MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

Modification process in of folic acid-rich dent corn (Zea mays identata) and its identification as smart food fortificant

Agustine Susilowati*, Puspa Dewi Lotulung, Yati Maryati, and Aspiyanto

Research Center for Chemistry – Indonesian Institute of Sciences, Kawasan PUSPIPTEK, Serpong 15314, South Tangerang, Banten, Indonesia.

Abstract. A modification on nixtamalization process of dent corn (Zea mays identata) was conducted in order to recover natural folic acid-rich corn. Nixtamalization process on varieties of white dent corn and yellow dent corn subsequently were performed by steeping solution of Ca(OH)2 at concentrations of 0, 10, 20 and 30 % (w/w corn dissolved protein) for 18 hours, and boiling at 90 °C for 15, 30, 45 and 60 minutes. Result of research showed that concentration of Ca(OH)2 solution becoming more and more high and long boiling time increased both folic acid and reducing sugar, dropped total solids and total sugar, and fluctuated dissolved protein for both types of corn. Nixtamalization optimalization of white dent corn and yellow dent corn were achieved at combination of Ca(OH)2 20 % (w/w corn dissolved protein) for 60 minutes of boiling and Ca(OH)2 30 % for 30 minutes of boiling and gave folic acid of 466.81 and 506.74 µg/mL, respectively. In this condition, it is occurred an increase of folic acid 192.3 % (1.9 folds) and 139.89 % (1.4 folds) when compared to initial material of corn. Identification on folic acid monomer and glutamic acid monomer of both nixtamalized dent corn and yellow dent corn at optimum operation condition displayed domination of folic acid monomer with molecular weight (MW) 442.56 Dalton (Da.) with relative intensity 25.51 %, and 441.73 Da. with relative intensity 100 %, while glutamic acid monomer of nixtamalized yellow dent corn and nixtamalized white dent corn were dominated by monomer with MWs of 148.27 Da. and 148.32 Da., and relative intensity 3.73 and 1.8 %.

1 Introduction will inhibit its occurrence of fractionation of amylose and amylopectin chain, so that they are binding and inhibiting Cereal grains have been the staple component of human its occurrence of retrogradation (snack products with high diet for thousands of years. Corn, wheat and rice together crispy level) or inhibiting its occurrence of syneresis (sauce accounted for about 89 % of total cereal production and pudding products). Nixtamalization on corn is a worldwide. Today, cereal grains are still the most process, in which corn is boiled directly in water solution important source of calories to the majority of the world of Ca(OH)2 without filtering, steeped overnight, and population. In developing countries, about 60 % of washed and cooled at room temperature (26 – 28 °C) in calories are derived from cereals, whereas in the order to obtain and improve nixtamal product with better developed world it is only 30 % [1-3]. Corn (Zea mays physical property (texture, color, flavor, aroma, taste, shelf identata) is one of the best cereal grains as potential life), maintain functional property (minimize loss of folic source of polysaccharides to produce energy and to build acid), ease gelatinization, increase nutritional value, body, which is supported by functional properties as adsorb water mass, and drain a portion of its skin and sufficient high source of protein (7 – 9 %). Due to high pericarp from corn grains [5, 6]. Nixtamalization process protein concentration, corn has the most important source on corn is done to soften corn grain and facilite absorbing of folic acid, in which folic acid is composed by glutamic Ca(OH)2 so that nixtamalized product undergoing a series acid expressed as dissolved protein [4]. In order to achieve of significant changes on the physical and chemical corn products as natural source of folic acid for smart food properties (color, aroma), and the morphological and and to diversify a wide variety and attractive products, it rheological properties of starch will be generated. can be conducted through nixtamalization process. Softening and swelling on the optimum gel (65 Nixtamalization process on corn is generally – 85 °C) is able to enlarge volume (22 folds) when performed to achieve corn flours with proved protein compared to corn starch granula at 95 °C. High content of quality, increase calcium content and better physical folic acid in corn (kernel) (26 µg) can be kept to select type properties in application on food products. Nixtamalization using suspension of calcium hydroxide * Corresponding author: [email protected]

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

[ S tive properties on sun’s light, oxygen, heat and high [],

2.2 Procedure

2.2.1 Nixtamalization process Fig. 1. Structure of folic acid. 2.2.2 Identification of folic acid through LC-MS ] 2 Experimental design m/z – 2.1 Materials and Equipments μ

