Development of Fortified Biscuits For Children Aged 6-59 Months Old and Sugar-Free Biscuits Enriched with Soy Flour For Diabetics

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INTERNSHIP REPORT

DEVELOPMENT OF FORTIFIED BISCUITS FOR CHILDREN AGED 6-59 MONTHS OLD AND SOY FLOUR

SUBSTITUTED SUGAR-FREE BISCUITS FOR DIABETICS

By

Valerie Valentina Dharmawan 19010142

Submitted to

i3L – Indonesia International Institute for Life Sciences School of Life Sciences

in partial fulfilment of the enrichment program for the Bachelor of Science in

Food Science and Nutrition

Internship Project Supervisor: Junaida Astina, S.Gz., Ph.D.

Internship Project Field Supervisor: Herawati Puspita Dewi, S.Farm., Apt

Jakarta, Indonesia 2022

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CERTIFICATE OF APPROVAL

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COPYRIGHT NOTICE

The author of this report is the owner of the copyright to the original work which she created.

Subsequent actions to the copyright owner’s property must be followed by the author’s prior consent.

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STATEMENT OF ORIGINALITY

Submitted to

Indonesia International Institute for Life Sciences (i3L)

I, Valerie Valentina Dharmawan, do herewith declare that the material contained in my report entitled:

“DEVELOPMENT OF FORTIFIED BISCUITS FOR CHILDREN AGED 6-59 MONTHS OLD AND SOY FLOUR SUBSTITUTED SUGAR-FREE BISCUITS FOR DIABETICS”

Is an original work performed by me under the guidance and advice of my field and academic advisor, Herawati Puspita Dewi, S.Farm, Apt. and Junaida Astina, S.Gz., Ph.D. I have read and do understand the definition and information on use of source and citation style published by i3L. By signing this statement I unequivocally assert that the aforementioned report conforms to published information.

i3L has my permission to submit an electronic copy of my thesis to a commercial document screening service with my name included. If you check NO, your name will be removed prior to submission of the document for screening.

✓ Yes No

Name of student : Valerie Valentina Dharmawan Student ID : 19010142

Study Program : Food Science and Nutrition

Signature : Date : Thursday, 22^ndDecember 2022

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ABSTRACT

Triple burden malnutrition is currently a problem faced by the public health across the world, where micronutrient deficiency, undernutrition, and overnutrition occurs at the same time.

Meanwhile, diabetes is also increasing in its prevalence due to change in lifestyle. To overcome this problem, several strategies could be done, such as food fortification and food substitution. Hence, in this internship program, two biscuits development projects were done with the aim to evaluate the physical properties (diameter, thickness, and spread factor) and sensory acceptance of: 1) micronutrient-fortified biscuits, and 2) sugar-free soy flour biscuits. In the fortified biscuits, no significant differences (p>0.05) were observed in both physical properties and sensory acceptance between unfortified (F1) and fortified biscuits (F2). The result from the sensory acceptance evaluation showed that both formulas were moderately liked by the panelists. For the sugar-free biscuits, 3 formulas were developed (T1 : 0%, T2 : 25%, and T3 : 50% substitution of wheat flour with soy flour). The diameter (p=0.022) and spread ratio (p=0.013) of T1 is significantly higher than T2 and T3. Sensory acceptance results showed that taste was significantly affected (p=0.022) with the highest score observed in T2. On the other hand, aroma, texture, and overall liking were not affected by the substitution of soy flour to the biscuits.

Keywords : food fortification, fortified biscuits, soy flour, sugar-free biscuits, sensory acceptance

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ACKNOWLEDGEMENTS

First and foremost, I would like to express my gratitude to the Almighty God for His guidance and blessings for me to be able to finish this internship at PT. Meprofarm Bandung. I also want to thank my family (papi, mami, dede, and ncus), who always supported me both mentally and financially.

I would like to give my warmest thanks to my field supervisor Ibu Herawati Puspita Dewi, S.Farm., Apt. for the guidance, support, and knowledge given throughout this internship. I am also grateful to have Ms. Junaida Astina, Ph.D as my academic advisor, who always gives insightful suggestions, guidance, encouragement, support for me, and also her patience in dealing with my problems. My gratitude also goes to Bapak Drs. Hendri, Apt. and Ibu Johanna T Sutrisno, S.Si., Apt.

for giving me the opportunity to do my internship at PT. Meprofarm.

Last but not least, I am very thankful to all of my friends that keep me sane during these semesters.

