the Even though okara is a by-product, it contains samples exhibited some differences in their char- many beneficial components from soybean. In acteristics (Table3). this study, it was determined that okara can be On the topic of the flavour, the snack without used as ingredient to prepare snacks with accept- Omega-3 was the one that most evidenced a taste able chemical and sensory qualities. The snacks of rice. The other snacks were not rated as rice fortified showed a free fatty acid profile with high flavoured. The emulsion-enriched snacks were content of EPA and DHA. Also, all the snacks the most difficult to qualify in terms of taste, formulated showed high levels of protein and the some panellists indicated this snack as sweet, ingredients used in the formulation allowed ob- others as salty and also undefined flavour was taining products with low caloric value. Even mentioned. The snack enriched with capsules though this study has been focused on okara was the only one that threw fishy taste, and in snacks fortification, the proposed approach could some cases without defining. The smell was sim- offer an interesting way to effectively incorporate ilarly qualified. In this case, the snack without oils rich in Omega-3 fatty acids into other kind enriching was described as smelling like rice, al- of cereal bars. According to the total sensory though in some cases the 3 snacks were rated as scores, the samples exhibited acceptable char- having a soy smell. The snack enriched with cap- acteristics among consumer panel members. In sules was the only one qualified with the smell of conclusion, it has been demonstrated that the in- fish, and in some cases the qualification with fried corporation of okara in snack foods fortified with odour was added. Omega-3 from fish by-products resulted in sig- Analysing the effect of snacks enrichment with nificant improvements in the nutritional value of Omega-3 on colour parameters change, results the products, whereas provide new alternatives have shown an increase the brown tonality but no for both by-products marketability and help in differences were detected between emulsion and reducing environmental wastes.

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IJFS February 2021 Volume 10 pages SI82–SI94 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI95–SI111

Study of Antimicrobial, Antioxidant and Cytotoxicity Properties of Selected Plant Extracts for Food Preservative Applications

Tania Islama, Md Nazrul Islama*, Wahidu Zzamanb, and Md Morsaline Billahc

a Forestry and Wood Technology Discipline, Khulna University, Khulna - 9208, Bangladesh b Department of Food Engineering and Tea Technology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh c Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna - 9208, Bangladesh *Corresponding author [email protected] Tel.: 880 41 730171-3, Ext: 2070

Received: 24 August 2019; Published online: 24 February 2021

Abstract

An attempt has been made to evaluate antimicrobial, antioxidant and cytotoxicity properties of extracts from onion (Allium cepa L.), garlic (Allium sativum), leaves of guava (Psidium guajava), papaya (Carica papaya), tea (Camellia sinensis), baen (Avicennia alba) and keora (Sonneratia apetala), respectively to apply as natural preservatives for tomatoes. The air-dried plant materials of the respective plant species were subjected to ethanol-methanol extraction, concentrated and stored at 4oC before use. The extracts were dissolved in 95% ethanol for analysis of antioxidant and antimicrobial properties. Of the extracts tested, tea extracts showed the highest zone of inhibition against several pathogenic bacteria (E. coli 35.0±3.2 mm; P. aeruginosa 29.3±2.6 mm; S. typhi 28.4±2.1 mm and S. pyogenes 27.7±3.7 mm) using the disc diﬀusion method. In regard to DPPH free radical scavenging assay, keora and guava extracts showed the highest percentage of radical scavenging activity with the values of 89.64± 0.18 and 89.39± 0.88, respectively, which were in agreement with higher total antioxidant capacity (TAC) of these extracts obtained by the phosphomolybdenum method. Brine shrimp lethality bioassay for cytotoxicity assessment showed LC50 of 132.54 ± 18.99 µg/mL for the leaf extract of keora which was found to be most toxic among all studied extracts. The initial results indicated that the extracts could be used for food preservative applications based on the antimicrobial, antioxidant and cytotoxicity properties of the tested extracts. However, eﬃcacy, stability and safety issues need to be addressed with both in vitro and in vivo studies. Keywords: Antimicrobial; Antioxidant; Cytotoxicity; Nutrient content; Natural food preservative

1 Introduction years for many purposes including food preservation and therapeutics (Hammer, Carson & Riley, Plants have been used for treating ailments and 1999). Plant-based foods are prone to deterior- different diseases since prehistoric times. Rem- ate at some rate after harvesting due to phys- nants from different civilizations still carry the ical interventions, and chemical and microbial re- evidence of plant usage for different purposes actions (Nakanishi, Irie & Murata, 2018; Thiel- (Hintz, Matthews & Di, 2015). Mankind is util- mann, Kohnen & Hauser, 2017), which lead to ising plant extracts and oils for thousands of loss of quality and shelf life reduction. Different

Nomenclature

FDA Food and Drug Administration BSA Bovine Serum Albumin AAE Ascorbic Acid Equivalent LC Lethal Concentration GAE Gallic Acid Equivalent DPPH 2,2-diphenyl-1-picrylhydrazyl DMSO Dimethyl Sulfoxide ZOI Zone of Inhibition

types of methods such as freezing, smoking, heat- variety of different compounds in plant extracts ing, sun drying, irradiation and chemical treat- possess antimicrobial activities, and several ex- ments are in place alone or in combination for tracts have been shown to have antimicrobial preservation of foods. A global estimate for food activity due to their phytochemical constituents loss has revealed that 10% of cereal and 40% (Ma et al., 2018; Shiekh, Malik, Al-Thabaiti & of vegetables and fruits, including 75% of peas Shiekh, 2013). The antibacterial activity could and beans are lost during storage every year. be attributed to the combined effects of adsorp- The estimate goes up if meat, poultry and fish tion by bacterial membranes of polyphenols, with are considered (Kumar & Kalita, 2017). There- membrane disruption leading to leakage of cellu- fore, loss during postharvest storage raises the lar contents and the formation of hydroperoxides price of food, affecting its availability and access, from polyphenols. Plant extracts also exhibit an- if proper control measures are not undertaken tifungal, antioxidant and antimutagenic activit- (Hac-Wydro, Flasinski & Romanczuk, 2017). In ies, and inhibit lipid oxidation in foods. Numer- addition, it also increases the potential for food ous in-vitro studies have been carried out to eval- contamination by food borne pathogens and in- uate the antimicrobial activity of plant extracts sects. As a result, contaminated food leads to (Donsi & Ferrari, 2016). However, very little in- an increased risk of human illnesses along with formation is available for food products. Most of a reduction in the nutritional quality of the food the studies employed crude extracts and resul- (Sarkar & Shetty, 2014). ted in reduced inhibition when compared with There are increasing consumer demands for less many of the pure compounds in foods. Diet- use of synthetic additives in processed food ary herbs and spices have traditional applications products. Although synthetic antimicrobials are as food additives to improve the sensory charac- approved in many countries, the recent trend to- teristics of foods and extend their shelf life by wards use of natural preservatives is due to the reducing or eliminating survival of pathogenic adverse health effect of synthetic ones (Khan, bacteria. These herbs and spice extracts usu- Ullah & Oh, 2016; King et al., 2017). Due ally possess antimicrobial activity against differ- to inherent chemical diversity and the unique ent bacteria, yeast and moulds as they are rich classes of compounds produced by plants as in phenolic compounds (Dwivedy, Kumar, Up- self-defence mechanisms against infectious organ- adhyay, Prakash & Dubey, 2016). isms, plant based natural products, either as Apart from antimicrobial properties, the extracts pure compounds or standardized extracts, of- are shown to preserve foods by reducing lipid ox- fer unlimited opportunities for control of micro- idation due to their significant antioxidant activ- bial growth. In addition, the use of plant ex- ity. Various phenolic substances derived from tracts in food applications has increased as sev- herbs and spices are reported to show several bio- eral are generally recognized as safe (GRAS) by logical activities which contribute to their preser- the Food and Drug Administration (FDA). A vative potential (Negi, 2012; Sumaiya, Jahurul &

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Zzaman, 2018). It has also been found that anti- lic were peeled carefully, and the inner portion oxidants may protect cell tissues from free radic- was kept for further use. The samples were then als and thus have a role in the prevention of dis- cut into small pieces, air dried for 24 h and sub- eases like cancer (Djeridane et al., 2006). With sequently dried in the oven for 24 h at 80 oC. evolving health consciousness and increasing con- Dried samples were ground later into a coarse cerns regarding the use of synthetic antioxidants powder (40-mesh) and placed in separate airtight to prevent oxidation reactions in food, the use of glass bottles. Powdered samples were stored in a safe plant-based antioxidants and antimicrobials refrigerator at 4 oC until they were removed for has drawn considerable interest and attention of further processing. scientists and industrialists. This has resulted in the use of plant based bioactive compounds 2.2 Extraction of plant extracts in food preservation (Thorat et al., 2013). Ag- ricultural and horticultural produce, especially Approximately 500 g of dried powdered sample plants and fruits which are rich in phenolic bio- was placed in a 5 L round bottom flask and active compounds, provide a mixture of phyto- soaked in 2.5 L of an ethanol (>99.5%)-methanol chemicals with potential health benefits. These (>95%) mixture (50% v/v basis). The flask con- bioactive phenolic compounds also can improve taining the mixture was kept airtight, using a storage quality and increase the shelf life of other cotton plug and aluminium foil, and magnetically fruits, vegetables, legumes, grains and other food stirred at 500 rpm at room temperature for 20 products (Thielmann et al., 2017). The present h. The extract was filtered through a fresh cot- study was undertaken to determine and com- ton plug, followed by Whatman No.1 filter paper, pare the potential of extracts of onion (Allium and concentrated using a rotary evaporator (Bu- cepa L.), garlic (Allium sativum), leaves of guava chi Labortechnik AG, Flawil, Switzerland). The (Psidium guajava), papaya (Carica papaya), tea resultant extracts were stored in a refrigerator (Camellia sinensis), baen (Avicennia alba) and until required for the experiments. The weight keora (Sonneratia apetala), respectively as nat- of the crude extract obtained from leaves was ural preservatives for tomatoes. found to be in the range of 5-10 g as oven dried.

2 Materials and Methods 2.3 Antimicrobial activity assessment 2.1 Materials Antibacterial activity of the extracts was evalu- The samples of onion and garlic were collected ated by the disc diﬀusion method (Bauer, Kirby, from the Gallamari Market near Khulna Univer- Sherris & Turck, 1966). Escherichia coli (ATCC sity campus at Khulna, Bangladesh. The leaves 8739), Pseudomonas aeruginosa (ATCC 27833), of guava (P. guajava) and papaya (C. papaya) Salmonella typhi (ATCC 6539) and Streptococ- were collected directly from the mature trees loc- cus pyogens (ATCC 19615) were collected as ated on the Khulna University campus. The ma- pure cultures from the Microbiology Laborat- ture tea (C. sinesis) leaves were collected from ory, Pharmacy Discipline, Khulna University, a tea garden located in Sylhet, Bangladesh. The Bangladesh. The bacterial isolates were cultured leaves of baen (A. alba) and keora (S. apetala) in nutrient broth at 37 oC for 2-4 h until the log were collected from Dhangmaree, Chadpai range, phase of growth. The sterile ﬁlter paper discs East zone of the Sundarbans, Bangladesh. The were prepared by adding the desired concentra- leaves of guava (P. guajava), papaya (C. papaya), tion (250 µg/disc) of extract onto the disc us- tea (C. sinesis), baen (A. alba) and keora (S. ing a micropipette. Bacterial broth culture was apetala) were washed thoroughly using distilled spread over the nutrient agar medium. Finally, water to remove dirt and other unwanted pol- the plates were incubated at 37 oC for 18 h and lutants and then kept at room temperature for then checked for the zone of inhibition, which surface drying. The outer layer of onion and gar- was measured in millimetres (mm). Erythromy-

IJFS February 2021 Volume 10 pages SI95–SI111 SI98 Islam et al. cin (10 µg/disc) was used as a control during the Cytotoxicity assessment study. The brine shrimp lethality bioassay was carried out for the cytotoxicity test and vincristine sulphate (0.52 µg/mL) was used as a positive Antioxidant activity assessment control. The eggs of the brine shrimp, Artemia salina, and sea water were collected from the The free radical scavenging properties of ex- BRAC prawn hatchery, Sreeghat, Bagerhat, tracts were evaluated by the 2,2-diphenyl-1- Bangladesh. 48 h after hatching, eggs become picrylhydrazyl (DPPH) assay method as mod- mature and are called nauplii. The water insol- ified by Brand-williams, Cuvelier and Berset uble extracts were dissolved in 50 µL of dimethyl (1995). Then, 2 mL of different concentrations sulfoxide (DMSO) and each test tube contained (1.75, 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 4 mL of sea water with different concentrations µg/mL) of the extracts were mixed with 3 mL of extracts (5, 10, 20, 40, 80, 160, 320 µg/mL). of 0.004% DPPH solution in ethanol. After a re- The final volume for each test tube was adjusted action period of 30 min at room temperature in to 10mL with artificial sea water and 10 living the dark, the absorbance was measured at 517 nauplii were transferred into each tube. After nm (Gupta et al., 2003). Ascorbic acid was used 24 h of incubation at room temperature (25 oC), as the standard. Each assay was carried out the number of live and dead nauplii were recor- in triplicate. The total antioxidant capacity of ded. The lethal concentration (LC50) values of the extracts was evaluated by the phosphomolyb- the plant extracts were obtained at 95% con- denum method (Prieto, Pineda & Aguilar, 1999). fidence interval and the slope calculated using Sulfuric acid, sodium phosphate and ammonium prohibit analysis by the Finney-1971 computer molybdate were supplied by Merck (Darmstadt, program (Mwangi, Wagacha, Nguta & Mbaria, Germany). A 0.3 mL of extract was combined 2015). with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the Nutrient analysis o reaction solution were incubated at 95 C for The different extracts having the concentration 90 min. Then, the absorbance of the solution of 0.5% were sprayed uniformly and separately was measured at 695 nm using a UV-vis spectro- on the collected tomatoes of uniform size, shape photometer (UV mini-1240, Shimadzu Corpora- and ripening condition with the help of a hand tion, Japan), against a blank, after it was cooled sprayer. The fruits were then dried at room tem- down to room temperature. Methanol (0.3 mL) perature and stored in an open and safe place in the place of extract was used as the blank. which was free from insects. The storage dur- The total antioxidant activity was expressed as ation was 12 days as reported by Islam et al. ascorbic (AAE) and gallic (GAE) acid equival- (2018). The moisture content of fruits was de- ents, and also as the percentage of radical scaven- termined by the oven drying method (AOAC, ging activity (Skrovankova, Sumczynski, Mlcek, 2005) before application of the extract and after Jurikova & Sochor, 2015) The percentage of ra- the storage time. Weight, as an indicator of the dial scavenging activity was calculated using the quality of fruits, was measured using a digital below mentioned formula: balance (Brishti, Misir & Sarker, 2013) before application of the extract and after the storage A − A I(%) = 100 × Blank Sample (1) time. The tomato juice was prepared by a di- ABlank gital blender (Vita-Prep 3, Vita-Mix Corpora- tion, UK) as reported by Islam et al. (2018), and where I (%) is the inhibition percentage, Ablank the pH of juice was measured by a benchtop pH is the absorbance of the control element and meter. Total carbohydrate content of the tomato Asample is the absorbance of the test sample before application of the extract and after the (Salawu et al., 2017). storage time was determined by the Lane-Eynon