2 MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

3.Results and Discussion arohydrate he hole opositions related to types [ and varietis o orn, partiularly lavonoid phenoli S pigent, suh as etaaroten, lutein, xanthins and 3.1 Characteristic of corn. ryptoxanthin pigent ith vitain in yello dent orn eletion on dent orns are ased on a aility to ahieve hih do not ontained in hite dent orn his atter and prepare the roader hen opared to others auses oth stronger aroa and taste o yello dent orn varieties oth types o orn sho dierene in olour, ierent trend is seeed on total sugars, in hih hite tive properties on sun’s light, oxygen, heat and high sie and unior o grains, as shon in igure , and dent orn yields loer ontent o total sugars oposition o dent orns are shoed in ale hite g opared ith yello dent orn g, dent orn displays high onentration o oli aid on the other hand, higher ontent o reduing sugars ug hen opared to yello dent orn g opared ith yello dent orn ug his atter is appropriate ith higher g otal sugars relating ith high ontent o starh onentration o dissolved protein g than that is ain oponent in oposing orn arohydrate yello dent orn g t had een non that his atter is related ith types, varieties o orns and post oli aid pteroyllutai aid is a derivative harvesting proesses eduing sugars are relating ith glutai aids so that it is solule in ater relating ith oiling proess o orn on dierene in oiling tie and aino aids in general alali onentration eore and ater nixtaaliation [], oposing siple sugars ith redution property during nixtaaliation has a role in onding oli aid o nixtaalied orn iilar trend sees at at onentration, in hih yello dent orn shos higher onentration o at hen opared to hite dent orn a b esides, due to variety o orn, this atter is also eeted y post harvesting drying proesses and at ontent in initial Fig. 2. a ello dent orn and hite dent orn Zea mays aterial hen harvesting orn at is enaled to have an identata iportant role in eulsiiation proess through hoogenation proess under oth optial rotation speed eanhile, suiient high onentration o orn protein and tie prior to drying proess perored in order to 2.2 Procedure , higher hen opared to other ereals rie, produe nixtaalied lour ith etter oil asorer in hoever under soys so that orn protein has appliation produts 2.2.1 Nixtamalization process potential use as soure o protein and energy

Fig. 1. Structure of folic acid. Table 1. oposition o yello dent orn and hite dent orn Zea mays identata oponent ind o dent oli aid otal solid issolved otal sugar eduing at orn g protein g sugar g g ello hite 3.2 Effect of nixtamalization process on ourrene o starh gelatiniation on orn seed lin n composition sae tie, solution o a ill e asored ausing its lined oponents, suh as oli aid hile, derease ixtaaliation proess is onduted y adding solution o oli aid is possiility aused y exess in solution o o a o , , and , dissolved protein a so that it is ourred oagulation o protein and o orn on yello dent orn and hite dent orn, and 2.2.2 Identification of folic acid through LC-MS lysis on urther proesses iltration, ooling, draining, oiling or , , and inutes, respetively reduing sie roperties o oli aid pteroyl ] glutai aid is dissolved in ater oagulated y alali or a solution and sensitive on light, oxigent, 3.2.1.Folic acid high teperature and proess treatents using pressure ixtaaliation proess on yello dent orn inreases , oli aid until aility level o inding olloed ierent trend sees on hite dent orn, in hih y dropping oli aid or all oiling tie, as shoed inrease o oli aid is still running to the highest igure ptiiation o oli aid as ahieved at onentration o a solution , although is 2 Experimental design solution o a orn dissolved protein lutuating, as displayed in igure o ahieve the or inutes oiling and gave ug optial oli aid, nixtaaliation rate deonstrated at m/z – opared ith oli aid o initial aterial higher onentration o a solution ill need 2.1 Materials and Equipments ug, the optial proess ondition inreases oli shorter ooing tie n other ords, adding a aid ro ug to ug or solution enales to strengthen orn starh gel y inding μ ties his inrease o oli aid is aused y its aylose hain during oiling proess ausing stronger

3 MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

struture o orn starh, in hih oli aid ill e inded 3.2.2 Total solids strong at starh gel at sae tie t had een non that orn has high ontent aylose o and aylopetin otal solids is an auulation or the hole o , so that they are easy to ulil sineresis and oponents in nixtaalied orn oth solule and retrogradation ptiiation o oli aid at insolule extrated aording to ravietri ethod nixtaalied hite dent orn is otained at a ixtaaliation proess on yello dent orn gave solution o or inutes o oiling ith result o lutuated total solids and as tend to drop, as ug opared ith oli aid in initial deonstrated in igure ropping total solids as aterial o hite dent orn ug, the optiu possility aused y its ourrene o ater ass proess ondition raises oli aid ro ug to adsorption at net o orn grains ausing selling starh ug or ties hile, derease gel to derease total solids ptiiation o total solids o oli aid is possiility aused y exess in solution o reahed at treatent o a solution o a so that it is ourred oagulation o protein and orn solule protein or inutes o oiling gave lysis on urther proesses iltration, ooling, draining, opared ith initial aterial o orn reduing sie roperties o oli aid pteroyl , the optiu proess ondition delined total solids glutai aid is dissolved in ater elit, et al, to 60 57.98 oagulated y alali or a solution and sensitive on 57.76 58 56.17 55.09 55.23 light, oxigent, high teperature and proess treatents 55 53.98 53.12 using pressure , . ierent trend sees on hite 53 52.62 50.31 50 51.89 dent orn, in hih inrease o oli aid is still running 49.45 47.58 47.40 Total Solid (%) Solid Total 48 to the highest onentration o a solution , 46.88 45 45.48 43.79

