Research Methodology The selection of iron type as fortificant in chocolate filled milk powder

required were; white PE Plastic bag, weighing Scale Mettler Toledo PE3600, stainless steel spoons, pilot scale-batch dry mixer with capacity approx 15 liter. For filling and packing preparation, the tools required were; aluminum foils sachet, vacuum pack sealer, sanitised glove and masker. 2. Materials and tools for sample analysis For sensory evaluation, the materials required was water at temp 15°C, 45°C, and 80°C and tools required were beaker glass 200mL, graduated cylinder 250mL, thermometer, stainless steel spoon, object glass and tasting cup. For chemical test, the materials required were reagent for Karl Fischer analysis, distilled water, ultra filtered water 18.2 MΩ, hydrochloric acid solution, approx. 3.7 vv, metaphosphoric acid solution 2 g100 mL, acetic acid solution 10 vv and 2,6-dichlorophenol-indophenol DCPIP solution, about 0.5 mgmL. And the tools required were Rotronic Hygrolab Instrument, Titrator Karl Fischer, Metrohm, KF Titrino 701, pH-meter Metrohm 691 or equivalent 0,01 pH, Atomic absorption spectrometer with Iron hollow cathode lamp, Beaker glass 100mL, magnetic stirrer, pipette and Metrohm Titrino. For microbiology test, the materials required were Sugar free agar as media, hydrogen peroxide solution 3 vv as reagent and tools required were Petri-dish, Incubation facility.

C. Research Methodology

1. Methodology This research was started by preparing bulk prototype of chocolate filled milk powder based on three variables, which were type of iron, fortification level of iron and temperature of water for reconstitution. These variables were important parameter which determine the taste and appearance of the finally consumed product, serving suggestions cold, warm or hot, as well as the claim of iron fortification which relate to level of iron. Types of iron that use in this research were Iron Sulphate FeSO 4 H 2 O, Iron Fumarate C 4 H 2 FeO 4 , and Iron Pyrophosphate Fe 4 P 2 O 7 3.8H 2 O. These irons cover low and high bioavailability as well as low and high solubility. The level of iron fortification was set on 10 and 20 of daily value DV, refer to Indonesian regulations so called “Pedoman Umum Pelabelan Produk Pangan” published by Direktorat Standardisasi Produk Pangan - Badan POM RI 2004 chapter 12.5 Klaim Perbandingan Gizi, with this level, one could claim “Contains Iron” and “Hi-Iron” respectively. Based on local regulation so called “Acuan Label Gizi Produk Pangan” published by Direktorat Standardisasi Produk Pangan - Badan POM RI 2007, for consumer group under 2000 kcal energy daily intake, the daily value for iron is 26 mg, therefore to deliver 10 DV, this product should contain Iron 10 of 26 mg per serving; 2.6 mg iron per serving. So the iron level in the finished product would be 2.6 mg per serving to fulfill 10 DV and 5.2 mg per serving to fulfill 20 DV. The temperature of water for reconstitution was proposed to adapt the consumer consumption behaviour, 15 o C for cold consumption, 45 o C for warm consumption and 80 o C for hot consumption. The selection of appropriate iron type at certain level of fortification in protototype chocolate filled milk powder that was reconstituted at different temperature was based on sensory evaluation, chemical analysis and keeping quality evaluation. Only the appropriate iron type would undergo keeping quality evaluation. The sensory evaluation was designed to test product in both powder and reconstituted form. The sensory evaluation was basically used as main basis to determine appropriate iron type. In powder form, it would be evaluated aroma and color. In reconstituted form, it would be evaluated taste and color at cold, warm and hot water reconstitution, all were observed at 0 and 15 min after reconstitution. The chemical and microbiological analysis would ensure other factor affecting product stability such as AW, TPC is monitored. The chemical analysis that designed to test the product were as follows : vitamin C content, to observe the impact of vitamin C degradation if any, which would be used as one of the basis to determine appropriate iron type, water activity and moisture Karl Fischer, to observe the shelf stability, fe content by Atomic Absorption Spectrometry AAS, to verify the designated iron level, pH, to observe pH of reconstituted chocolate milk at certain water temperature. The objective of keeping quality was to see the stability of the prototype undergone NRT storage condition for at least 6 months periods of time, where it was represent the normal product age sold in the store. Chocolate filled milk powder without iron fortification was provided as a reference control for sensory evaluation, as well as to verify other factor affecting the keeping