IJFS February 2021 Volume 10 pages SI95–SI111 Plant extracts for food preservative applications SI99 method (Okoye & Ugwu, 2008). The protein con- (more than 30% of dry weight) as reported earlier tent of tomato was estimated by Lowry’s method (Reto, Figueira, Filipe & Almeida, 2007). In using a standard curve of Bovine Serum Albumin the present investigation, other plant extracts (BSA) solution (20-100 mg/mL) and a double also showed strong activity against the tested beam UV-Visible spectrophotometer to measure bacteria irrespective of their types (Dorman & absorbance at a wavelength of 660 nm (Lowry, Deans, 2000). However, the extracts also showed Rosebrough, Farr & Randall, 1951). varying levels of inhibitory properties, which could be regarded as one of key findings in this study and allowed us to investigate their prop- 2.4 Assessment of antimicrobial erties for fruit preservation. With regard to po- and antioxidant properties of tential antimicrobial activity, the plant extracts preserved tomato juice could be ranked as tea (C. sinensis)> garlic (A. sativum)> baen (A. alba)> keora (S. apetala)> To evaluate the efficacy of preserved tomatoes in papaya (C. papaya)> guava (P. guajava)> onion regard to antimicrobial and antioxidant proper- (A. cepa). ties, tomato juice was prepared from different to- It is worth mentioning that several previous stud- matoes which had been coated with an extract as ies showed the efficacy of plant extracts as an- described earlier. In addition, fresh tomato juice timicrobial agents for preventing the growth of was prepared to compare the effects of extract food borne and spoilage bacteria (Calo, Crandall, application. Antimicrobial activity assessment, O’Bryan & Ricke, 2015; Cleveland, Montville, free radical scavenging assay and total antioxid- Nes & Chikindas, 2001). It is well documented ant capacity were conducted as stated earlier. that plant extracts possess a number of antimicrobial compounds, e.g. terpenoid, alkaloid and phenolic compounds. These compounds usually 3 Results and Discussion interact with enzymes and proteins of the microbial cell membrane and subsequently lead to 3.1 Antimicrobial activities of the its disruption. These events in turn induce cell extracts death or inhibit enzymes essential for amino-acid biosynthesis (Burt, 2004; Gill & Holley, 2006). In Among seven different plant extracts, the leaf addition, several researchers have reported that extract of tea showed the strongest inhibition the inhibitory effect of these plant extracts could against E. coli (35.0±3.2 mm), P. aeruginosa be correlated with their hydrophobic nature as (29.3±2.6 mm), S. typhi (28.4±2.1 mm) and S. well as the cell membrane of targeted bacteria pyogenes (27.7±3.7 mm) (Table1). Garlic ( A. (Gill & Holley, 2006; Tiwari et al., 2009). This sativum) extract and the leaf extract of Baen attribute would facilitate interaction of the plant (A. alba) produced approximately similar zones extracts with microbial cell membrane protein of inhibition followed by the leaf extract of tea. and mitochondria, deform their structures and The assay results were interpreted using the scale ultimately alter their permeability (Friedman, of measurement described by Carovic-Stanko et Henika, Levin & Mandrell, 2004; Tiwari et al., al. (2010) which considered zones of inhibition 2009). The present study suggested that the se- greater than 15 mm as strongly inhibitory, 10-15 lected plant extracts, which proved to be poten- mm as moderately inhibitory and smaller than tially effective, could be used as natural preser- 10 mm as not inhibitory, respectively. Based vatives and alternatives to chemical preservatives on this scale, the extracts used in this study which have become an increasing concern due to showed strong inhibition at 250 µg/Disc against potential health hazards. the bacteria under investigation. However, the onion (A. cepa) extract showed the lowest zone of inhibition. The strong inhibitory activity of the extracts could be attributed to the presence of a variety of flavonoids in tea (C. sinensis)

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Table 1: Mean values of diameters for zone of inhibition (ZOI) of diﬀerent plant extracts (expressed in mm, presented as mean± standard deviation; n=3) and standard, Erythromycin

ZOI diameter Erythromycin Bacteria Type of extracts (mm) (mm) Keora 25.0±1.2a Tea 35.0±3.2b Baen 27.0±2.3a 29.5±1.29a E. coli Papaya 21.0±1.9c Guava 20.0±2.3d Garlic 27.0±1.7a Onion 20.0±2.6e Keora 19.5±2.3b Tea 29.3±2.6a Baen 21.4±3.1c d a P. aeruginosa Papaya 19.2±2.8 27.75±0.96 Guava 18.7±1.7e Garlic 25.0±1.2a Onion 18.3±2.8f Keora 20.5±2.9b Tea 28.4±2.1a Baen 22.9±2.5c a a S. typhi Papaya 23.5±2.7 28.75±0.5 Guava 19.4±2.0d Garlic 24.5±1.8a Onion 19.2±2.7e Keora 24.6±3.5b Tea 27.7±3.7a Baen 25.5±3.9c d a S. pyogenes Papaya 24.5±2.9 32.75±0.98 Guava 23.3±3.1e Garlic 26.3±2.7f Onion 23.6±2.1g Dissimilar small letters in a row indicate significant differences (p< 0.005) of the antimicrobial effect of different extracts, respectively when compared to standard, while similar small letters in a row indicate the lack of significant differences (p< 0.05) of the same.

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Table 2: Nutrient analyses of fresh tomatoes at day 0, tomatoes without any extract coating at day 12 and tomatoes coated with diﬀerent extracts at day 12.

Type of Moisture Changes in pH of the Total Protein extracts content (%) weight (%) fruit juice carbohydrate (%) content(%) Fresh Tomato at Day 0 89.93±0.15a - 4.08±0.02a 2.42±0.03a 0.82±0.02a∗ Tomato at day 12 45.85±0.27b 45.52±1.56a 6.59±0.89b∗ 0.87±0.15b∗ 0.46±0.17a∗ Keora extract coated tomato 80.61 ±0.38c 11.24±0.24b 4.16±0.06a 2.04±0.06a 0.73±0.02a∗ Tea 79.86±0.18c 15.89±0.18c 5.34±0.16c 1.42±0.04c∗ 0.62±0.03a∗ Baen 77.64±0.58d 17.92±0.12d 5.48±0.13d 1.32±0.02d∗ 0.57±0.03a∗ Papaya 80.31±0.63e 19.12±0.12e 5.25±0.11e 1.72±0.05a 0.52±0.02a∗ Guava 72.15±0.55f 19.63±0.27f 5.40±0.10f 1.59±0.03e∗ 0.54±0.02a∗ Garlic 69.18±0.63g 20.02±0.05g 6.17±0.06g∗ 0.94±0.05f∗ 0.49±0.01a∗ Onion 67.47±0.19h 20.28±0.07h 6.56±0.06h∗ 0.87±0.03g∗ 0.49±0.02a∗ Means with the same letter in a column are not significantly different (p<0.005) when compared to fresh tomato at day 0, while * indicates the values which are not significantly different when compared to tomatoes kept for 12 days without any extract application. Significant values (p<0.005) for tomatoes kept 12 days with different extract applications are shown without any symbol.

3.2 Antioxidant properties of the scavenging activity due to their redox properties, extracts i.e. through their electron transfer or hydrogen donating ability (Lee, Hwang, Ha, Jeong & Kim, In the DPPH free radical scavenging assay, the 2003; Rahman, Islam, Biswas & Alam, 2015). leaf extract of keora (S. apetala) and guava (P. In terms of total antioxidant capacity, the leaf guajava) showed the highest percentage of free extract of guava showed the highest antioxid- ± radical scavenging activity (89.64 ± 0.17 and ant capacity which was 257.10 14.97 mg AAE/g ± 89.39± 0.88, respectively) (Fig.1). With regard and 311.19 20.22 mg GAE/g, respectively fol- to free radical scavenging activity the plant ex- lowed by the leaf extract of keora (S. apetala) ± tracts were ranked as keora (S. apetala) > guava which showed values of 135.17 0.78 mg AAE/g ± (P. guajava) > tea (C. sinensis)> papaya (C. and 163.61 0.94 mg GAE/g, respectively (Fig. papaya) > onion (A. cepa) > baen (A. alba). 2). The antioxidant potential of the different ex- The ability of antioxidants to donate hydrogen tracts was determined by their ability to reduce is a guide to DPPH free radical scavenging. In the reduction of Mo (VI) to Mo (V) and sub- the DPPH assay, the violet color of DPPH solu- sequent formation of a green phosphate/Mo (V) tion is reduced to a yellow colored product, di- complex at acidic pH. The presence of bioact- phenylpicryl hydrazine, with the introduction of ive compounds, such as polyphenols, carotenoids, the extracts in concentration-dependent manner. and vitamin E and C in the extracts influences The ethanolic plant extracts showed a similar their antioxidant activity (Mwangi et al., 2015). type of free radical scavenging activity when The presence of bioactive compounds and their compared with the standard, ascorbic acid. It concentration in the extracts is important for has been reported that polyphenol compounds conferment of antioxidant activity. Thus, higher and tocopherols scavenge the DPPH radicals concentrations of extract show higher antioxid- by their hydrogen donating ability (Amarowicz, ant activity. These results comply with previ- Pegg, Rahimi-Moghaddam, Barl & Weil, 2004). ous findings, which suggested that the antioxid- In addition, total polyphenols content and rad- ant capacity could be attributed to the chemical scavenging antioxidant activity are found to ical composition and polyphenol content of the be highly correlated. The results obtained in extract and not just the total phenolic content this study suggest that the extracts show radical (Amarowicz et al., 2004; Rahman et al., 2015;

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Table 3: Mean values of diameters for zone of inhibition (ZOI) of fresh tomato juice (TJ) at Day 0, tomato juice at Day 12 and diﬀerent juices prepared from extract coated tomatoes at Day 12 (expressed in mm, presented as mean± standard deviation; n=3) and standard, Erythromycin

ZOI diameter Erythromycin Bacteria Types of extracts (Mean ± SD, mm) (Mean ± SD, mm) Fresh TJ 24.5±0.5b TJ without extract 13.3±2.4c Keora +TJ 31.0±1.2a Tea + TJ 38.0±1.4d 32.5±1.34a a E. coli Baen + TJ 29.0±2.7 Papaya + TJ 26.0±1.3e Guava + TJ 23.0±2.8f Garlic + TJ 37.0±1.4g Onion + TJ 23.0±1.2h Fresh TJ 25.7±0.3a TJ without extract 12.5±2.1b Keora +TJ 26.5±1.3a Tea + TJ 39.3±2.4c 29.47±0.87a a P. aeruginosa Baen + TJ 30.4±1.4 Papaya + TJ 25.2±1.3d Guava + TJ 21.7±1.7e Garlic + TJ 33.0±1.6a Onion + TJ 20.3±1.3f Fresh TJ 26.5±0.7a TJ without extract 15.8±1.8b Keora +TJ 27.4±1.4a Tea + TJ 36.3±1.9c a a S. typhi Baen + TJ 29.7±1.6 29.55±0.53 Papaya + TJ 23.5±1.8d Guava + TJ 22.4±1.3e Garlic + TJ 34.5±0.7f Onion + TJ 21.2±1.3g Fresh TJ 27.8±0.5b TJ without extract 12.7±2.3c Keora +TJ 27.6±1.6d Tea + TJ 32.7±1.8a e a S. pyogenes Baen + TJ 28.5±1.6 33.43±0.87 Papaya + TJ 25.5±1.6f Guava + TJ 24.3±1.1g Garlic + TJ 29.3±1.9h Onion + TJ 24.6±1.8i Dissimilar small letters in a row indicate significant differences (p< 0.05) of the antimicrobial effect of juice prepared from different extract coated tomatoes, respectively when compared to the standard, while similar small letters in a row indicate the lack of significant differences (p< 0.05) of the antimicrobial effect of the same.