although is lutuating, as displayed in igure 43 0 10 20 30 40 525 466.81 Cocentration of Ca(OH)2 (% w/w dissolved protein) 450 383.04 15 ' 30' 45 ' 60 ' 357.85 375 368.74 342.89 300 271.04 344.07 305.70

281.48 225 250.07 Fig. 5 elationship eteen a onentration and

119.78 150 96.44 oiling tie at on reovery o total solids in nixtaalied

Folic acid (ug/mL) 94.89 75 72.44 79.93 64.89 yello dent orn 0 0 10 20 30 40

Ca(OH)2 (% w/w dissolved protein corn) 15' 30' 45' 60' ierent trend seeed at nixtaalied hite dent orn, in hih raising total solids is ourred at treatent Fig. 3 elationship eteen a onentration and oiling tie at on oli aid in nixtaalied yello dent o a solution o orn solule protein orn and alost all oiling tie olloed y dereasing lutuation relating ith inreasing a

60 57.98 onentration, as shoed in igure 57.76 58 56.17 65 63.57 55.09 55.23 55 53.98 60 53.12 53 52.62 55.20 50.31 54.36 51.89 55 50 49.45 53.90 51.35 52.90 47.58 47.40 Total Solid (%) Solid Total 53.21 48 51.39 46.88 50 47.84 50.55 50.19 45 50.02 45.48 43.79 46.37 Total Solid (%) 47.38 47.07 43 45 0 10 20 30 40 44.32 Cocentration of Ca(OH)2 (% w/w dissolved protein) 40

15 ' 30' 45 ' 60 ' 0 10 20 30 40 Cocentration of Ca(OH)2 (% w/w dissolved protein) 15 ' 30' 45 ' 60 ' Fig. 4 elationship eteen a onentration and oiling tie at on oli aid in nixtaalied hite dent Fig. 6 elationship eteen a onentration and orn oiling tie at on reovery o total solids in nixtaalied hite dent orn. o ahieve the optial oli aid, nixtaaliation rate deonstrated at higher onentration o a ptiiation o total solids as ahieved at solution ill need shorter ooing tie n other ords, treatent oination o a solution o or adding a solution enales to strengthen orn inutes o oiling, and eae ore and ore lo starh gel y inding aylose hain during oiling relating ith inreasing a solution onentration proess ausing stronger struture o orn starh, in hen opared to total solids o initial orn aterial hih oli aid ill e inded strong at starh gel at , the optiu proess ondition dropped total sae tie t had een non that orn has high ontent solids ro to his atter aylose o and aylopetin o , so that they displayed that nitaaliation rate as tend to loer are easy to ulil sineresis and retrogradation total solids relating ith long ooing tie, although ptiiation o oli aid at nixtaalied hite dent adding solution o a eae ore and ore orn is otained at result o ug opared uh aount xess in onentration o a ith oli aid in opared ith oli aid in initial solution o orn solule protein, oiling aterial o hite dent orn ug, the optiu proess syste as not ale to retain gelatiniation in proess ondition raises oli aid ro ug to order to dissolve oponents o orn, so that is dropped ug orties total solids