quality, i.e. packaging performance, residual oxygen content. The keeping quality KQ that applies to this product was designed based on empirical data of keeping quality of similar product produced in PT. Nestle Indonesia, Kejayan factory. per 100gr Vit C, min 50mg, AW Max 0.2 Total Plate Count max 3000 cfugr Powder color and aroma is acceptable as per reference Selected iron Sensory evaluation Taste, color and flavor is acceptable as per reference Powder Analysis Reconstitution Analysis 0 and 15 min Prepare prototype with level of iron fortification 10 and 20 DV Chemical Analysis Taste, color and flavor is acceptable as per reference Sensory evaluation Reconstitution Analysis Powder color and aroma is acceptable as per reference 0 and 15 min Selection of Iron candidate 3 types as an object of research Chemical Analysis Microbiology Analysis Sensory evaluation Powder Analysis Microbiology Analysis Sensory evaluation per 100gr Vit C, min 50mg, Fe min 8,7mg or 17,3mg, AW Max 0.2 Total Plate Count max 3000 cfugr KQ Stability for 6 W 37°C Figure 1 Research plan flow chart The research plan flow chart is shown in Figure 1, which composed of prototype preparation, selection of iron and stability test of iron fortified product. 2. Working procedure a. Bulk preparation procedure of chocolate filled milk powder Bulk chocolate filled milk powder which was made fix for all kind of tested iron, composed of filled milk powder, sucrose, cocoa 10-12 fat, CaCO 3 , vitamin A C and Iron 0.02-0.08, to reach min. 2.6mg 10DV min. 5.2mg 20DV per serving. Reference bulk chocolate filled milk powder had the same composition as above, but without iron fortification. There were six main processing steps to produce prototype of chocolate filled milk powder, which was shown at Figure 2. Sugar milling Generally sugar has the highest particle size 200 - 700 micron than other major ingredient. To improve mixing homogeneity and avoid materials separation, it is necessary to mill sucrose prior to mixing. Sucrose milling was performed in sugar milling available in the factory. Weighing each ingredient Each ingredient was weighed to nearest one digit for major materials; filled milk powder, sucrose, cocoa, and to the nearest two digit for minor ingredient; Calcium Carbonate Micronised, Vitamin premix, Iron SulphateFumaratePyrophosphate. Preweighed ingredient is labelled with identification of ingredient name, weight, lot nr and when is prepared. Pre-mixing for minor ingredient Minor ingredient like Calcium Carbonate Micronised, Vitamin premix, and Iron SulphateFumaratePyrophosphate will tend to be hardly homogenous because they are small in percentage normally 1. Pre-mixing would help to improve the homogeneity of these small ingredients when they finally mix with other major ingredient. Mixing of all ingredient There is several type of dry mixing equipment. To do this small experimental and due to availability mixer, it was chosen a batch ribbon mixer with capacity 15 liter. All preweighed material was manually fed into this mixer, and mixing in certain minutes, then discharged it. Based on operational experience in Kejayan factory, the batch dry mixer has optimal mixing time at 6 - 7 minutes, a homogeneity test must be performed to ensure homogeneous result. Homogeneity test To perform homogeneity test, 10 samples must be taken from different point of area in mixing chamber. These samples must undergo chemical analysis duplo analysis i.e. Vit C content, then a homogeneity calculation should be made. Refer to MI.18.348-1, an internal guideline on how to perform dry mixing, made by Nestle, the homogeneity parameter is Coefficient of Variation CV. If CV is below 1, the product is considered homogeneous and if the CV is 1 - 2 the product is considered marginally homogeneous, which is also acceptable. The formula is expressed as follows: CV = Std. Dev. for chemical value i.e. Fe content of samples Average for chemical value ie. Fe content of samples A nutritional fact of above product per serving 30g this could be expected as follows; macronutrient: total fat 4.2 g, protein 4.3 g, Sugar 12.3 g and micronutrient: Vitamin A 162 mg RE, Vitamin C 15.0 mg, Calcium 225 mg and Iron 2.6 or 5.2 mg b. FillingPacking procedure Bulk chocolate filled milk powder must undergo these below test before filing and packing activity was started: AW test maximum 0.2, iron content target 8.67 mg100g or 17.33 mg100g and sensory evaluation. The bulk was manually filled into aluminium foil sachet, with net weight 30g, then it sealed under vacuum to minimise residual oxygen content. D. Method of Analysis 1. Accelerated keeping quality procedure Through linear regression of KQ data taken from Attachment 1, at temperature NRT and 37°C, it could be established an order 1 equation, as follows : Y1 