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Thielmann et al., 2017). weight observed was in the onion (A. cepa) extract treated tomato (20.28%), followed by the garlic (A. sativum) treated samples (20.02%), 3.3 Cytotoxicity of the extracts while the lowest value was obtained in the leaf extract of keora (S. apetala) treated samples. How- In the brine shrimp lethality bioassay, the plant ever, the changes in weight were significantly dif- extracts showed brine shrimp larvicidal activ- ferent when plant extract treated samples were ity in a concentration dependent manner. The compared to tomatoes stored without any plant leaf extract of keora (S. apetala) showed the extract application (control) at day 12. The lowest LC of 132.54 ± 18.99 µg/mL which 50 control displayed a rapid decline in weight (%) was the most toxic among all tested extracts and the highest changes in weight percentage at (Fig.3). The leaf extract of guava ( P. day 12 as compared to all other treatments at guajava) exhibited the next lowest LC value 50 that stage. This could be attributable to uncon- of 137.89± 22.21 µg/mL. The other extracts trolled ripening of tomatoes, as ripening in to- could be ranked based on toxicity as garlic (A. matoes could be regarded as climacteric with a sativum) (234.58± 22.0 µg/mL) > onion (A. sudden increase in ethylene production and res- cepa) (245.84± 12.54 µg/mL) > tea (C. sin- piration rate. This higher respiration rate also ensis) (324.52±20.15 µg/mL) > baen (A. alba) resulted in higher transpiration of water from the (453.21± 18.9 µg/mL). It has been reported fruit surface which led to an increase in percent- that LC values in the range of 100-500 µg/mL 50 age of changes in weight during storage (Sabir, are considered moderately toxic (Salawu et al., Shah & Afzal, 2004; Zagory & Kader, 1988). 2017). Therefore, LC values obtained in this 50 Thus, physiological changes in weight of toma- study indicate moderate toxicity for the plant toes were influenced by different extract applic- extracts. In addition, it was suggested that, if ations. Moreover, as cited by Proulx, Cecilia, crude extracts possessed LC values less than 50 Nunes, Emond and Brecht (2005), high storage 250 µg/mL they could be considered significantly temperature led to accelerated water loss and active and have the potential for further invest- subsequent shrivelling and softening of the fruit. igation (Nair, Anju & Hatha, 2017). Therefore, Furthermore, lower changes in weight of toma- the preliminary cytotoxic activity exhibited by toes could be due to a slow rate of ripening and the extracts was promising and this clearly indic- prevention of excessive moisture loss on applic- ated the presence of potent bioactive compounds. ation of different extracts to tomatoes (Haile, 2018; Seyoum & Woldetsadik, 2004). Zhang and 3.4 Nutrient analysis Quantick (1997) mentioned similar results for litchi coated with an edible coating of chitosan. Analytical results of moisture, changes in weight, The pH value is a measure of acidity or alkalinity pH, total carbohydrate and protein content be- of water-soluble substances. The pH value is one fore and after treatment of the tomato samples of the most important factors which influences are shown in Table2. Moisture content in- food properties. The pH of tomatoes is determ- fluences the physical, chemical and microbiolo- ined primarily by the acid content of the fruit, gical aspects of food, and the keeping qualities termed titratable acidity (TA). The acidity of of freshness and firmness during the storage of the fruit also contributes to the flavor of tomato the food for a long period of time. The moisture products. The pH value of the fresh tomato was content of the fresh tomato was 89.93%. The 4.08 and the pH value increased after treatment leaf extract of keora (S. apetala) treated tomato of the sample. The lowest pH value of 4.16 was retained the highest moisture content of 80.61% found in the tomato coated with the leaf extract whereas the lowest value was found to be 67.47% of keora (S. apetala) whereas the highest value of for onion (A. cepa) extract treated samples. The 6.56 was observed in the tomato coated with the moisture content of plant extract treated samples onion extract. However, the tomato without any was found to be significantly different from each extract coating showed the highest change in pH other. The highest percentage of change in on comparing samples at day 0 and after 12 days

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Figure 1: Percentage free radical scavenging of diﬀerent plant extracts (n=3).

of storage. The rise in pH could be related to a pounds occurring in foods and living tissues, and decline in TA due to a loss of citric acid. Citric include sugars, starches and cellulose. The total acid is the most abundant organic acid in toma- carbohydrate and protein contents of the con- toes. The decrease in TA could be associated trol sample were 2.42 and 0.82 %, respectively. with increased fruit ripening and subsequent de- The highest total carbohydrate content was es- cay which would lead to loss of citric acid from timated as 2.04% in tomatoes coated with the tomatoes. It has been reported that during to- leaf extract of keora (S. apetala) while the lowest mato ripening, as in other fruits, a decline in value determined was 0.87% for tomatoes coated acid levels is accompanied by an increase in sug- with onion (A. cepa) extract. The highest pro- ars. At least a portion of this change may be due tein content was 0.73% for tomatoes coated with to the metabolic conversion of acids into sugars the leaf extract of keora (S. apetala) whilst toma- by gluconeogenesis. In addition, the loss of or- toes coated with extracts of onion (A. cepa) and ganic acids from tomatoes could occur through garlic (A. sativum) showed the lowest value of respiration during storage (Anthon, LeStrange & 0.49% after storage for 12 days. Nutrient results Barrett, 2011). It has been suggested that 4.4 is for carbohydrates and protein were supported by the maximum desirable pH for safety and the op- the work of Jiang, Li and Jiang (2005) who in- timum target pH should be 4.25 (Monti, 1979). vestigated the effects of an edible chitosan coat- The present finding is supported by the result ing on the litchi fruits at ambient temperature observed on apple samples by Coseteng and Lee and Martinez-Romero et al. (2006) who studied (1987). the influence of aloe-vera on the storage of sweet Carbohydrates are a large group of organic com- cherries.

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Figure 2: Total antioxidant capacity of diﬀerent plant extracts, both ascorbic acid equivalent (AAE) and gallic acid equivalent (GAE), respectively in mg/g.

Figure 3: LC50 values of diﬀerent plant extracts and control (n=3).

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Figure 4: Free radical scavenging assay of diﬀerent juices prepared from extract coated tomatoes at day 0 and 12, fresh tomato juice at day 0, tomato juice without any extract at day 12.

3.5 Antimicrobial activities of (p<0.005). The results indicate that a syner- preserved tomatoes gistic effect of both extracts and tomato juice could contribute towards appearance of larger Tomato juice prepared from different tomatoes zones of inhibition than seen for both free fresh which had been coated with an extract showed tomato juice and erythromycin, respectively. In varying levels of antibacterial properties after addition, the leaf extract of baen (A. alba), ke- storage for 12 days (Table3). Of these, juice pre- ora (S. apetala) and garlic (A. sativum) showed pared from tea extract treated tomatoes showed strong antibacterial activities against P. aeru- the highest level of activity against the tested ginosa, S. typhi and S. pyogenes, respectively. bacteria. It should be noted that fresh tomato In all cases, juice prepared from both onion (A. juice also showed strong antibacterial properties cepa) and the leaf extract of guava (P. guajava) when compared to values obtained from the con- coated tomatoes showed poorest antibacterial trol which is in agreement with previous find- properties, and from papaya (C. papaya) coated ings (Maz’uma, Dadah & Uba, 2018). Juices tomatoes showed moderate antibacterial proper- from extract coated tomatoes were able to pro- ties. duce a zone of inhibition close to erythromycin. For E. coli, P. aeruginosa, S. typhi and S. pyogenes, juice from extract of garlic (A. sativum) and leaf extract of tea (C. sinensis) coated tomatoes produced a larger zone of inhibition than control values which were statistically significant

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Figure 5: Total antioxidant capacity of diﬀerent juices prepared from extract coated tomatoes at day 0 and 12, fresh tomato juice at day 0, tomato juice without any extract at day 12; expressed in both Ascorbic Acid Equivalent (AAE, mg/g) and Gallic Acid Equivalent (GAE, mg/g).

3.6 Antioxidant properties of of extract applied was 0.5%, the free radical scav- preserved tomatoes enging did not increase substantially when compared with the results obtained for the extracts The phenolic content of tomato fruits has been alone. However, the total antioxidant capacity correlated with their antioxidant capacity. Sim- of juice prepared from tomatoes without any ex- ilar to free radical scavenging assay findings, tract increased slightly from day 0 to day 12, al- DPPH free radical scavenging assay findings for though this was not statistically significant (Fig. juices from different extract coated tomatoes 5). The increase in antioxidant capacity could be showed varying levels of activity (Fig.4). Juice related to the lower moisture content at day 12. from leaf extract of keora (S. apetala) and extract The leaf extract of guava (P. guajava) showed of guava (P. guajava) coated tomatoes showed the highest antioxidant capacity for both AAE the highest free radical scavenging activity. Fresh and GAE values (mg/g) which was highly sig- tomato juice (day 0) also showed a substan- nificant (p<0.0001). Statistically significant val- tial level of activity as reported by de Abreu, ues were also shown in terms of both AAE and Piccolo Barcelos, de Barros Vilas Boas and da GAE values (mg/g) for the extracts of onion (A. Silva (2014) and Balaswamy (2015), however, the cepa) and garlic (A. sativum) and the leaf ex- activity declined after storage for 12 days. Ex- tract of keora (S. apetala). Storage of tomatoes tract treated tomatoes were able to retain their coated with different extracts favors the libera- activity as evident from Figure1. As the amount tion of phenolic compounds and lycopene, the

IJFS February 2021 Volume 10 pages SI95–SI111 SI108 Islam et al. main antioxidant compound present in tomatoes ies. Food Chemistry, 84 (4), 551–562. from the cellular matrix. Thus, phenolic com- doi:10.1016/S0308-8146(03)00278-4 pounds and lycopene were more bioavailable in Anthon, G. E., LeStrange, M. & Barrett, D. M. tomatoes subjected to storage and dehydration (2011). Changes in ph, acids, sugars and in the process of preservation (de Abreu et al., other quality parameters during extended 2014; Toor, Savage & Lister, 2009). It is con- vine holding of ripe processing tomatoes. sidered that these phenolic compounds improve Journal of the Science of Food and Agri- the nutritional value and functional properties of culture, 91 (7), 1175–1181. doi:10.1002/jsfa. tomatoes. 4312 AOAC. (2005). Offcial Methods of Analysis. 14th 4 Conclusion ed. Washington, D.C.: Association of Off- cial Analytical Chemists. The studied plant extracts showed different levels Balaswamy, K. (2015). Antioxidant activity of of antimicrobial, antioxidant and cytotoxic activ- tomato (lycopersicon esculentum l.) of low ity due to differences in phenolic compounds, and soluble solids and development of a shelf the amount and concentration of extracts applied stable spread. Int J Food Sci Nutr Diet, to tomatoes. Although the study is preliminary 4 (4), 202–207. in nature, it has provided valuable insights about Bauer, A. W., Kirby, W. M. M., Sherris, J. C. the use of plant extracts in food preservation. & Turck, M. (1966). Antibiotic suscept- The current study indicates that leaf extracts of ibility testing by a standardized single tea (C. sinensis), keora (S. apetala) and guava disk method. American Journal of Clinical (P. guajava) and the extract of garlic (A. sat- Pathology, 45 (4), 493–&. ivum) can be used for potential applications in Brand-williams, W., Cuvelier, M. & Berset, C. preventing the adverse effects of food spoilage. (1995). Use of a free-radical method to eval- Chemical and synthetic preservatives are still uate antioxidant activity. LWT- Food Sci- used for preventing food spoilage in the food in- ence and Technology, 28 (1), 25–30. dustry. The adverse effects of these chemical pre- Brishti, F. H., Misir, J. & Sarker, A. (2013). servatives on human health necessitate the search Effect of biopreservatives on storage life for potentially effective, healthier, safer and nat- of papaya (carica papaya l.) International ural food preservatives based on plant sources. It Journal of Food Studies, 2 (1). is a common belief that biologically active pure Burt, S. (2004). Essential oils: Their antibac- compounds are more effective than crude ex- terial properties and potential applications tracts. However, to get the overall view of plant in foods—a review. International journal of extracts for prevention of food deterioration and food microbiology, 94 (3), 223–253. extension of shelf-life, it is essential to consider Calo, J. R., Crandall, P. G., O’Bryan, C. A. crude extracts. As food spoilage is usually as- & Ricke, S. C. (2015). Essential oils as sociated with the growth of different pathogenic antimicrobials in food systems-a review. bacterial strains and oxidation of food compon- Food Control, 54, 111–119. doi:10.1016/j. ents, further study is required to investigate the foodcont.2014.12.040 application of these extracts in food preservation. Carovic-Stanko, K., Orlic, S., Politeo, O., Strikic, F., Kolak, I., Milos, M. & Satovic, Z. (2010). Composition and antibacterial References activities of essential oils of seven ocimum taxa. Food Chemistry, 119 (1), 196–201. Amarowicz, R., Pegg, R. B., Rahimi- doi:10.1016/j.foodchem.2009.06.010 Moghaddam, P., Barl, B. & Weil, J. A. Cleveland, J., Montville, T. J., Nes, I. F. & Chi- (2004). Free-radical scavenging capa- kindas, M. L. (2001). Bacteriocins: Safe, city and antioxidant activity of selected natural antimicrobials for food preserva- plant species from the canadian prair- tion. International journal of food microbiology, 71 (1), 1–20.