4 MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

struture o orn starh, in hih oli aid ill e inded 3.2.2 Total solids 3.2.3 Dissolved protein 0.05 0.041 strong at starh gel at sae tie t had een non that 0.04 0.037 otal solids is an auulation or the hole issolved protein o nixtaalied orn is all aino aids, 0.038 0.039 orn has high ontent aylose o and aylopetin 0.04 0.035 0.032 0.034 0.034 0.034 0.034 oponents in nixtaalied orn oth solule and peptides, and protein derivative, hih is solule in 0.033 o , so that they are easy to ulil sineresis and 0.034 0.033 0.03 retrogradation ptiiation o oli aid at insolule extrated aording to ravietri ethod ater and is inded y other oponents as a result o ixtaaliation proess on yello dent orn gave nixtaaliation proess issolved protein o 0.03 0.024 nixtaalied hite dent orn is otained at a 0.023 0.022 lutuated total solids and as tend to drop, as nixtaalied orn eoes a ain proess reerene (mg/mL) protein Dissolved 0.02 solution o or inutes o oiling ith result o 0 10 20 30 40 ug opared ith oli aid in initial deonstrated in igure ropping total solids as guide eause o oli aid oli aid is protein Concentration of Ca(OH)2 (% w/w dissolved protein) 15' 30' 45' 60' aterial o hite dent orn ug, the optiu possility aused y its ourrene o ater ass derivative oning heial struture o oination adsorption at net o orn grains ausing selling starh ro pteridin heteroyli, paraainoenoat aid proess ondition raises oli aid ro ug to Fig. 8 elationship eteen a onentration and ug or ties hile, derease gel to derease total solids ptiiation o total solids and pteroylglutai aid ts presene o oiling tie at on reovery o dissolved protein in o oli aid is possiility aused y exess in solution o reahed at treatent o a solution o high protein o orn – eoes reerene guide nixtaalied hite dent orn a so that it is ourred oagulation o protein and orn solule protein or inutes o oiling gave to selet this ereals, esides high ontent o oli aid opared ith initial aterial o orn g and its potential use as soure o energy lysis on urther proesses iltration, ooling, draining, 3.2.4 Total sugar reduing sie roperties o oli aid pteroyl , the optiu proess ondition delined total solids ixtaaliation proess on yello dent orn gave to optiiation g ahieved at treatent glutai aid is dissolved in ater elit, et al, otal sugars on nixtaalied orn is a guidelines ro 60 57.98 oination o onentration o a o oagulated y alali or a solution and sensitive on 57.76 all arohydrates aording to henol – ulphate etode 58 56.17 55.09 55.23 orn dissolved protein and oiling tie o inutes, light, oxigent, high teperature and proess treatents 55 n oiling proess o orn , it is happened 53.98 53.12 as displayed in igure using pressure , . ierent trend sees on hite 53 52.62 50.31 rationation o aylose and aylopetin 50 51.89 dent orn, in hih inrease o oli aid is still running 49.45 47.58 47.40 0.038 ontained in orn to or gel at orn granular net and Total Solid (%) Solid Total 0.04 48 0.036 to the highest onentration o a solution , 46.88 0.032 0.035 45 dissolve gluose expressed as total sugars reatent on although is lutuating, as displayed in igure 45.48 43.79 0.03 0.032 43 0.029 0.029 yello dent orn shoed lutuated nixtaaliation 0 10 20 30 40 525 0.03 0.023 466.81 Cocentration of Ca(OH) (% w/w dissolved protein) 0.027 2 0.025 0.021 proess rate relating ith inrease o solution o 450 0.022 383.04 15 ' 30' 45 ' 60 ' 0.020 357.85 0.02 0.020 375 a reatent o oiling tie o inutes raises 368.74 342.89 0.016 Dissolved protein (mg/mL) protein Dissolved 300 271.04 344.07 305.70 0.015 0.01 total sugars until axiu onentration o a 281.48 225 250.07 Fig. 5 elationship eteen a onentration and 0 10 20 30 40 Concentration of Ca(OH) (% w/w dissolved protein) orn dissolved protein to produe higher 119.78 2 150 96.44 oiling tie at on reovery o total solids in nixtaalied

Folic acid (ug/mL) 94.89 15' 30' 45' 60' 75 72.44 onentration o total sugar hen opared to all 79.93 64.89 yello dent orn 0 treatents olloed y dropping to axiu 0 10 20 30 40 Fig. 7 elationship eteen a onentration and Ca(OH)2 (% w/w dissolved protein corn) ierent trend seeed at nixtaalied hite dent onentration o a orn dissolved 15' 30' 45' 60' oiling tie at on reovery o dissolved protein in orn, in hih raising total solids is ourred at treatent protein n other proesses displays an inrease o total Fig. 3 elationship eteen a onentration and nixtaalied yello dent orn oiling tie at on oli aid in nixtaalied yello dent o a solution o orn solule protein sugars, although axiu onentration o a orn and alost all oiling tie olloed y dereasing opared ith dissolved protein o initial orn orn dissolved protein and oiling tie lutuation relating ith inreasing a aterial, the optiu proess ondition loered longer than inutes, as shon in igure