37°C = -0.0137X + 1.0594 Y2 NRT = -0.0021X + 1.0262 Filled Milk Powder Sucrose Cocoa Powder Filled Milk Powder CaCO 3 Vitamin Iron Weighing Weighing Weighing Weighing Milling Pre-Mixing Weighing Weighing Weighing Weighing Chocolate Filled Milk Powder 30g Final Mixing Homogeneity Test based on Fe content Manual Filling 30g in Sachet Vacuum Sealing Figure 2 Processing step of preparation prototype chocolate filled milk powder Since the KQ data of taste and appearance was almost the same see Attachment 1, therefore above equation could be considered valid for both taste and appearance prediction. From Attachment 3, it showed the correlation between keeping quality at Normal Room Temperature NRT and at 37 o C, there was a correlation between result of keeping quality 37 o C and NRT that was every 1 week at 37 o C similar to 1 month at NRT. However, the condition was only valid until 6 weeks at 37°C that was similar to 6 months at NRT. Refer to technical instructions GI-31.355, May 2006 established by Nestle with title Best Practices for Sensory Evaluation and shelf life assesment, the accelerated keeping quality procedure was developed. The procedure was based on empirical data of keeping quality of similar product produced in PT. Nestle Indonesia, Kejayan factory and considering the average storage time of this similar product sold in the market, which has normally 6 months. It was proposed to perform keeping quality evaluation as follows 1, 3, 6 weeks at 37 o C and 1 month at NRT. Both KQ at temperature NRT and 37 o C were performed under special room, with temperature control. Each KQ period, the same sensory and chemical test must be performed as it was performed in fresh sample evaluation. 2. Sensory evaluation procedure Refer to technical instructions GI-31.107-1, September 2007, established by Nestle with title The “InOut” test method for sensory quality control, a sensory evaluation procedure applicable for this research is made. General rule to performed sensory evaluation : performed by trained panellist, minimum panellist 6 person, using descriptive InOut test, reference sample to define in and out must be available, a cceptable sensory if “In” above 80. Reference sample is chocolate filled milk powder composed as describe in “bulk preparation procedure”, page 27 but without any Iron fortification. Sample preparation : 30 g samples were weighed or pre-weighed 30g sachet open and pour in beaker glass, then 150 mL boiled water was prepared in beaker glass, with temperature as follows : 15 o C for cold reconstitution, 45 o C for warm reconstitution and 80 o C for hot reconstitution. Test before reconstitution, for powder color evaluation, the powder was put in object glass and the color was evaluated, against reference. For powder aroma evaluation, the powder placed in beaker was sniffed and the aroma was evaluated, against reference. Test after reconstitution, pre-weighed 30 g samples was poured in to prepared water 150mL. For color evaluation at fresh and after 15 minutes of reconstitution, the color was evaluated, against reference. And for taste evaluation at fresh and after 15 minutes of reconstitution, the taste was evaluated milky taste, chocolate taste, metallic taste, rancid taste against reference. Sensory evaluation evaluation sheet could be seen in attachment 4 Chemical analysis procedure a. Water activity AW Refer to technical instructions LI-00.015-1, November 2007, established by Nestle with title Water activity using hygrolab from rotronic, an aw test procedure applicable for this research was made. The measurement procedure: the sample cup is filled with approximately three quarters of their volume with sample. Care should be taken not to overfill the cups, as this may contaminate the sensing element and the sample compartment of the instrument measuring chamber. On the other hand, too small an amount of sample in a cup means a large headspace which also can affect the results. The cup is placed inside the measuring cell, as rapidly as possible and the measurement is started. The equilibration time with the sample in place must be allowed before recording a W value Approximately 30 minutes. The temperature value displayed by the instrument is recorded. The aw of all samples at 25 ±1 °C is measured, in duplicate and note the readings individually. Then, the recorded a W value must be corrected by using the calibration value. The picture of AW measurement device is shown in Figure 4. b. Value of pH at fresh and 15 minutes after reconstitution Refer to technical instructions LI-00.222-2, August 2000, established by Nestle with title General pH and Acidity, a pH test procedure applicable for this research was made. Sample preparation : the test portion is weighed in a 150 ml beaker. Recently boiled distilled water