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IJFS February 2021 Volume 10 pages SI95–SI111 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI112–SI120

The Eﬀect of Seaweed (Eucheuma cottonii) Flour Addition on Physicochemical and Sensory Characteristics of an Indonesian-Style Beef Meatball

Aris Sri Widatia, Djalal Rosyidib*, Lilik Eka Radiatib, and Happy Nursyamc

a Post Graduate Program, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang 65145, Indonesia b Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang 65145, Indonesia c Faculty of Fisheries and Marine Science, Universitas Brawijaya, Jl. Veteran, Malang 65145, Indonesia *Corresponding author djalal [email protected] Tel.: +62-341-553513

Received: 19 November 2019; Published online: 24 February 2021

Abstract

The objective of this work was to study the effect of seaweed (Eucheuma cottonii) flour addition on physicochemical and sensory characteristics of an Indonesian-style beef meatball. Seaweed flour (SF) was added to meatball batter at 0% (CON), 2.5% (SF2.5), 5.0% (SF5) and 7.5% (SF7.5) in weight/meat weight (w/w) basis. Proximate composition (moisture, crude protein, crude fat, crude fiber and ash content), pH, cooking loss, hardness, water-holding capacity, instrumental color and sensory properties (color, texture, aroma, taste and overall acceptability) of the boiled meatballs were evaluated. The effect of seaweed flour addition was very significant (P < 0.01) on moisture, crude fat, crude fiber, ash, cooking loss, hardness, water-holding capacity, lightness (L*), yellowness (b*) and all sensory properties, and was significant (P < 0.05) on crude protein, pH and the redness (a*) of a boiled Indonesian-style beef meatball. The addition of seaweed flour at more than 2.5% resulted in darker meatballs, with less protein and fat, and a stronger seaweed aroma. The results suggest that seaweed flour could be added at 2.5% (w/w) to produce an Indonesian-style beef meatball with enhanced fiber content. Keywords: Eucheuma cottonii; Fiber; Meatball; Seaweed flour

1 Introduction pitojo, 2019). From the nutritional point of view, the Indonesian-style beef meatball is a source of An Indonesian style-meatball or bakso is a energy and protein. However, it lacks dietary boiled-type of meatball and among the most fa- fiber as it is made from meat, starch (tapioca), voured traditional restructured meat products egg white and seasonings. Tapioca functions as a found in the Indonesian market (Purnomo & Ra- filler and egg white is used as a binder (Purnomo, hardiyan, 2008). The name bakso originated 2012). The high-quality meatballs should meet from the word, Hokkien for finely chopped meat the national standard set by (Badan Standard- mixed with starch and seasonings (Tan, 2002). It isasi Nasional, 1995). The meatballs should con- has a short shelf life (max. 1 day) when stored at tain a maximum of 70% moisture, 2.0% fat and 13oC but can be stored frozen in order to extend 3.0% ash, and a minimum of 9.0% protein, and its shelf life (Rifqie Mariana, Hidayati & Soeko-

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a9 SI112 Widati et al. have a meaty flavour, umami taste, normal ap- of Eucheuma cottonii flour in the formulation of pearance (light brown and light grey) and chewy an Indonesian-style beef meatball. texture. The physicochemical and sensory characteristics of Indonesian-style beef meatballs are influenced 2 Materials and Methods by the ingredients and methods of processing and packaging (Purnomo, 2012). A higher propor- 2.1 Seaweed flour preparation tion of meat in the formulation of the meatballs Seaweed (Eucheuma cottonii) flour was made ac- increases the proportion of protein and fat, and cording to a method described by Manuel, Corne- reduces the chewiness. In contrast, a lower pro- lia and Wijaya (2015), with modifications in dry- portion of meat (replaced by starch) increases ing temperature and time. Semi-dried seaweed chewiness but lowers the protein and fat content. was purchased from a local market, washed to re- Sodium tripolyphosphate (E451) has been widely move sand, soaked in water for 12 h and drained used to overcome water-holding capacity and tex- for 60 min at room temperature. Seaweed was ture issues in restructured meat products (Lee, cut into 3 cm lengths, dehydrated at 60oC for Hendricks & Cornforth, 1998). Even though this 24 h (YTK ST-02, Yason General Machinery, additive is categorized and generally recognized Guangdong, China), ground using a food miller as safe by the US FDA, it is preferable to use (FGD230, Ramesia Mesin Indonesia, Jakarta, In- a natural ingredient. To attain the required nu- donesia), sieved through an 80 mesh-sieve, air- tritional value and chewy texture, the natural packed and stored at room temperature prior to ingredient added to the meatballs’ formula must use. function as a binder and a filler. Eucheuma cottonii is an edible seaweed species that is categorized as Rodhopycheae. This 2.2 Meatball preparation seaweed is rich in k-carrageenan which functions as a stabilizer and a water-binder, and A beef round cut and other ingredients were pur- strengthens protein gel development in heat- chased from a local market. Indonesian-style induced emulsion-type food products (MacAr- beef meatballs were manufactured according to tain, Gill, Brooks, Campbell & Rowland, 2007). a method described by Rohman, Erwanto, Man The k-carrageenan extracted from red seaweed et al. (2011), with modifications in beef cube size has been widely used within the meat pro- and chopping time. First, excess fat and vis- cessing industry as a water-binder (Campo, ible connective tissue in the beef were trimmed. Kawano, da Silva & Carvalho, 2009). The k- Then, beef was cut into 4Ö4 cm cubes and carrageenan from Eucheuma cottonii has been minced using a meat chopper for 1 min (MGB80, used in the production of chicken and catfish Ramesia Mesin Indonesia, Jakarta, Indonesia). meatballs to improve water-holding capacity, Minced beef was divided into four groups and cooking yield and texture, and increase fiber in mixed with seaweed flour at different addition those products (Kurniawan, Al-Baarri & Kusra- levels, salt, sugar, spices, tapioca, egg white and hayu, 2012; Yakhin, Wijaya & Santoso, 2015). ice (Table1). Each mixture from the four treat- Hartati and Endang (2011) reported the addition ment groups was chopped for 2 min. Three of ground and raw Eucheuma cottonii at 2.5% batches of each treatment were prepared. The of meat weight improved the acceptability of batter was shaped into balls (approximately 11 Indonesian-style beef meatballs. However, there g) and cooked in 70oC-water and 96oC-water for is no information regarding the use of Eucheuma 5 min each (until the core temperature reached cottonii flour in Indonesian-style beef meatball 72oC). The core temperature of sampled meat- manufacture. Raw seaweed is more susceptible balls was checked using a digital thermometer to quality degradation and has a shorter shelf-life (DTH128, Shenzen Tonglixing Technology, Shen- than the flour (Guerrero, Campos & Alzamora, zen, China). The cooked meatballs were drained 2002). Therefore, the objective of the present and then chilled at 4oC overnight prior to ana- study was to determine the optimal addition level lysis. Meatballs were ground to determine prox-

IJFS February 2021 Volume 10 pages SI112–SI120 Seaweed ﬂour-added Indonesian-style beef meatball SI113 imate composition and pH using a food blender slurry was then determined using a calibrated pH (HR7627, Philips Indonesia, Jakarta, Indonesia). meter in duplicate (PHS-3DW, Shaoxing Worner Lab Equipment, Zhejiang, China).

Table 1: Indonesian-style beef meatball formula Physical characteristics Cooking loss was determined in triplicate based Treatment 1 on the differences in weight of meatball samples Ingredient (g) SF0 SF2.5 SF5 SF7.5 before and after cooking at 80oC for 30 min, and Beef 300 300 300 300 expressed as a percentage (%). Water-holding Tapioca 60 60 60 60 capacity was measured in triplicate using the fil- Seaweed flour 0 7.5 15 22.5 ter paper press method (Grau & Hamm, 1953). Salt 10.5 10.5 10.5 10.5 A cooked sample was placed on filter paper and Sugar 10.5 10.5 10.5 10.5 pressed at 35 kg for 5 min. The area of water Pepper 0.75 0.75 0.75 0.75 release was calculated, and the results were ex- Garlic 7.5 7.5 7.5 7.5 pressed as a percentage (%). The hardness of Egg white 9.0 9.0 9.0 9.0 the sample was measured using a Digital Force Ice 90 90 90 90 Gauge Imada ZP-200 N (Imada Co., Ltd., Ai- chi, Japan) according to the method of Utama, 1 Treatments: SF0, seaweed flour 0%; SF2.5, Jeong, Kim, Barido and Lee (2019) with modific- seaweed flour 2.5%; SF5, seaweed flour 5.0%; ations. The sample was compressed to 1 cm from SF7.5, seaweed flour 7.5%. The addition the surface, with a test speed of 1.0 mm/s, using level was weight per meat weight basis. a 35-mm cylinder probe. The first peak was identified as hardness and expressed in Newton (N). The measurement of hardness was performed in triplicate. The instrumental surface color of the 2.3 Analysis methods meatball was recorded at five random locations, in triplicate, based on the International Commis- Proximate composition and pH sion on Illumination’s system for lightness (CIE determination L*), redness (CIE a*) and yellowness (CIE b*) using a CR-10 Plus color reader (Konica Minolta Proximate composition; moisture, crude fat, Inc., Tokyo, Japan). The light source of illumin- crude protein, crude fiber and ash content were ant D65 (10o observer) with 8 mm aperture and determined according to AOAC official methods attached-closed cone was calibrated using a white (AOAC, 2005). Ground samples were dried in plate (Y = 93.6, X = 0.3134, y = 0.3194). an oven at 105oC for 24 h to determine moisture content. Ether extraction was performed to Sensory evaluation analyze the crude fat content using the Soxhlet system. Nitrogen content was determined using A total of 5 trained panelists were asked to eval- the Kjeltec system (2200 Kjeltec Auto Distilla- uate the samples. Panelists were faculty mem- tion Unit, Foss, Hillerod, Denmark). Crude pro- bers who had been trained for three sessions us- tein was then calculated as nitrogen content mul- ing commercial Indonesian-style beef meatballs. tiplied by 6.25. Crude fiber was determined by This study was approved by the research eth- acid hydrolysis with 1.25% H2SO4 and followed ics committee (REC) of Universitas Brawijaya. by alkaline hydrolysis with 1.25% NaOH. Ash Drinking water to clean the palate and remove content was determined by burning the samples residual flavors was provided. Samples were re- in a muffle furnace at 550oC for 8 h. heated using a microwave for 1 min, put on For pH determination, the ground sample (5 g) white dishes which were individually labeled with was added to 10 mL distilled water and homo- 3-digit random numbers, and served to panel- genized at 2500 rpm for 2 min. The pH of the ists in random order. Three sessions were per-

IJFS February 2021 Volume 10 pages SI112–SI120 SI114 Widati et al. formed using two samples per session. Color, 3 Results and Discussion texture, aroma, taste and overall acceptance of the samples were assessed. The 5-scales ad- 3.1 Proximate composition apted from Watts, Ylimaki, Jeffery and Elias (1989) were used for sensory evaluation. Color: 1 The addition of seaweed (Eucheuma cottonii) (dark brown and dark grey), 2 (dark brown and flour affected the proximate composition of grey), 3 (brown and grey), 4 (light brown and the Indonesian-style beef meatball significantly grey), 5 (light brown and light grey). Texture: 1 (Table2). The fat, protein and ash contents of (mushy), 2 (moderately mushy), 3 (moderately the cooked Indonesian-style beef meatball with chewy), 4 (chewy), 5 (very chewy). Aroma: 1 added seaweed flour were <=2.0%, >=9.0% and (very dominant seaweed aroma), 2 (dominant <=3.0%, respectively, in accordance with the na- seaweed aroma), 3 (balance seaweed and meat tional standard (Badan Standardisasi Nasional, aroma), 4 (dominant meat aroma), 5 (very dom- 1995). However, the moisture content did not inant meat aroma). Taste: 1 (very dominant sea- comply with the standard as it was greater than weed taste), 2 (dominant seaweed taste), 3 (bal- 70%. An increasing level of seaweed flour ad- ance seaweed and meat taste), 4 (dominant meat ded to meatballs reduced the moisture content taste), 5 (very dominant meat taste). (P < 0.01) as the flour increased the proportion of dry matter. The moisture content of seaweed flour itself was 13.83%. The moisture content of the Indonesian-style beef meatball was over the Microstructure standard of 70% because ice used in this study was 30% of the meat weight and the beef was ex- A scanning electron microscope (SEM, TM3000, tremely lean with fat, protein and moisture con- Hitachi High-Technologies Corp., Tokyo, Ja- tents of 4.23%, 18.70% and 74.5%, respectively. pan) was used to observe the microstructure The use of lean beef and the addition of seaweed of the Indonesian-style beef meatballs. The flour also reduced protein (P < 0.05) and fat (P method was adapted from Hashemi and Jafarp- < 0.01) content of the cooked Indonesian-style our (2016). A 1-2 mm thick slice of each sample beef meatball. was fixed with 2.5% glutaraldehyde in 0.2 M The presence of fiber in seaweed flour contrib- phosphate buffer (pH 7.0) for 2 h, washed and uted positively (P < 0.01) to the crude fiber soaked in distilled water for 1 h, and serial de- and ash content of the cooked Indonesian-style hydrated with ethanol in distilled water (50%, beef meatball. The crude fiber and ash con- 60%, 70%, 80%, 90% and 100%, v/v) for 1 h. tent of the seaweed flour used in this study were The dried sample was put onto a holder, coated 16.52% and 11.46%, respectively. Therefore, sea- with gold and visualized under SEM at a magni- weed flour added fiber and dry matter to the Ö fication of 2000. meatballs. Hartati and Endang (2011) reported that the unprocessed Eucheuma cottonii contains 26.83% moisture, 0.46% fat, 2.75% protein, 2.4 Statistical analysis 5.07% crude fiber and 17.86% ash. After oven- drying at 90-100oC for 12 h and milling, the Eucheuma cottonii flour comprised 87.99% dry This study to determine the effect of addition matter of which 38.77% was water-soluble diet- level of seaweed employed a completely random- ary fiber and 43.17% was insoluble dietary fiber ized design. There were four seaweed addition (Wresdiyati, Hartanta & Astawan, 2008). levels (0%, 2.5%, 5.0% and 7.5%) and three rep- lications. One-way analysis of variance (AN- OVA) was employed to determine the effect of ad- 3.2 Physical characteristics dition level of seaweed. Significant differences (P < 0.05) of mean values among treatments were The addition of seaweed (Eucheuma cottonii) determined using Duncan’s multiple range test. flour affected physical characteristics of the

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Table 2: Proximate composition of an Indonesian-style beef meatball made with diﬀerent addition levels of seaweed (Eucheuma cottonii) ﬂour

Treatment1 Significance Variable SF0 SF2.5 SF5 SF7.5 level2 Moisture (%) 72.22c ± 0.20 72.15bc ± 0.38 71.37ab ± 0.29 71.25a ± 0.18 ** Crude fat (%) 0.27b ± 0.04 0.12a ± 0.03 0.08a ± 0.01 0.07a ± 0.01 ** Crude protein (%) 12.00b ± 1.11 9.67a ± 1.12 9.90a ± 0.50 10.31a ± 0.15 * Crude fiber (%) 0.21a ± 0.02 0.33b ± 0.03 0.45c ± 0.01 0.58d ± 0.05 ** Ash 0.44a ± 0.01 0.62ab ± 0.07 0.80bc ± 0.18 0.94c ± 0.05 ** Data are presented as mean ± standard deviation (n=3). 1 Treatments: SF0, seaweed flour 0%; SF2.5, seaweed flour 2.5%; SF5, seaweed flour 5.0%; SF7.5, seaweed flour 7.5%. The addition level was weight per meat weight basis. 2 Significance level: * P < 0.05; ** P < 0.01.