60 57.98 onentration, as shoed in igure dissolved protein ro g to g 57.76 450 428.31 58 56.17 65 63.57 55.09 55.23 or ties iultaneously derease o 55 53.98 375 60 343.09 53.12 dissolved protein is taen plae at onentration 53 52.62 330.59 50.31 55.20 54.36 300 51.89 55 50 49.45 53.90 a solution o orn dissolved protein 51.35 52.90 47.58 47.40 250.51 Total Solid (%) Solid Total 53.21 48 51.39 225 206.03 46.88 50 47.84 his atter is possiility aused y oagulation to ind 50.55 50.19 176.25 190.96192.65 45 50.02 146.84 45.48 43.79 46.37 168.82 Total sugar (ug/mL) sugar Total Total Solid (%) 47.38 150 45 47.07 a solution, in hih long oiling tie ill 123.24 114.63 43 118.60 0 10 20 30 40 44.32 105.59 116.25 104.85 eoe ore and ore lo dissolved protein his 75 Cocentration of Ca(OH)2 (% w/w dissolved protein) 40 0 10 20 30 40 15 ' 30' 45 ' 60 ' 0 10 20 30 40 atter sees or the longest oiling tie inutes Cocentration of Ca(OH)2 (% w/w dissolved protein) Concentration Ca(OH)2 ( % w/w dissolved protein) 15 ' 30' 45 ' 60 ' ther possiilities ere aused y proesses relating 15' 30' 45' 60'

Fig. 4 elationship eteen a onentration and ith ater oiling proess, suh as hoogeniation oiling tie at on oli aid in nixtaalied hite dent Fig. 6 elationship eteen a onentration and rp, inutes or sie reduing iilar trend seeed Fig. 9 elationship eteen a onentration and orn oiling tie at on reovery o total solids in nixtaalied at nixtaalied hite dent orn, in hih rate o oiling tie at on reovery o total sugars in hite dent orn. nixtaalied yello dent orn o ahieve the optial oli aid, nixtaaliation nixtaaliation reation as lutuative and as tend to raise relating ith high onentration o a rate deonstrated at higher onentration o a ptiiation o total solids as ahieved at ptiiation proess as ahieved y adding solution nreasing is reverse proportional ith oiling solution ill need shorter ooing tie n other ords, treatent oination o a solution o or solution o a o orn dissolved tie, in hih oiling tie eoing ore and ore adding a solution enales to strengthen orn inutes o oiling, and eae ore and ore lo protein or inutes o oiling and gave total sugars long ill drop dissolved protein, as deonstrated in starh gel y inding aylose hain during oiling relating ith inreasing a solution onentration onentration o ug n this ondition, it igure proess ausing stronger struture o orn starh, in hen opared to total solids o initial orn aterial shoed an inrease o total sugars onentration ro ptiiation proess as ahieved at onentration hih oli aid ill e inded strong at starh gel at , the optiu proess ondition dropped total ug to ug initial aterial o o a solution o orn dissolved sae tie t had een non that orn has high ontent solids ro to his atter yello dent orn ties hereas, hite protein or inutes o oiling tie and gave solule aylose o and aylopetin o , so that they displayed that nitaaliation rate as tend to loer dent orn deonstrated a dierent trend o reation rate, protein g olloed y loering are easy to ulil sineresis and retrogradation total solids relating ith long ooing tie, although in hih delining total sugars is taen plae g relating ith long oiling tie or inutes ptiiation o oli aid at nixtaalied hite dent adding solution o a eae ore and ore siultaneously at all proess treatents, as shoed in hen opared to dissolved protein o initial hite dent orn is otained at result o ug opared uh aount xess in onentration o a igure orn aterial g, the optiu proess ith oli aid in opared ith oli aid in initial solution o orn solule protein, oiling ondition drops dissolved protein ro g to aterial o hite dent orn ug, the optiu proess syste as not ale to retain gelatiniation in g or ties proess ondition raises oli aid ro ug to order to dissolve oponents o orn, so that is dropped ug orties total solids

5 MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

900 4.82 5.0

750 700.74 743.01 3.66 582.65 3.8 600 3.15 2.80 443.27 450 367.13 351.40 2.5 348.75 323.46 327.06 1.74 300 1.37 1.48 308.31 178.01 1.72 276.25 200.81 1.80 Total Sugar (ug/mL) Sugar Total 1.3 150 0.40 139.20 168.82 0.49 1.60 120.72 0.35 0.86 0.54 0.34 0 0.0 Reducing sugari (mg/mL) sugari Reducing 0 10 20 30 40 0 10 20 30 40 Concentration of Ca(OH)2 (% w/w dissolved protein) Concentration of Ca(OH)2 (% w/w dissolved protein) 15' 30' 45' 60' 15' 30' 45' 60'

Fig. 10 Fig. 12

3.2.5 Reducing sugar a b 2.5 Fig. 13. 2.04 2.0 1.73 1.5 1.58 0.84 3.2 Identification of folic acid and glutamic acid 1.0 1.24 0.63 0.56 1.15 monomer in nixtamalized corn 0.48 0.44 0.5 0.34 0.31 0.29 0.27 0.28

Reducing sugar (mg/mL) sugar Reducing 0.0 0.18 0 10 20 30 40 Concentration of Ca(OH)2 (% w/w dissolved protein) 15' 30' 45' 60' – Fig. 11 m/z –

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BPI= > NR(2.00)