is added and cooled to 40-50 °C then a 10 solution 10 g sample + 90 ml water is prepared. Stir until complete dissolution and cool it to room temperature. Measurement with potentiometric method: the calibrated electrode 4.5 is dipped in the sample solution. Slightly stir, and read the pH when the reading is stable for 30 seconds. Picture of water activity meter could be seen in Figure 3. Figure 3 Water activity meter c. Moisture Karl Fischer Refer to technical instructions LI-08.055-1, March 1999, established by Nestle with title Moisture determination according to Karl Fischer, a moisture KF test procedure for this research was applied. The method is based on the ability of iodine to react with water in the presence of sulfur dioxide sulphurous anhydride. Sample is prepared by weighing about 0.5 g sample, to the nearest 0.0001 g into weighing boat, sample is introduced rapidly into the titration vessel and the empty boat is weighed. The difference in mass is the test portion. Measurement : «mode» is pressed, until «KFT» appears, about 40 ml methanol is introduced by aspiration, «start» is pressed to condition the installation, the product to be analysed is rapidly introduce and the titration vessel is closed. The required data is entered in order to start the titration, the concentration result is displayed in percentage and printed. After each determination, the titration cell is emptied by aspiration and the installation is conditioned with clean solvent and the measurement is repeated. At the end of the second titration, the average of the two determinations is calculated. The apparatus for moisture KF is shown in Figure 4. Calculation of moisture content, in g100 g product = v.t m.10 v = volume of KF reagent used, in millilitres t = titer of KF reagent, in mg waterml m = mass of the test portion, in grams 10 = conversion factor of mgml into g100 g d. Iron Fe Refer to technical instructions LI-00.814-1, July 1999, established by Nestle with title Iron by atomic absorption spectroscopy acid hydrolysis method, an Iron test procedure applicable for this research was used. The principle of method is acid hydrolysis of food products with nitric acid for Ca, Na, K and Mg and hydrochloric acid for Fe and Zn. Determination of sodium, potassium, calcium, magnesium, iron and zinc by flame atomic absorption spectroscopy. Figure 4 Apparatus for Moisture Karl Fischer determination Sample preparation: sample is grinded in a suitable sample mill if necessary and ensure that samples are thoroughly mixed before analysis. Acid hydrolysis with hydrochloric acid 3.7 vv : into a 50 ml PP or glass volumetric flask, 1 g dry product is weighed or 5 – 10 g liquid product, to the nearest 1 mg. And then 5 ml concentrat hydrochloric acid is added carefully, mix well and allow standing for 15 minutes. Then 5 ml pure water is added and swirl gently, and it is heated for 30 minutes in rapidly boiling water bath. Sample is removed from water bath and allowed cooling to room temperature. With pure water, make up to volume and mixed well. Sample is filtered through an ashless filter paper, the first five ml of filtrate is discarded and the remaining filtrate is collected for analysis. This solution should be analysed on the day of preparation. Calibration procedure : the atomic absorption system is auto-zero with the 0 calibration solution. Using the auto sampler system or manually, each calibration solution is aspirated starting with the lowest concentration. Three readings per solution are taken and the average absorbance of each solution is calculated. Using the instrument software, the calibration line is calculated by plotting the absorbance in the y axis and the concentration of the element in μgml on the x axis. The linearity of the calibration must be checked. Blank test :a blank test should be carried out in parallel using the same reagents and conditions as described under acid hydrolysis. Measurement : using the auto sampler of the AAS, each product solution and blank solution is aspirated into the AAS. Three readings are taken per solution and the average of three readings is calculated. The absorbance measured is within the calibration range must be ensured. The concentration of the element to be measured should be near to the middle of the calibration range if possible. The concentration of the element to be determined in the analytical solution must not be higher than the highest calibration point. After a rinse solution pure water is aspirated between each analytical solution, the concentration of the element is calculated in each product solution C and blank B using the calibration curve and the data station of the AAS. The apparatus for Iron determination is shown in figure 5. The concentration of iron is calculated in the product solution in