Table 3: Physical characteristics of an Indonesian-style beef meatball made with diﬀerent addition levels of seaweed (Eucheuma cottonii) ﬂour

Treatment1 Significance Variable SF0 SF2.5 SF5 SF7.5 level2 pH 5.94a ± 0.10 6.00abc ± 0.03 6.03bc ± 0.08 6.07c ± 0.05 * Cooking loss (%) 1.67c ± 0.19 1.36bc ± 0.20 0.99ab ± 0.03 0.74a ± 0.08 ** Water-holding capacity (%) 50.67a ± 5.41 59.63abc ± 3.71 63.80bc ± 1.20 64.11c ± 3.23 ** Hardness (N) 12.00a ± 0.43 13.83a ± 0.20 16.83b ± 1.20 20.83c ± 1.34 ** CIE L* 48.30b ± 1.32 46.83ab ± 1.24 44.63a ± 0.49 43.93a ± 1.16 ** CIE a* 20.07c ± 0.67 19.10bc ± 0.52 17.47ab ± 0.90 16.60a ± 2.04 * CIE b* 16.00b ± 0.65 14.93ab ± 0.11 14.00a ± 0.61 13.53a ± 0.66 ** Data are presented as mean ± standard deviation (n=3). 1 Treatments: SF0, seaweed flour 0%; SF2.5, seaweed flour 2.5%; SF5, seaweed flour 5.0%; SF7.5, seaweed flour 7.5%. The addition level was weight per meat weight basis. 2 Significance level: * P < 0.05; ** P < 0.01.

Indonesian-style beef meatball (Table3). The of prerigor beef is usually above 5.90 (Utama et pH and water-holding capacity of the cooked al., 2017). However, the pH of the Indonesian- Indonesian-style beef meatball were enhanced by style beef meatball with added seaweed flour was addition of seaweed flour. In contrast, cooking higher than that of the control. The pH increas- loss, L* value (lightness), a* (redness) and b* ing effect of seaweed flour was caused by the (yellowness) declined as the addition level of sea- presence of alkaline minerals such as sodium, po- weed flour was increased. tassium, calcium and magnesium (Fitri, 2012). The pH of the control meatballs was lower than The pH of the seaweed flour used in this study normal Indonesian-style beef meatballs (6.05- was 7.89±0.59. 6.07) according to Purnomo (2012), as the beef Seaweed flour improved texture, particularly used in this study was purchased after 24-h hardness, and water-holding capacity of the postmortem with a pH of 5.64±0.10. In gen- Indonesian-style beef meatball resulting in an eral, an Indonesian-style meatball is made from enhanced cooking yield. These were caused by early postmortem beef but the quality is com- k-carrageenan in Eucheuma cottonii that pos- parable with that made from late postmortem sesses good water-binding properties and thus beef (Purnomo & Rahardiyan, 2008). The pH strengthens the gel matrix in heat-induced meat

IJFS February 2021 Volume 10 pages SI112–SI120 SI116 Widati et al.

Table 4: Sensory characteristics of an Indonesian-style beef meatball made with diﬀerent addition levels of seaweed (Eucheuma cottonii) ﬂour

Treatment1 Significance Variable SF0 SF2.5 SF5 SF7.5 level2 Color 4.44c ± 0.51 4.22bc ± 0.55 3.89ab ± 0.90 3.56a ± 1.15 ** Texture 3.11a ± 1.28 3.61ab ± 1.04 4.17bc ± 0.71 4.33c ± 0.84 ** Aroma 3.61c ± 0.98 3.17bc ± 0.62 2.67ab ± 0.85 2.28a ± 0.96 ** Taste 4.11c ± 0.83 3.67bc ± 0.77 2.72a ± 0.83 2.11a ± 1.13 ** Data are presented as mean ± standard deviation (n=3). Color: 1 (dark brown and dark grey), 2 (dark brown and grey), 3 (brown and grey), 4 (light brown and grey), 5 (light brown and light grey). Texture: 1 (mushy), 2 (moderately mushy), 3 (moderately chewy), 4 (chewy), 5 (very chewy) Aroma: 1 (very dominant seaweed aroma), 2 (dominant seaweed aroma), 3 (balanced seaweed and meat aroma), 4 (dominant meat aroma), 5 (very dominant meat aroma). Taste: 1 (very dominant seaweed taste), 2 (dominant seaweed taste), 3 (balanced seaweed and meat taste), 4 (dominant meat taste), 5 (very dominant meat taste). 1 Treatments: SF0, seaweed flour 0%; SF2.5, seaweed flour 2.5%; SF5, seaweed flour 5.0%; SF7.5, seaweed flour 7.5%. The addition level was weight per meat weight basis. 2 Significance level: ** P < 0.01.

emulsion products and increases cooking yield to emulsion-type meat products to improve yield (Purnomo, 2012). DeFreitas, Sebranek, Olson and texture, and also offer nutritional benefits and Carr (1997) mentioned that k-carrageenan (Talukder, 2015). has one ester sulfate and one negative charge The decrease in color variables caused by the ad- between sulfate chains, thus enhancing water- dition of seaweed (Eucheuma cottonii) flour res- binding properties of the meat batter. Mi- ulted in a darker appearance of the Indonesian- crographs of the Indonesian-style beef meatball style beef meatball. The brown appearance of (Fig.1) show that i) seaweed ( Eucheuma cot- the meatball is the result of globin hemochro- tonii) flour could interact with beef protein, ta- mogen denaturation during thermal processing pioca and water to build a compact gel matrix (Pearson & Gillett, 2012). In addition, the color and ii) the increasing level of addition reduced of seaweed flour used in this study was light the amount and size of the pores. The pore size brown. Therefore, the L*, a* and b* values of found in the control group was larger than that the treatment groups were lower than those of of treatment groups. A previous study also re- the control and the effect was level dependent. ported that k-carrageenan in Eucheuma cottonii Veronika, Mappiratu and Sumarni (2017) repor- improved gel strength and water-holding capa- ted that the pigment extracted from Eucheuma city of catfish sausage (Yakhin et al., 2015). Car- cottonii was light brown. The use of Eucheuma rageenan could also be used as a fat replacer in cottonii flour at more than 1% in a fish meatball emulsion-type meat products to reduce cooking also resulted in a darker appearance (Manuel et loss and improve texture (Cierach, Modzelewska- al., 2015). Kapitula & Szacilo, 2009). Eucheuma cottonii as a source of dietary fiber could be used to add fiber in processed food (Kurniawan et al., 2012). 3.3 Sensory characteristics The capillary-like structure of dietary fiber has a The addition of seaweed (Eucheuma cottonii) function to absorb water thus it could be added flour affected (P < 0.01) the sensory characterist-

IJFS February 2021 Volume 10 pages SI112–SI120 Seaweed ﬂour-added Indonesian-style beef meatball SI117

Figure 1: Microstructure of an Indonesian-style beef meatball with added seaweed flour. SF0 (seaweed flour 0%), SF2.5 (seaweed flour 2.5%), SF5 (seaweed flour 5.0%), and SF7.5 (seaweed flour 7.5%). G = seaweed flour granule, M = matrix, and P = pore.

IJFS February 2021 Volume 10 pages SI112–SI120 SI118 Widati et al. ics of the Indonesian-style beef meatball (Table Acknowledgements 4). Trained panelists revealed that higher addition levels of seaweed flour resulted in darker The authors would like to thank the Director- meatballs. The normal and acceptable color ate General of Higher Education of the Republic of an Indonesian style-meatball should combine of Indonesia (DIKTI) for Beasiswa Pendidikan light brown and light grey with a pink note Pascasarjana and the Faculty of Animal Science, (Badan Standardisasi Nasional, 2014). In this Universitas Brawijaya for financial support. study, the color of the Indonesian-style beef meatball ranged from 3.56 (brown and grey) to References 4.44 (light brown and grey). Among treatments, the color of the SF2.5 group was comparable AOAC. (2005). Official Methods of Analysis. with that of the control. Seaweed flour increased 18th ed, Gathersburg (MD): Association of chewiness and the dominancy of seaweed aroma Official Analytical Chemists International and taste. Pramuditya and Yuwono (2014) re- (AOAC). ported that the most favorable Indonesian-style Badan Standardisasi Nasional. (1995). SNI beef meatball has a chewy texture and is not Bakso Daging. SNI 01-3818-1995. Badan mushy. Cierach et al. (2009) mentioned that k- Standardisasi Nasional, Jakarta. carrageenan (10% addition level) improves the Badan Standardisasi Nasional. (2014). Standar texture and color of frankfurters. In this study, Nasional Indonesia-Bakso Daging. SNI panelists noted that the seaweed aroma was more 3818. Badan Standardisasi Nasional, dominant in samples with more than 2.5% SF. A Jakarta. previous study reported that the addition level Campo, V. L., Kawano, D. F., da Silva, D. B., Jr. of Eucheuma cottonii up to 1.0% (the maximum & Carvalho, I. (2009). Carrageenans: Bio- level used in the study) did not affect the sensory logical properties, chemical modifications characteristics including color, aroma and taste and structural analysis-a review. Carbo- of catfish meatballs (Yakhin et al., 2015). Among hydrate Polymers, 77 (2), 167–180. doi:10. treatments, the sensory characteristics of the 1016/j.carbpol.2009.01.020 SF2.5 group were comparable with those of the Cierach, M., Modzelewska-Kapitula, M. & Sza- control. These results suggest that seaweed flour cilo, K. (2009). The influence of car- could be added at 2.5% of meat weight to pro- rageenan on the properties of low-fat duce Indonesian style-beef meatballs with sens- frankfurters. Meat Science, 82 (3), 295–299. ory characteristics comparable to original ones doi:10.1016/j.meatsci.2009.01.025 and enhanced fiber content. DeFreitas, Z., Sebranek, J. G., Olson, D. G. & Carr, J. M. (1997). Carrageenan effects on thermal stability of meat proteins. Journal of Food Science, 62 (3), 544–547. doi:10 . 1111/j.1365-2621.1997.tb04426.x 4 Conclusion Fitri, M. (2012). Kajian sifat fisiko-kimia karaginan dari rumput laut jenis eucheuma sp. di perairan sulawesi selatan. Jurnal Galung Seaweed (Eucheuma cottonii) flour addition in- Tropika, 2 (2). creased hardness, water-holding capacity and Grau, R. & Hamm, R. (1953). Eine ein- fiber content of an Indonesian-style beef meat- fache methode zur bestimmung der wasser- ball. It also increased the seaweed aroma when bindung im muskel. Naturwissenschaften, added at more than 2.5%. Therefore, the ad- 40 (1), 29–30. dition of seaweed flour at 2.5 % of meat weight Guerrero, S. N., Campos, C. A. & Alzamora, could be applied to produce Indonesian-style beef S. M. (2002). Development of shelf stable meatballs with enhanced fiber content and sens- seaweed by hurdle processing. Food Science ory properties comparable to the original ones.