T3.0 900 4.82 100 445.4 5.0 90 700.74 750 80 743.01 3.66 582.65 3.8 70 600 3.15 2.80 60 – 443.27 450 50 367.13 351.40 2.5 % Intensity 40 348.75 323.46 327.06 1.74 300 30 1.37 1.48 308.31 178.01 1.72 276.25 200.81 1.80 20 Total Sugar (ug/mL) Sugar Total 1.3 0.40 BPI=>NR(2.00) 150 0.49 1.60 10 139.20 168.82 T1.5 167.9 0.35 0.86 100 120.72 0 0 0.54 0.34 0 2 4 6 8 10 0 0.0 Retention Time (Min) 90 Reducing sugari (mg/mL) sugari Reducing 0 10 20 30 40 0 10 20 30 40 80 Concentration of Ca(OH)2 (% w/w dissolved protein) 70 Concentration of Ca(OH)2 (% w/w dissolved protein) 15' 30' 45' 60' Fig. 14. 60 15' 30' 45' 60' 50 Intensity % 40

30 Fig. 10 Fig. 12 20 10 Mariner Spec /78:79 (T /2.97:3.01) -55:57 (T -2.97:3.01) ASC=>NR(2.00)[BP = 148.1, 469] 148.1479 100 469.2 0 0 0 2 4 6 8 10 90 Retention Time (Min) 80

70

60 166.1490 Fig. 18. 50 % % Intensity 40 162.1737 – 144.1524 30 20 170.1485 10 148.4662 130.1236 184.1788 145.1527 167.1499 158.8098 175.8903 190.6868 0 0 116.0 134.2 152.4 170.6 188.8 207.0 Mass (m/z) m/z – Fig. 15. m/z – m/z – – 3.2.5 Reducing sugar – m/z – m/z – BPI=>NR(2.00) T1.7 100 115.0 – – 90 80 70 60 50 Intensity % 40 30 20 10 Mariner Spec /38:39 (T /1.43:1.46) -28:32 (T -1.43:1.46) ASC=>NR(2.00)[BP = 159.2, 148] 0 0 159.22 0 2 4 6 8 10 100 148.5 Retention Time (Min) 191.30 90 80 70 Fig. 16. 60 50

Intensity % 40 163.26 30 20 131.18 219.34 Mariner Spec /44:46 (T /1.66:1.73) -29:34 (T -1.66:1.73) ASC=>NR(2.00)[BP = 337.6, 116] 442.76 10 129.22 261.48 365.69 100 4.3 453.87 543.16 0 0 9 9 .0 319.2 539.4 759.6 979.8 1200.0 90 Mass (m/z) 80 70 60 a b 50 Fig. 19. Intensity % 40 30 443.70 m/z – 20 10 457.10 435.93 441.48 449.35 Mariner Spec /37:40 (T /1.39:1.50) -4:16 (T -1.39:1.50) ASC[BP = 159.2, 124] 0 0 425.0 439.6 454.2 468.8 483.4 498.0 148.27 4.6 Mass (m/z) 100 2.5 Fig. 13. 90 80 2.04 Fig. 17. m/z – 70 2.0 60 1.73 50 148.37 1.5 1.58 40

%Intensity 147.94 148.44 0.84 3.2 Identification of folic acid and glutamic acid 30 148.72 148.62 1.0 20 1.24 148.04 148.55 148.93 0.63 0.56 1.15 monomer in nixtamalized corn 10 0.48 0.44 0.5 147.83184 148.07588 148.31992 148.56396 148.80799 149.05203 0.34 0.31 0.29 Mass (m/z) 0.27 0.28

Reducing sugar (mg/mL) sugar Reducing 0.0 0.18 0 10 20 30 40 Fig. 20. Concentration of Ca(OH) (% w/w dissolved protein) 2 15' 30' 45' 60' – m/z – Fig. 11 m/z – 3.3.1 Nixtamalized yellow dent corn

7 MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

Mariner Spec /56:61 (T /2.12:2.31) -48:51 (T -2.12:2.31) ASC[BP = 391.8, 8] Mariner Spec /39:41 (T /1.46:1.54) -27:33 (T -1.46:1.54) ASC[BP = 159.2, 149] 442.56 2.2 148.32 100 100 2.7 90 90 148.26 80 80 441.49 70 442.45 70 60 60 443.51 50 441.02 443.29 50 442.69 148.05 148.21 148.54 40 40 148.42 148.60 148.76

%Intensity 441.89 30 442.20 %Intensity 441.66 442.99 30 148.69 148.86 442.08 20 20 148.48 441.19 442.31 442.79 10 441.40 10 147.92 440.88250 441.44944 442.01639 442.58333 443.15027 443.71721 0 0 147.89835 148.11013 148.32192 148.53370 148.74548 148.95726 Mass (m/z) Mass (m/z) Fig. 21. Fig. 24. m/z m/z – –