mg100 g using the following equation = [C. D – B V] [M.10] C = concentration of Fe in solution of test portion, in gml D = dilution factor, if required B = concentration Fe in solution of blank, in gml V = volume of solution of test portion, in ml M = mass of product analysed, in g 10 = factor to convert gg to mg100g e. Vitamin C Refer to technical instructions LI-00.678, May 2009, established by Nestle with title Ascorbic Acid by Visual Titration, a vitamin C test procedure applicable for this research is used. The method corresponds to the AOAC Official Methods 985.33 and 967.21 with small modifications on test sample preparation. Sample preparation: the whole laboratory sample original container or at least 75 g must be brought to room temperature. It must be homogenized by mixing or grinding. Extraction: into a 100 mL amber glass volumetric flask, 10 - 30 g ± 0.01 g of sample is weighed, depending on the expected vitamin C content. The sample is added with 40 mL water at 40 °C ± 5 °C. and mixed well. If the product contains starch e.g. infant or breakfast cereals products, 100 mg of α-amylase or 0.5 - 1.0 g takadiastase must be added. Figure 5 AAS aparatus for Iron determination Sample must be mixed well, flushed with nitrogen and incubated 15 min ± 1 min at 40 °C ± 5 °C. Twenty mL metaphosphoric acid solution 10 g100 mL must be added and shaken vigorously during 5 min. With water, made up to volume. Through a 597½ folded paper filter, filtered rapidly into an amber glass Erlenmeyer flask. Note : solutions that filter slowly must be centrifuged before filtering. This activity must be proceeded rapidly and do not let stand longer than 30 min. Test solution analysis: into a 100 mL beaker, an aliquot V a of the extract containing 0.5 mg to 2 mg of ascorbic acid is pipetted. 30 mL ± 2 mL of metaphosphoric acid solution 2 g100 mL and 5 mL of acetic acid solution 10 vv are added. Sample is titrated with the DCPIP 2,6-dichlorophenol-indophenol solution 0.5 mgml while stirring with a magnetic stirrer. The end point is reached when a slight pink coloration of the solution persists for at least 15 seconds. The volume of DCPIP solution used V p must be recorded to the nearest 0.02 mL. The apparatus for Vitamin C determination is shown in figure 6. Calculations : the mass fraction, w, of ascorbic acid content in the sample is calculated, in mg100 g of product, as: W = Vp.C.Vo.Vs.100 Vf.Va.m where: V p : volume of DCPIP solution used for the titration of an aliquot of the test solution in mL V f : volume of DCPIP solution used for the titration of the standard solution in mL C : concentration of the standard solution in mgmL V : volume of the standard solution titrated for calibration in mL; 2 V s : volume in which the sample is dissolved in mL; 100 V a : volume of the aliquot part of sample extract in mL m : mass of the test portion in g 100 : factor to express the result per 100 g Figure 6 Titrimetri aparatus for Vitamin C determination 3. Microbiology test procedure Refer to technical instructions LI-00.736, September 1998, established by Nestle with title Enumeration of contaminating micro organism with sugar free agar, a TPC test procedure applicable for this research was used. This method was based on the IDF standard 153:1991; Enumeration of contaminating microorganism. Total plate count is contaminant aerobic mesophilic micro-organism, catalase positive, non-lactic acid bacteria, yeasts and moulds that forms countable colonies. The principal method of this test is basically a test portion or series of decimal dilutions is surface plated on sugar free agar and incubated aerobically at 30oC for 72 hours. For confirmation a catalase test is performed. Sugar free agar composition : peptone from casein 7.5g, peptone from gelatine 7.5g, sodium chloride NaCl 5.0g, agar-agar 9 – 18g, distilled water 1000 mL, pH 7.5 ± 0.2 at 25 o C Sample preparation: 10 g of sample is weighed, to be analysed in 90 mL of tryptone salt ts. The sample is rehydrated or homogenised as appropriate and further decimal dilutions is prepared as necessary. Inoculation: 0.1 mL of the appropriate dilutions is transferred to the surface of Petri- dishes containing sugar free agar and spread the liquid evenly over the surface. The time elapsed between preparations of the samples and spreading should not exceed 15 minutes. Incubation : the inverted plates are incubated for 72 ± 4 hours at 30 ± 1 o C. Stack maximum 6 plates. Reading: after incubation, all colonies grown on the plates are counted except pinpoint colonies The catalase test with at lease 5 colonies must be performed from the sugar free agar plates. Only if positive reaction is obtained the colony is considered as contaminant micro-organism. IV. RESULT AND DISCUSSION

A. Prototype of chocolate filled milk powder