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and Technology International, 8 (2), 95–99. syarat tambahan dalam sni dan pengaruh doi:10.1106/108201302024588 lama pemanasan terhadap tekstur bakso Hartati, H. & Endang, M. (2011). Effect of [in press oktober 2014]. Jurnal Pangan dan Euchema cottonii seaweed as the natural Agroindustri, 2 (4), 200–209. gelling agent for quality beef meatball. Ber- Purnomo, H. & Rahardiyan, D. (2008). Review ita Litbang Industri: Media Publikasi dan article: Indonesian traditional meatball. In- Komunikasi Peneliti Industri, 2 (47), 54– ternational Food Research Journal, 15 (2), 65. Retrieved from https://lib.atmajaya. 101–108. ac . id / default . aspx ? tabID = 61 & id = Purnomo, H. (2012). Teknologi pengolahan dan 240579&src=a pengawetan daging. Universitas Brawijaya Hashemi, A. & Jafarpour, A. (2016). Rheolo- Press. gical and microstructural properties of beef Rifqie Mariana, R., Hidayati, L. & Soekopitojo, sausage batter formulated with fish fil- S. (2019). Implementing the haccp sys- let mince. Journal of Food Science and tem to the production of bakso malang- Technology-mysore, 53 (1), 601–610. doi:10. indonesia. Journal of Culinary Science & 1007/s13197-015-2052-4 Technology, 17 (4), 291–312. Kurniawan, A. B., Al-Baarri, A. N. & Kusra- Rohman, A., Erwanto, Y., Man, Y. B. C. et hayu, K. (2012). Kadar serat kasar, daya al. (2011). Analysis of pork adulteration ikat air, dan rendemen bakso ayam dengan in beef meatball using fourier transform penambahan karaginan. Jurnal Aplikasi infrared (ftir) spectroscopy. Meat Science, Teknologi Pangan, 1 (2). 88 (1), 91–95. Lee, B. J., Hendricks, D. G. & Cornforth, D. P. Talukder, S. (2015). Effect of dietary fiber (1998). Effect of sodium phytate, sodium on properties and acceptance of meat pyrophosphate and sodium tripolyphos- products: A review. Critical Reviews in phate on physico-chemical characteristics Food Science and Nutrition, 55 (7), 1005– of restructured beef. Meat Science, 50 (3), 1011. doi:10.1080/10408398.2012.682230 273–283. doi:10 . 1016 / S0309 - 1740(98 ) Tan, M. G. (2002). Chinese dietary culture in 00002-3 indonesian urban society. The globalization MacArtain, P., Gill, C. I. R., Brooks, M., Camp- of Chinese food, 152 (1997), 33–65. bell, R. & Rowland, I. R. (2007). Nutri- Utama, D. T., Jeong, H. S., Kim, J., Bar- tional value of edible seaweeds. Nutrition ido, F. H. & Lee, S. K. (2019). Fatty Reviews, 65 (12, 1), 535–543. doi:10.1301/ acid composition and quality properties nr.2007.dec.535-543 of chicken sausage formulated with pre- Manuel, J., Cornelia, M. & Wijaya, W. (2015). emulsified perilla-canola oil as an animal Utilization of eucheuma cottonii and euch- fat replacer. Poultry Science, 98 (7), 3059– euma spinosum flour in narrow-barred 3066. doi:10.3382/ps/pez105 spanish mackerel meatballs. In R. Sety- Utama, D. T., Lee, S. G., Baek, K. H., Chung, obudi, J. Burlakovs, M. Mel, P. Adinurani W. S., Chung, I. A., Jeon, J. T. & Lee, S. K. & Z. VincevicaGaile (Eds.), International (2017). High pressure processing for dark- symposium on aquatic product processing firm-dry beef: Effect on physical properties (isapprosh) 2013 (Vol. 1, pp. 12–18). KnE and oxidative deterioration during refriger- Life Sciences. 1st International Symposium ated storage. Asian-australasian Journal of on Aquatic Product Processing, Safety and Animal Sciences, 30 (3), 424–431. doi:10 . Health (ISAPPROSH), Bogor, INDONE- 5713/ajas.16.0175 SIA, NOV 13-14, 2013. doi:10.18502/kls. Veronika, H. H., Mappiratu, M. & Sumarni, v1i0.78 N. K. (2017). Ekstraksi dan karakterisasi Pearson, A. M. & Gillett, T. A. (2012). Processed ekstrak zat warna rumput laut (eucheuma meats. Springer. cottonii). KOVALEN: Jurnal Riset Kimia, Pramuditya, G. & Yuwono, S. S. (2014). Pen- 3 (1), 7–16. entuan atribut mutu tekstur bakso sebagai

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Watts, B. M., Ylimaki, G. L., Jeffery, L. E. & Elias, L. G. (1989). Basic sensory methods for food evaluation. IDRC, Ottawa, ON, CA. Wresdiyati, T., Hartanta, A. B. & Astawan, M. (2008). The effect of seaweed eucheuma cottonii on superoxide dismutase (sod) liver of hypercholesterolemic rats. HAYATI Journal of Biosciences, 15 (3), 105–110. Yakhin, L. A., Wijaya, K. M. & Santoso, J. (2015). The effect of seaweed powder (eucheuma cottonii) addition in catfish sausage. In R. Setyobudi, J. Burlakovs, M. Mel, P. Adinurani & Z. Vincevica- Gaile (Eds.), International symposium on aquatic product processing (isapprosh) 2013 (Vol. 1, pp. 1–5). KnE Life Sciences. 1st International Symposium on Aquatic Product Processing, Safety and Health (IS- APPROSH), Bogor, INDONESIA, NOV 13-14, 2013. doi:10.18502/kls.v1i0.76

IJFS February 2021 Volume 10 pages SI112–SI120 International Journal of Food Studies IJFS February 2021 Volume 10 pages SI121–SI132

Potential for Development of Novel Food Products from Azanza garckeana Tree Fruit: A Review

Johnson Mwove

Department of Human and nutrition sciences, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000 – 00200, Nairobi, Kenya Department of Plant Sciences, Chuka University, P.O. Box 109 – 60400, Nairobi, Kenya [email protected]

Received: 22 October 2018; Published online: 24 February 2021

Abstract

Azanza garckeana is among the least utilized indigenous wild fruit trees of interest in the arid and semi-arid regions of Africa. The tree’s fruit and seeds have found their importance as food while their barks and leaves as medicine because of their vast nutritional and functional components. This paper reviews the utilization of the fruit in food processing demonstrating the potential this species has in the preparation of novel foods. There are few reports on macronutrients and micronutrient composition of the fruit and the seeds, and their utilization in food processing. Some researchers have identiﬁed key functional ingredients in the fruits as well as their seeds that could be of beneﬁt when incorporated in the production of value-added food products. This paper not only advocates for the production of value-added food products from this fruit, but also its integration into farming systems to enhance nutritional security and provide ready income for communities in the dry areas in sub Saharan Africa. Keywords: Wild tree fruits; Value addition; Functional ingredients; Dryland resources

1 Introduction most developing countries, in especially mothers and children (Kehlenbeck, Asaah & Jamnadass, 2013; Motlhanka & Makhabu, 2011; Simitu et Indigenous fruit trees for which research is lim- al., 2009). ited in Sub-Saharan Africa have accumulated Among the most widely used indigenous wild considerable interest for their role in poverty re- fruit trees of interest is A. garckeana (Bélair, duction (Akinnifesi, Ajayi, Sileshi, Kadzere & Ichikawa, Wong & Mulongoy, 2010; Legwaila, Akinnifesi, 2007; Awodoyin et al., 2015; Shum- Mojeremane, Madisa, Mmolotsi & Rampart, sky, Hickey, Pelletier & Johns, 2014; Simitu, 2011; Packham, 1993). It is also referred by Jamnadass, Kindt, Kimiywe & Kungu, 2009). its synonym, Thespesia garckeana. It is a semi- Many countries in this region have vast numbers deciduous tree, producing fruits with a rough and of these trees that are mostly underexploited. hairy bark. The fruit is greyish brown in col- Simitu et al. (2009), reported that Kenya has our and fibrous with longitudinal fissures. The some 400 such indigenous fruit plants. These fruit is sweet and edible, containing 15-30 seeds trees show great potential for providing food, with brownish and woolly floss. The ripe fruit is micronutrients as well as income for smallholder available from December to March mostly from farmers and their families thereby advancing the eastern and southern Africa from Sudan to South fight against the existing micronutrient deficien- Africa. The plant is common in dry areas with cies and food insecurity among populations in

Copyright ©2021 ISEKI-Food Association (IFA) 10.7455/ijfs/10.SI.2021.a10 SI122 Johnson Mwove limited rainfall ranging from 600-800 mm (Jam- zinc (Abass & Ahmed, 2017; Danbature, Yirank- nadass et al., 2013; Maundu & Tengnas, 2005; inyuki, Magaji & Ibrahim, 2015; Jacob, Shehu Michael, Onyia & Jidauna, 2015). The fruit, et al., 2016; Nkafamiya et al., 2016; Saka & bark and leaves are considered to be of great Msonthi, 1994; Suliman, Difa & Salih, 2012) importance (Ochokwu, Dasuki & Oshoke, 2015). as well as a vast amount of various vitamins, For instance, the rural semi-arid areas of Kenya vitamin C (Danbature et al., 2015; Nkafamiya rely on these indigenous fruits to supplement et al., 2016), vitamin A (Michael et al., 2015; their diets (Kehlenbeck et al., 2015; Shumsky Nkafamiya et al., 2016), vitamin B1, B2, and et al., 2014; Simitu et al., 2009). In addition, E (Nkafamiya et al., 2016). Furthermore, the marketing surveys have revealed that A. garck- fruit has been reported to have a remarkable eana already has commercial potential as it is composition of phytochemicals, including, alkal- sold in local markets (Akinnifesi et al., 2007; oids, flavonoids, tannins (Ahmed, El Hassan & Krog, Theilade, Hansen & Ruffo, 2005; Muok, El Hadi, 2016; Dikko, Khan, Tor-Anyiin, Anyam Owuor, Dawson, Were et al., 2000). Similarly, in & Linus, 2016; Nkafamiya et al., 2016), steroids, Sudan, the fruit is processed and marketed for terpenoids, saponins (Dikko et al., 2016), triter- household income (Abass & Ahmed, 2017). In penes and cumarins (Ahmed et al., 2016), most Botswana, it is a valuable edible indigenous fruit of which have a reported health effect. Further- widely distributed and used to supplement local more, Michael et al. (2015) and Nkafamiya et food (Legwaila et al., 2011; Ochokwu et al., 2015) al. (2016) reported A. garckeana seed to have whereas, in South Africa, the ripe fruit carpels 3.40% carotenoids and 75.00±0.23 mg/100 g Vit- are widely used for food as well as food addit- amin A, respectively. Therefore, their consump- ives (Maroyi, 2017; Mojeremane & Tshwenyane, tion has potential of preventing a wide range of 2004). Moreover, in Nigeria, it has a high social deficiency diseases (Simitu et al., 2009). These and economic value (Jacob, Shehu, Danbature results show the ability of this tree fruit to not & Karu, 2016; Ochokwu et al., 2015) while in only advance the fight against micronutrient defi- Malawi, the nutritional value of the fruit is highly ciencies but also its ability to reduce PEM which valued (Saka & Msonthi, 1994). Thus, this tree has plagued the residents of sub-Saharan Africa can be a great resource for the people living in and most developing countries around the world. the range lands. Nevertheless, there is limited Few researchers have tried to study the fruit’s po- information on its composition as well as its util- tential for application in the food industry. The ization in value addition of foods. production of juice has been attempted (Suliman These fruits are excellent sources of both mac- et al., 2012) while its potential in the produc- ronutrients and micronutrients. They have been tion of caramel colour has been demonstrated reported to be high in fibre, total carbohydrates, (Benhura, Mbuya & Machirori, 1999). Further- protein level, ash and low in fat (Abass & Ahmed, more, its functional ingredients (Table3) have 2017; Nkafamiya, Ardo, Osemeahon & Akinter- been described showing a considerable array of inwa, 2016; Saka & Msonthi, 1994). Moreover, nutrients (Ahmed et al., 2016; Danbature et al., the amino acid profile presented by Nkafamiya 2015; Dikko et al., 2016; Nkafamiya et al., 2016; et al. (2016) showed that A. garckeana can sup- Ochokwu et al., 2015; Simitu et al., 2009) that ply nine non-essential amino acids (alanine, ar- are useful for supplementing food products, and ginine, aspartic acid, cysteine, glycine, glutamic also a range of antimicrobial constituents (Dikko acid, proline, serine and tyrosine) and eight es- et al., 2016) that are very useful and effective sential amino acids (histidine, isoleucine, leu- in food preservation. However, more research is cine, lysine, methionine, phenylalanine, threon- needed to provide adequate nutrient databases ine and valine). This shows that this fruit may to promote its utilization in food. According be important in the fight against protein en- to Grivetti and Ogle (2000), the lack of ad- ergy malnutrition (PEM). The fruits have been equate nutrient databases, due to limited and reported to have a variety of minerals includ- uneven compositional data, limits educational ef- ing phosphorus, calcium, magnesium, iron, po- forts to improve diets in many developing coun- tassium, sodium, cobalt, copper, manganese and tries. Therefore, enhanced value addition and

IJFS February 2021 Volume 10 pages SI121–SI132 Novel food products from Azanza garckeana SI123 support of agroforestry is important. However, sential amino acid (histidine, isoleucine, leucine, the promotion of wild trees for food has not been lysine, methionine, phenylalanine, threonine and adequately fostered by countries and thus, as a valine). In their work, they reported a very high consequence, they remain underutilized (Simitu level of protein (12%). Therefore, A. garckeana et al., 2009). Farming A. garckeana will not only has potential to advance the fight against PEM enhance the effort towards nutrition security but which has plagued the sub-Saharan Africa for will also advance the battle against food insec- generations (Bain et al., 2013). Most populations urity through creation of value-added products in these regions rely on highly deficient sources as well as the creation of alternative sources of of food that has resulted in high and increasing income for the people living in range lands. The prevalence of deficiency diseases (Harika, Faber, aim of this review was to explore the nutritional Samuel, Mulugeta et al., 2017). A. garckeana value as well as value addition potential for A. fruit can significantly contribute to reduced PEM garckeana in the food industry to guide efforts by contributing significantly to the protein con- aimed at not only determining the quality char- tent and quality of the daily diets. acteristics of A. garckeana tree fruit, but also utilizing it among other edible wild fruit tree species in value addition of food products. 2.2 Micronutrients