3.3.2 Nixtamalized white dent corn – m/z – Mariner Spec /39:40 (T /1.46:1.50) -27:33 (T -1.46:1.50) ASC[BP = 159.2, 150] 441.73 100 3.4 90 m/z – 80 – 70 60 442.12 50 442.52 442.00 40 441.22 442.39 442.82 %Intensity 30 442.27 440.92 441.59 442.96 20 441.86 443.14 442.69 10 443.23 0 0 440.81171 441.32793 441.84414 442.36035 442.87656 443.39278 m/z – Mass (m/z) m/z – – Fig. 25. – m/z –

BPI=>NR(2.00) T1.5 100 153.9 90 4 Conclusions 80

70 60 50

% Intensity % 40 30

20 10 0 0 0 2 4 6 8 10 Retention Time (Min) Fig. 22. – Mariner Spec /39:40 (T /1.46:1.50) -31:33 (T -1.46:1.50) ASC=>NR(2.00)[BP = 159.2, 150] 159.22 100 150.1 191.30 90 80 70 60 50

% Intensity % 40 163.26 30 20 131.20 145.20 219.34 10 261.48 161.21 365.67 441.94 568.82 734.77 0 0 9 9 .0 319.2 539.4 759.6 979.8 1200.0 Mass (m/z) Fig. 23. m/z –

8 MATEC Web of Conferences 154, 01017 (2018) https://doi.org/10.1051/matecconf/201815401017 ICET4SD 2017

Mariner Spec /56:61 (T /2.12:2.31) -48:51 (T -2.12:2.31) ASC[BP = 391.8, 8] Mariner Spec /39:41 (T /1.46:1.54) -27:33 (T -1.46:1.54) ASC[BP = 159.2, 149] 12. 442.56 2.2 148.32 Hamner, H. C. and Tinker, S. C. Fortification of corn 100 100 2.7 90 90 148.26 flour with folic acid in the United States: an 80 80 441.49 70 442.45 70 overview of the evidence, ANNALS OF THE NEW 60 60 443.51 50 441.02 443.29 50 YORK ACADEMY OF SCIENCES. ISSN 0077- 442.69 148.05 148.21 148.54 40 40 148.42 148.60 148.76

%Intensity 441.89 30 442.20 %Intensity 8923. 1312, p.8 – 14. New York Academy of 441.66 442.99 30 148.69 148.86 442.08 20 20 148.48 441.19 442.31 442.79 Sciences (2014). 10 441.40 10 147.92 440.88250 441.44944 442.01639 442.58333 443.15027 443.71721 0 0 147.89835 148.11013 148.32192 148.53370 148.74548 148.95726 Mass (m/z) 13. Onggo, D. General Principles in Electrospray Mass Mass (m/z) Spectrometry : A New Technique in Mass Spectral Fig. 21. Fig. 24. m/z Analysis. Journal of Mass Spectrum, Vol. 3, No. 2, m/z – References pp.115 – 131, Wikipedia. Accessed at 3 February – 2013 (1998). 1. Anonymous. Recommendations for the use of folic 14. Dass, Chreh. Dasar-dasar Spektrometri Massa 3.3.2 Nixtamalized white dent corn acid to reduce the number of cases of spina bifida Kontemporer. John Wiley & Sons, Inc., pp.151 – and other neural tube defects. MMWR Recomm. 194. doi : 10.1002/9780470118498.ch5. ISBN Rep., 41 (No. RR.14) (1992). 9780470118498. Accessed at 20 April 2017 (2007). 2. De Wals, P., Rusen, I. D., Lee, N. S., Morin, P., and 15. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Niyonsenga, T. Trend in prevalence of neural tube Randall, R. J. Protein measurement with Folin defects in Quebec. Birth Defects Res A Clin Mol – phenol reagent. J. Biol. Chem. 193 (1), pp.265 – 276. Teratol., 67, p.919 – 23 (2003). m/z – PMID 14907713 (1951). 3. Anonymous. EUROCAT. Special Report : 16. AOAC. Official Methods of Analysis. Association of Mariner Spec /39:40 (T /1.46:1.50) -27:33 (T -1.46:1.50) ASC[BP = 159.2, 150] 441.73 Prevention of Neural Tube Defects by Official Analytical Chemists International, 20th. 100 3.4 90 Periconceptional Folic Acid Supplementation Edition, Washington D. C. (2016). m/z – 80 – 70 4. Wronkowska, M. Wet-Milling of Cereals, Journal of 17. 60 Ruengsitagoon, W. and Hattanat, N. Simple 442.12 50 442.52 Food Processing and Preservation ISSN 1745-4549, spectrophotometric method for determination of 442.00 40 441.22 442.39 442.82

%Intensity th 30 442.27 40, p.572 – 580. doi:10.1111/jfpp.12626 (2016). folic acid. The 4 Annual Northeast Pharmacy 440.92 441.59 442.96 20 441.86 443.14 442.69 10 Research Conference, Thailand (2012). 443.23 5. Rooney, L.W. and Serna-Salvidar, S.O. Food uses 0 0 440.81171 441.32793 441.84414 442.36035 442.87656 443.39278 whole corn and dry milled factions. in Corn : 18. Eichhorn, P. and Knepper, T. P. Electrospray Mass (m/z) m/z – Chemistry and Technology (Watson, S. A. and ionization mass spectrometric studies on the m/z – – Fig. 25. Ramsted, P. E., ed.). ACCC. St. Paul, M. N. (1987), amphoteric surfactant cocamidopropylbetaine. – m/z – F. (ed.), Molecular Structure and Function or Food Journal of Mass Spectrum, June : 36 (6), pp.677 – Carbohydrate. Appl. Sci. Publ. Ltd., London (1973). 684. DOI : 10.1002/jms.170 (2001). BPI=>NR(2.00) 6. T1.5 Argun, M. S. and Dogan, I. S. Effects of varying 19. Susilowati, A., Iskandar, Y. M., Melanie, H., 100 153.9 90 4 Conclusions nixtamalization conditions on the calcium Maryati, Y., Lotulung, P. D., Aryani, D. G. 80 70 absorption and pasting properties of dent and flint Pengembangan konsentrat sayuran hijau dan 60 corn flours, Journal of Food Process Engineering, 50 kacang-kacangan terfermentasi pada jagung (Zea

% Intensity % 40 ISSN 1745–4530, 40 : e12436. Wiley Periodicals, mays L.) pramasak sebagai sumber asam folat untuk 30 20 Inc., p.1 – 7. doi:10.1111/jfpe.12436 (2017). formula pangan pintar. Laporan Hasil Penelitian, 10 0 0 7. Program Tematik, Kedeputian IPT, Tahun Anggaran 0 2 4 6 8 10 Banks, W., Greenwood, C. T. and Muir, D. D. The Retention Time (Min) Structure of Starch in Birch, G. G. and Green, L 2015, Pusat Penelitian Kimia – LIPI, Kawasan 8. PUSPIPTEK, Serpong, Tangerang Selatan (2015). Fig. 22. Nilsen, R. M., Leoncini, E., Gastaldi, P., Allegri, V., – 20. Verlinde, P., Oey, I., Deborggraeve, W., Me, J., Agostino, R., Faravelli, F., Ferrazzoli, F., Finale, E., Hendrickx, M. E., Van Loey, A. M. Mechanism and Ghirri, P., Scarano, G., and Mastroiacovo, P. Mariner Spec /39:40 (T /1.46:1.50) -31:33 (T -1.46:1.50) ASC=>NR(2.00)[BP = 159.2, 150] Prevalence and determinants of preconception folic related kinetics of 5-methyltetrahydrofolic acid 159.22 100 150.1 degradation during combined high hydrostatic 191.30 acid use : an Italian multicenter survey, Italian 90 80 Journal of Pediatrics, Published online July 13, 42 : pressure–thermal treatments. Journal of 70 60 65, p.1 – 10. DOI 10.1186/s13052-016-0278-z. Agricultural and Food Chemistry, 57 (15) : 6803 – 50

% Intensity % 40 163.26 Open Access. CrossMark (2016). 14. Accessed at 25 April 2017 (2009). 30 20 131.20 21. Delchier, N., Ringling, C., Maingonnat, J. F., 145.20 219.34 9. Belitz, H. D., Grosch, W., and Schieberle, P. Food 10 261.48 161.21 365.67 441.94 568.82 734.77 th 0 0 Rychlik, M., Renard, C. M. G. C. Mechanisms of 9 9 .0 319.2 539.4 759.6 979.8 1200.0 Chemistry. 4 revised and extended edition, Mass (m/z) Springer-Verlag, Berlin Heidelberg. ISBN 3-540- folate losses during processing: diffusion vs heat Fig. 23. 15043-9 (2009). degradation. Food Chem., 157: pp.439 – 47.

m/z – 10. Anonymous. Folic acid, Semolus. Accessed at 8 Accessed at 25 April 2017 (2014). December 2014 (2008). Fleche, G. Chemical Modification and Degradation 11. Anonymous. FDA approves folic acid fortification of Starch in G.MA. Van Beynum and J. A. Rolls of corn masa flour, U. S. Food and Drug (eds.), Marcel Dekker Inc., New York, U. S. A. Administration News Release, April 14 (2016). (1985).

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