The fruits have been reported to have a variety 2 Fruit Composition of minerals including phosphorus, calcium, magnesium, iron, potassium, sodium, cobalt, cop- 2.1 Macronutrients per, manganese and zinc (Abass & Ahmed, 2017; Danbature et al., 2015; Jacob, Shehu et al., 2016; There is limited research on the macronutrients Nkafamiya et al., 2016; Saka & Msonthi, 1994; composition of this fruit as shown in Table1. Suliman et al., 2012) as shown in Table2. In Nevertheless, the fruits have been reported to addition, the fruit has also been reported to be high in fibre, total carbohydrates, protein have a vast amount of various vitamins includ- level, ash and low in fat. These values are ing vitamin A (Michael et al., 2015; Nkafam- comparable to other wild fruits; Strychnos spin- iya et al., 2016), ascorbic acid (23.664mg/100g) osea, Detarium microcarpum, Diospyros mespili- (Danbature et al., 2015), and vitamin B1,B2,C formis, Dialium guineese, and Gardenia terni- and E at 1.28±0.97, 1.18±0.45, 319.09±0.45 and folia whose moisture contents ranged between 3.08±0.55 mg/100 g, respectively (Nkafamiya et (6.17-10.70%); crude fat (2.04-8.85%); crude pro- al., 2016). Furthermore, Michael et al. (2015) tein (5.16-6.80%); crude fiber (7.23-19.65%); Ash and Nkafamiya et al. (2016) reported A. garck- (3.46-5.56%); and carbohydrate (57.77-69.79%) eana seed to have 3.4% carotenoids and 0.075% (Jacob, Mann, Adeshina & Ndamitso, 2016). Vitamin A, respectively. Both mineral and vit- Nevertheless, despite the great variability, A. amin analysis shows that A. garckeana could garckeana can provide more dietary fiber ( 36.0 contribute a considerable amount of both micro - 45.5% dry weight), (Abass & Ahmed, 2017; and macro elements to human nutrition. The Nkafamiya et al., 2016; Saka & Msonthi, 1994). results show that the fruit has potential to help The fibre content is also higher than that of in the battle against the prevalence of vitamin baobab (Adansonia digitate L.) fruit pulp, an- A deficiency, as well as mineral deficiencies in other commonly consumed wild fruit, with a sub-Saharan Africa. Based on RDI values (Joint crude fibre of 8.68% (dry weight) recorded in FAO/ WHO expert committee, 2005), 100g of Kenya (Muthai et al., 2017). Moreover, the the fruit is able to supply sufficient levels of Vit- amino acid profile presented by Nkafamiya et amin B1, B2, and C to infants, adolescents and al. (2016) showed that A. garckeana can sup- adult males and females per day. According to ply nine non-essential amino acids (alanine, ar- Jamnadass et al. (2013) consuming 100 g of A. ginine, aspartic acid, cysteine, glycine, glutamic garckeana fruit pulp can contribute approxim- acid, proline, serine and tyrosine) and eight es- ately 50% of the vitamin C daily requirement of

IJFS February 2021 Volume 10 pages SI121–SI132 SI124 Johnson Mwove iei 013.310----8.3-Jcb hh,DnaueadKr ( 2016 ) Karu ( 2017 ) and ( 2012 ) Danbature Ahmed Salih Shehu, and and Jacob, Abass Difa Suliman, - - - ( 2016 ) ( 1994 ) Akinterinwa Msonthi and and Osemeahon Saka Ardo, Nkafamiya, 84.33 120.2 4.3 - - - 4.3 0.2 - - - - 4.5 - 0.2 - 52 - - 600 - 202 1860 13600 - 40 - 26190 60 120 120 84 1833.33 218 - 96250 293 1453 40 560 1270 columns. respective the in available values the from 913.7 95 obtained was Average 965 * D D Average* - 300 D - Sudan D D Nigeria 1476 Nigeria D ( Minerals Sudan Malawi Region iei .0-671.0114.02.0-Naaia ro smao n kneiw ( 2016 ) Akinterinwa and Osemeahon ( 2017 ) Ardo, Ahmed Nkafamiya, ( 1994 ) and ( 2017 ) Msonthi Abass Ahmed and Saka and Abass ( 2012 ) Salih and Difa Suliman, 52.02 61.39 - - 74.44 - 87.65 28.40 35.2 30.58 22.444 36.00 45.30 45.3 1.55 45.524 1.81 1.1 1.1 1.04 5.65 6.65 12.00 10.05 3.43 12.0 4.04 6.7 7.3 6.3 68.86 84.92 - Reference - % Sugar Total 52.8 % 15.07 - CHO Total - 6.50 % 13.542 Fiber - columns. respective the in % available Fat values the from obtained % was protein Average Crude ** speciﬁed, Not * W % D Ash D Average** % matter Dry * Sudan W % Sudan Moisture D Nigeria basis (D) Dry (W)/ Sudan Wet Malawi Region 2 0. 8 4 884 . . bs n he ( 2017 ) Ahmed and Abass - 3.7 3.7 - 44 1808 340 185 204.9 820 W aM eKN oC nZn Mn Cu Co Na K Fe Mg Ca P 14.31 W ± 8. 451.6 589.7 µ g/g) ± 0. 24938.6 500.8 ± .8-5.37 - 1.08 al 1: Table ± ± 27 5.68 22.71 74. 84.8 47545.9 al 2: Table znagarckeana Azanza ± ± .47.85 2.74 .310.22 1.43 ± znagarckeana Azanza 5713883.3 35.7 ± ± .11.30 3.11 .91.34 3.09 ± 26. 284.7 12167.5 ri arntin Composition Macronutrient Fruit ± ± .638.05 0.36 .142.20 0.41 ri iea Composition Mineral Fruit ± 8. 4.4 - 283.2 ± ± 05 48.44 10.57 .750.42 5.37 ± . 29.6 0.1 ± ± 67 - 36.77 24 - 32.42 ± 74- 47.4 Reference

IJFS February 2021 Volume 10 pages SI121–SI132 Novel food products from Azanza garckeana SI125 an adult man. In addition, the fruits may con- umented. Tannins have also been reported to tribute significantly to the daily requirements of accelerate blood clotting process, reduce blood both vitamins A and E. Nevertheless, the results pressure, decrease the serum lipid level, and in indicate a wide variation in fruits composition, modulating immune responses (Chung, Wong, possibly because of the varied regions from where Wei, Huang & Lin, 1998). On the other hand, these studies were carried out as well as the pos- steroids possess many documented medicinal, sibility of analysis being carried out on fruits at pharmaceutical and agrochemical activities like different stages of maturity. Furthermore, the anti-tumour, immunosuppressive, antibacterial, range of data determined for each one of the as well as cytotoxic activity (Patel & Savjani, studies is also highly varied. There is therefore 2015). Generally, most of these phytochemicals the need for more research on the composition of have found great application in the design and fruits of different regions, at similar harvesting manufacture of medicines (Adamu, Ushie, Lawal or maturity periods for ease of comparison. Op- & Oga, 2013; Maroyi, 2011). Besides their ap- timization of the harvesting period to facilitate plication in medicine, in the food industry, these the selection for and extraction of various fruit substances with antimicrobial effects can be util- constituents is also important. ized to achieve preservation in food products. According to Chung et al. (1998), the tannic acid has antimicrobial property and can therefore be 2.3 Functional components used in food processing to increase the shelf-life of foods such as catfish fillets. Functional foods contain physiologically active constituents that provide an additional health benefit beyond basic nutrition. This may include some plants products containing proven 2.4 Microbial quality and well documented physiologically active compounds that may improve human health and re- Research has revealed the potential of A. gar- duce the risk of chronic diseases (Choudhary & ckeana fruit extracts to act as antimicrobi- Tandon, 2009; Goldberg, 2012; Hasler, 2000; als. In a recent study, methanol and ethyl Milner, 1999; Reynolds & Martirosyan, 2016). acetate extracts containing flavonoids, steroids, These compounds with physiological activity are terpenoids, saponins, alkaloids, reducing sugars called phytochemicals (Johnson & Williamson, and tannins were found to inhibit methicillin 2003). The fruit (flesh and seeds) of A. garck- resistant Staphylococcus aureus, Staphylococcus eana have been reported to have significant con- aureus, Pseudomonas aeruginosa, Candida kru- tent of a variety of these substances (Table3). sei and Escherichia coli (Dikko et al., 2016). Most researchers have reported presence of al- Pathogenic ubiquitous microorganisms such as S. kaloids, flavonoids and tannins in the fruit ex- aureus have been attributed to many infections tracts (Ahmed et al., 2016; Dikko et al., 2016; and intoxications involving many food products. Nkafamiya et al., 2016). In addition, steroids, Chung et al. (1998), reported that tannic acid terpenoids and saponins (Dikko et al., 2016), could be used in preservation to increase the triterpenes and cumarins (Ahmed et al., 2016) shelf-life of foods such as catfish fillets. There- have also been identified. Various researchers fore, utilization of this fruit in food has poten- have reported their importance. For instance, tial to keep the food unadulterated or contam- alkaloids are used in the design of modern medi- inated with pathogens. This has great potential cine as natural or modified compounds due to in extending shelf life as well as enhancing food their proven enzyme inhibitory effects as well safety. Nevertheless, there is need to study the as their anticancer, anti-inflammatory, and an- effectiveness of these antimicrobial substances in timicrobial activity (Aniszewski, 2007; Wansi, whole fruits as well as whole fruit formulations to Devkota, Tshikalange & Kuete, 2013). In ad- determine their activities. Moreover, the charac- dition, tannins’ antimicrobial activity on fungi, terization of the microbial profile of these fruits yeasts, bacteria, and viruses has been well doc- is required.

IJFS February 2021 Volume 10 pages SI121–SI132 SI126 Johnson Mwove

Table 3: Functional ingredients in Azanza garckeana tree fruit

Phytochemicals Reference Alkaloids∗+ Adamu, Ushie, Lawal and Oga (2013), Ahmed, El Hassan and El Hadi (2016), Dikko, Khan, Tor-Anyiin, Anyam and Linus (2016), Michael, Onyia and Jidauna (2015), Nkafamiya, Ardo, Osemeahon and Akinterinwa (2016) Tanninss∗+ Adamu, Ushie, Lawal and Oga (2013), Ahmed, El Hassan and El Hadi (2016), Dikko, Khan, Tor-Anyiin, Anyam and Linus (2016), Michael, Onyia and Jidauna (2015), Nkafamiya, Ardo, Osemeahon and Akinterinwa (2016) Steroids∗ Dikko, Khan, Tor-Anyiin, Anyam and Linus (2016), Nkafam- iya, Ardo, Osemeahon and Akinterinwa (2016) Terpenoids∗ Adamu, Ushie, Lawal and Oga (2013), Ahmed, El Hassan and El Hadi (2016), Dikko, Khan, Tor-Anyiin, Anyam and Linus (2016), Nkafamiya, Ardo, Osemeahon and Akinterinwa (2016) Saponinss∗+ Adamu, Ushie, Lawal and Oga (2013), Ahmed, El Hassan and El Hadi (2016), Dikko, Khan, Tor-Anyiin, Anyam and Linus (2016), Michael, Onyia and Jidauna (2015) Cumarins* Ahmed, El Hassan and El Hadi (2016) Flavonoidss∗+ Adamu, Ushie, Lawal and Oga (2013), Ahmed, El Hassan and El Hadi (2016), Dikko, Khan, Tor-Anyiin, Anyam and Linus (2016), Michael, Onyia and Jidauna (2015), Nkafamiya, Ardo, Osemeahon and Akinterinwa (2016) Cardlac glycosides∗ Nkafamiya, Ardo, Osemeahon and Akinterinwa (2016) Phenolss∗+ Adamu, Ushie, Lawal and Oga (2013), Michael, Onyia and Jidauna (2015) Cyanogenic glucosides+ Michael, Onyia and Jidauna (2015) Carotenoidss+ Michael, Onyia and Jidauna (2015) *+ Reported in both fruit and seeds; + reported only in seeds

3 Protein energy malnutrition high. Moreover, high prevalence of anaemia, vit- and Micronutrient deficiencies amin A, zinc, and iodine deficiencies are of public health significance in children living in these areas. The fruit was reported to contain a vast Due to the vast levels of macro and micronutri- amount of ascorbic acid (28.5 mg/100g) (Jacob, ents in wild fruits (Grivetti & Ogle, 2000), their Shehu et al., 2016) which is important in the fight role in value addition to deficient foods cannot be against scurvy. The utilization of these fruits in underestimated. The fruit is highly nutritious food processing with the aim of providing de- with high reported levels of beta carotene and ficient nutrients in processed food has poten- iron. Some of these nutrients are deficient in the tial in alleviating malnutrition especially PEM diet for most of the population that live in the which is a major problem in developing coun- arid and semi-arid areas of sub-Saharan Africa. tries (Bain et al., 2013). The amino acid pro- According to Harika, Faber, Samuel, Kimiywe et file presented by Nkafamiya et al. (2016), shows al. (2017), in women aged between 15–49 years that the fruit’s protein profile contains all essen- and pregnant women in Kenya, Ethiopia, Nigeria tial amino acids except Tryptophan as well as 9 and South Africa, the prevalence of iron, zinc, non-essential amino acids including alanine, ar- iodine, folate and vitamin A deficiencies are very

IJFS February 2021 Volume 10 pages SI121–SI132 Novel food products from Azanza garckeana SI127 ginine, aspartic acid, cysteine, glycine, glutamic 4.3 Production of juice acid, proline, serine and tyrosine. Nevertheless, since the addition of substances in the processing Studies to determine the suitability of this fruit of foods may impact on the functionality of the for juice production have been successfully done various components of the food, more research is with promising results. According to Suliman et needed on the potential of utilizing this fruit in al. (2012) this fruit was successfully used to make food formulations. juice having a pH value, total soluble solid and ascorbic acid contents of 5.48, 4.5% and 21.13 mg/100 g, respectively. In their research, sensory 4 Food processing evaluation revealed that the overall acceptance was notable for A. garckeana fruits juice while 4.1 Production of caramel colours moulds and yeasts were absent in the juices. However, despite the promising results, the total Controlled heat treatment of carbohydrates pro- bacterial count found in the juice ranged from duces caramel, which can be utilized in various 1.20x107 to 1.72x106 CFU mL−1. This calls for applications. Caramel colours have application strict methodologies for processing to enhance in processed foods such as baked products and safety as well as the shelf life of the product. As drinks (Sengar & Sharma, 2014). The mucilage there is limited data on the utilization of this from A. garckeana fruit can be extracted with for juice making, more research inclined towards water and the extract utilized in various ways. quality as well as safety of A. garckeana fruits According to Benhura et al. (1999), the extract drink is needed. In addition, more research is re- can be heated in the presence of ammonium salts quired on the microbial quality and safety of the causing the sugars present to form colours de- resulting product. pending on the initial concentration of the mucilage. The result is a soluble caramelized material that can be used to impart colours ranging 4.4 Production of pectin from pale golden to dark brown to liquids and semisolids. This has potential for wide applic- Pectins are naturally occurring biopolymers that ation in juices, baked foods as well as in gums can be used in various food applications. They and candies not only for the colour but also the have gelling, emulsifying, and ﬁlm forming prop- taste and nutritional composition. The utiliza- erties that are ideal for various processing sys- tion of caramel colours will reduce the utiliza- tems and applications such as in the making of tion of synthetic ones which are associated with jams. The yield of pectin from A. garckeana many complications (Sengar & Sharma, 2014). fruits both dried and wet samples were in the Furthermore, according to Benhura et al. (1999), ranges of 4.65 to 24.38 % and 9.82 to 26.75 %, re- the insoluble material obtained when mucilage spectively (Joel, Barminas, Riki, Yelwa & Edeh, from A. garckeana fruit is heated can be used to 2018). Compared to other fruits such as Adan- impart shades of brown colours to powders. sonia digitate L whose highest pectin content in fruits was reported as 2.56 %, A. garckeana 4.2 Production of sweets fruit has very high levels. This makes it suitable for the production of pectin for commercial use. A. garckeana fruit has been used in the manufac- The addition of this fruit to jam formulations has ture of sweets with promising results. According the potential to enhance the gelling properties of to Abass and Ahmed (2017) it was used to pro- such formulations as well as the nutritional qual- duce a Turkish delight (Lokum) type sweet, a ity of these products. In addition, being resist- product made from starch and sugar. Owing to ant to degradation in the upper gastrointestinal its ability to produce caramel colours (Benhura tract, it may be ideal for use in enhancing the et al., 1999), the mucilage can be used in sweet growth of probiotic bacteria in the body as a pre- formulations to achieve the desirable caramel col- biotic (Gomez, Gullon, Yanez, Schols & Alonso, our and ﬂavour. 2016). Therefore, A. garckeana fruit can be ideal

IJFS February 2021 Volume 10 pages SI121–SI132 SI128 Johnson Mwove in the design of symbiotic products. Few studies have been launched to characterize the edible wild tree products to determine their commercial potential. Thus, 5 Integration into farming there is lack of adequate nutrient database systems for A. garckeana tree fruit. The data on fruit composition is very minimal and highly variable and influenced by the region. Re- Despite their wide natural occurrence in dry searchers who have analysed this fruit tar- areas, A. garckeana trees can be integrated into geted certain nutrients. As a result, data farming systems to support the nutritional se- on other nutrients is highly limited or un- curity and income of communities (Simitu et available. Thus, there is need to analyse the al., 2009). This is because these trees have po- fruit from different regions to provide suffi- tential for domestication. In Nigeria, an at- cient data to inform the probable fruit nutri- tempt has been made to integrate this plant tional quality. Furthermore, to obtain uni- into farming systems (Ochokwu et al., 2015). form data for ease of comparison, the period However, the promotion of wild trees has not of harvest or maturity has to be taken into been adequately fostered by agricultural and account during sampling of fruits. forestry institutions and thus, as a consequence, they remain underutilized (Simitu et al., 2009). The development of any tree fruit occurs Thus, there is need to promote the domestica- gradually with many physiological changes tion and commercialization of under-utilized tree taking place. As a result, the concentration products (Leakey, 1999). The domestication of of different fruit components also changes A. garckeana trees for agroforestry has potential gradually over time with each component for not only poverty alleviation but also envir- reaching its highest concentration at a par- onmental rehabilitation in sub-Saharan Africa. ticular time. The optimal time for extrac- However, this will largely depend on the expan- tion of these components may therefore not sion of the market demand for its value-added be the same. The determination of the ideal products. Hence, the need for value addition of time for harvesting and processing is also A. garckeana tree resources. important in the extraction of different components. A study targeting the extraction of these functional substances and determining 6 Recommendations and future the optimal harvesting time for highest yield prospects for each one is therefore important. Despite the promising nutritional and func- Millions of people in especially the developing tional quality of the fruit, minimal research countries depend on food from wild tree species exists on value added products containing to supplement their diets. Thus, wild tree spe- these fruits. Moreover, the potential for con- cies contribute significantly to human nutrition. sumption of A. garckeana fruit seeds as well However, many of these tree species are still un- as tree leaves has received minimal atten- known or underutilized despite their potential for tion. Only production of juice has been at- value addition and commercialization. With the tempted although potential for use as a col- increasing world population, more sources of hu- ouring agent has been demonstrated. In ad- man food are required. One important source is dition, there were no studies found on en- the wild fruit trees that have not been utilized or richment of food despite the vast nutrient commercialized. Besides food security, nutrition composition reported. Utilisation of this security is also a global problem that will require fruit in many processes in food will inform the exploration of new sources of food nutrients. on the functionality of the various nutrients Listed below is a summary of the challenges and during processing. Furthermore, some of way forward in enhancing utilization of A. gar- the constituents that have functional prop- ckeana tree fruit: erties may be investigated to determine their

IJFS February 2021 Volume 10 pages SI121–SI132 Novel food products from Azanza garckeana SI129

mechanisms of action and safety in food tion in the food industry. Currently published re- products. This information may be use- search on the A. garckeana fruit shows promising ful for advancing research studies on applic- results as some of the nutrients identiﬁed may ation of A. garckeana fruit in other food be useful in the battle against food and nutrition products. Discovering new avenues for util- insecurity in the developing countries. Further ization of this fruit will not only help in the research is required to characterize A. garckeana ﬁght against food and nutrient insecurity fruit to unravel its utility as a source of important but also help in improving the livelihoods food nutrients and functional constituents useful for actors in the fruit value chain through in the food industry. increased household incomes.

The contribution of wild food tree species References including A. garckeana to human nutrition Abass, A. A. & Ahmed, G. A. (2017). Assess- among the rural poor in different development of the suitability of using jakjak fruits ing countries should be quantified. There is (azanza garcheana) for production of lokum need to determine the contribution of these candy (Doctoral dissertation, Sudan Uni- tree species to daily nutrient intake to better versity of Science and Technology). determine their potential in reducing food Adamu, H. M., Ushie, O. A., Lawal, D. S. & and nutrition insecurity. Oga, I. A. (2013). Phytochemical screen- Despite the usefulness of wild fruit trees as ing of fruit of azanza garckeana and root of sources of nutrition for millions of people acacia macrothyrsa. International Journal in the developing countries, many of these of Traditional and Natural Medicines, 3 (1), tree species are still harvested from the wild, 19–25. with little attempts on on-farm manage- Ahmed, R. H., El Hassan, M. S. & El Hadi, H. M. ment. Despite its usefulness, limited at- (2016). Potential capability of azanza gar- tempts have been made towards domestic- ckeana fruits aqueous extract on enhance- ation of A. garckeana. Furthermore, many ment of iron absorption in wistar albino of these tree species are only known locally rats. Int. J. Adv. Res. Biol. Sci, 3, 245– in their respective regions with minimal util- 250. ization and commercialization. Thus, there Akinnifesi, F. K., Ajayi, O. C., Sileshi, G., is need for increased public awareness on Kadzere, I. & Akinnifesi, A. I. (2007). Do- wild fruit trees that can potentially influence mesticating and commercializing indigen- the lives of people living in these arid and ous fruit and nut tree crops for food secur- semi-arid areas. Research targeting food ity and income generation in sub-saharan tree domestication is therefore encouraged. africa. On-farm management and improvement of Aniszewski, T. (2007). Alkaloids - secrets of life these wild tree species has potential for en- (Elsevier, Ed.). doi:10.1016/B978- 0- 444- hanced utilization and quality of these tree 52736-3.X5000-4 resources. Furthermore, they may contrib- Awodoyin, R. O., Olubode, O. S., Ogbu, J. U., ute significantly to increased plant cover on Balogun, R. B., Nwawuisi, J. U. & Orji, earth as well as in soil conservation, which K. O. (2015). Indigenous fruit trees of trop- are important in climate change adaptation ical africa: Status, opportunity for develop- and mitigation. ment and biodiversity management. Agri- cultural Sciences, 6 (01), 31. Bain, L. E., Awah, P. K., Geraldine, N., Kindong, N. P., Siga, Y., Bernard, N. & Tanjeko, 7 Conclusions A. T. (2013). Malnutrition in sub–saharan africa: Burden, causes and prospects. Pan Clearly, there is limited information on A. gar- African Medical Journal, 15 (1). ckeana tree fruit despite its potential for utiliza-

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Bélair,C., Ichikawa, K., Wong, B. Y. L. & Mu- Harika, R., Faber, M., Samuel, F., Kimiywe, J., longoy, K. J. (2010). Sustainable use of Mulugeta, A. & Eilander, A. (2017). Mi- biological diversity in socio-ecological pro- cronutrient status and dietary intake of duction landscapes. In Background to the iron, vitamin a, iodine, folate and zinc satoyama initiative for the benefit of biod- in women of reproductive age and preg- iversity and human well-being. secretariat nant women in ethiopia, kenya, nigeria and of the convention on biological diversity, south africa: A systematic review of data montreal. techni-cal series (52, p. 184). from 2005 to 2015. Nutrients, 9 (10). doi:10. Benhura, M. A. N., Mbuya, N. & Machirori, E. 3390/nu9101096 (1999). Facile formation of caramel colours Harika, R., Faber, M., Samuel, F., Mulugeta, A., using the polysaccharide material that is Kimiywe, J. & Eilander, A. (2017). Are low extracted from the fruit of azanza garck- intakes and deficiencies in iron, vitamin a, eana. Food chemistry, 65 (3), 303–307. zinc, and iodine of public health concern Choudhary, R. & Tandon, R. V. (2009). Con- in ethiopian, kenyan, nigerian, and south sumption of functional food and our health african children and adolescents? Food and concerns. Pakistan Journal of Physiology, Nutrition Bulletin, 38 (3), 405–427. doi:10. 5 (1). 1177/0379572117715818 Chung, K. T., Wong, T. Y., Wei, C. I., Huang, Hasler, C. M. (2000). The changing face of Y. W. & Lin, Y. (1998). Tannins and hu- functional foods. Journal of the Amer- man health: A review. Critical Reviews in ican College of Nutrition, 19 (5, S), 499S– Food Science and Nutrition, 38 (6), 421– 506S. Conference on Nutritional and Func- 464. doi:10.1080/10408699891274273 tional Roles of Eggs in the Diet, AMELIA Danbature, W. L., Yirankinyuki, F. F., Magaji, ISL, FL, FEB 25-27, 2000. doi:10 . 1080 / B. & Ibrahim, Z. (2015). Comparative De- 07315724.2000.10718972 termination of Vitamin C and Iron in Ten Jacob, C., Shehu, Z., Danbature, W. L. & (10) Locally Consumed Fruits in Gombe Karu, E. (2016). Proximate analysis of State, Nigeria. 2 (8). the fruit azanza garckeana (goron tula). Dikko, Y. J., Khan, M. E., Tor-Anyiin, T. A., An- Bayero Journal of Pure and Applied Sci- yam, J. V. & Linus, U. A. (2016). In vitro ences, 9 (2), 221–224. antimicrobial activity of fruit pulp extracts Jacob, J. O., Mann, A., Adeshina, O. I. & Ndam- of azanza garckeana (f. hoffm.) exell & hillc. itso, M. M. (2016). Nutritional composition and isolation of one of its active principles, of selected wild fruits from minna area of betulinic acid. Journal of Pharmaceutical niger state, nigeria. International Journal Research International, 1–10. of Nutrition and Food Engineering, 10 (1), Goldberg, I. (2012). Functional foods: De- 37–42. signer foods, pharmafoods, nutraceuticals. Jamnadass, R., Place, F., Torquebiau, E., Springer Science & Business Media. Malézieux,E., Liyama, M., Sileshi, G., . . . Gomez, B., Gullon, B., Yanez, R., Schols, H. & Dawson, I. (2013). Agroforestry, food and Alonso, J. L. (2016). Prebiotic potential of nutritional security. pectins and pectic oligosaccharides derived Joel, J. M., Barminas, J. T., Riki, E. Y., from lemon peel wastes and sugar beet Yelwa, J. M. & Edeh, F. (2018). Extraction pulp: A comparative evaluation. Journal and characterization of hydrocolloid pectin of Functional Foods, 20, 108–121. doi:10 . from goron tula (azanza garckeana) fruit. 1016/j.jff.2015.10.029 World Scientific News, 101, 157–171. Grivetti, L. E. & Ogle, B. M. (2000). Value of Johnson, I. & Williamson, G. (2003). Phytochem- traditional foods in meeting macro- and mi- ical functional foods. CRC Press. cronutrient needs: The wild plant connec- Kehlenbeck, K., Asaah, E. & Jamnadass, R. tion. Nutrition Research Reviews, 13 (1), (2013). Diversity of indigenous fruit trees 31–46. doi:10.1079/095442200108728990 and their contribution to nutrition and livelihoods in sub-saharan africa: Examples

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