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LIST OF FIGURES

Figure 1.1. Organizational Structure of PT. Meprofarm

Figure 1.2. Organizational Structure of R&D Department PT. Meprofarm

Figure 3.1. Experimental design of fortified biscuits for children aged 6-59 months old project Figure 3.2. Production flow of fortified biscuits

Figure 3.3. Experimental design of sugar-free biscuits project Figure 3.4. Procedure for the sugar-free biscuits production

Figure 3.5. Illustration of sensory acceptance of fortified biscuits with and without premix

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LIST OF TABLES

Table 3.1. Composition of F1 and F2 for fortified biscuits Table 3.2. Composition of T1 for sugar-free biscuits Table 3.3. Composition of T2 for sugar-free biscuits Table 3.4. Composition of T3 for sugar-free biscuits

Table 3.5. Measurement of diameter, thickness, and spread factor of fortified biscuits Table 3.6. Sensory acceptance of fortified biscuits

Table 3.7. Concentration of thiamine Table 3.8. Concentration of riboflavin

Table 3.9. Measurement of diameter, thickness, and spread factor of sugar-free biscuits Table 3.10. Sensory acceptance of sugar-free biscuits

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LIST OF ABBREVIATIONS

R&D Research and Development BusDev Business Development NPD New Product Development

PerBPOM Peraturan Badan Pengawas Obat dan Makanan Permenkes Peraturan Kementrian Kesehatan

H0 Null Hypothesis

H1 Alternative Hypothesis ANOVA Analysis of Variance

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I. INTRODUCTION

1.1. Brief History of the Company

PT. Meprofarm is a pharmaceutical company located in Soekarno Hatta 789 Street Bandung. It has been operating since 1973 in Citarum street Bandung and moved to Soekarno-Hatta street in 1995. PT. Meprofarm puts quality as a sole key that must be achieved and cannot be compromised in producing goods in accordance with the regulations and the latest standard available.

PT. Meprofarm has 4 main buildings, which are MEPRO-1, MEPRO-2, beta lactam, and cephalosporin. MEPRO-1 has been operating since 1995 with a land area of 40,000 m² and a building area of 25,000 m². The production facility is built and designed according to the current Cara Pembuatan Obat yang Baik (Indonesia’s Good Manufacturing Practices). It is used to produce tablets, capsules, and liquid capsules. On the other hand, MEPRO-2 was built in 2011 and focused to produce liquid injection, liquid syrups, creams and suppositories/ovules. Beta lactam and cephalosporin buildings are used to produce antibiotics, either in dry injection or dry syrup form.

Research and development (R&D) department was also located in MEPRO-2 and has

“state of the art” facilities to accommodate a better development of new products along with the marketing strategies and the improvement of the products itself. Both facilities are not only to meet PT. Meprofarm’s needs but also accommodate other pharmaceutical companies' toll manufacturing requests.

1.2. Vision and mission

PT. Meprofarm has a vision to promote products and health services to improve the living standards and a mission to be one of the biggest health companies that keeps making continuous improvement and a good quality of health services. Besides vision and mission, PT Meprofarm also has ‘MEPRO Values’ consisting of mature and responsible, efficient in any action, passionate, honest and communicative, robust, and optimistic to achieve excellent results.

1.3. Main Tasks

PT. Meprofarm produced a wide range of products, such as antibiotics, antivirals, hormones, antidepressants, antihistamines, vitamins and minerals, surgical dressing and wound care, etc. PT. Meprofarm aimed to expand their health services by starting to produce

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healthy food and beverages along with medical devices. Thus, the R&D department is planning to produce vitamin and mineral fortified biscuits for children aged 6-59 months and sugar-free biscuits enriched with soy flour for diabetics.

1.4. Organizational Structure

The organizational structure of PT Meprofarm is shown in Figure 1.1. PT. Meprofarm is led by a president commissioner and helped by a president director. The president director is in charge of 4 departments, which are Sales and Marketing, Business Development (BusDev), Manufacturing, as well as Finance and Operations. Each department is headed by a vice president, who is responsible for the appointed functional departments.

The manufacturing department consists of the most sub-departments with 2 general managers, plant operations and quality operations general manager. R&D, Production 1, and Production 2 is supervised under the plant operations, while the Quality Service and Compliance (QSC), Quality Control (QC) and Quality Assurance (QA) is supervised under the quality operations. Engineering department is positioned directly under the manufacturing vice president.

a. Department of Placement

The organizational structure of R&D department is shown in Figure 1.2.

Meprofarm’s R&D Department. The internship program took place in the R&D department, specifically in the new product development sub-department. This department is the sub-department under the manufacturing department. There is one R&D manager, who is in charge of the whole R&D department along with 4 assistant managers. All of the assistant managers were assisted with R&D pharmacist, R&D senior analyst (for method of analysis and existing product, transfer formulation, and stability), and R&D analyst.

The R&D assistant manager for method of analysis (MoA) is responsible for developing and improving the methods used in raw materials and drug testing, while the R&D assistant manager for Uji Disolusi Terbanding (UDT), BE (Bioequivalent), and Sample supervises and developed products that needed to be tested by UDT (in-vitro testing with dissolution method) and BE (clinical trial). UDT, BE, and Sample also searched for second sources of raw materials needed for production. The R&D assistant manager for New Product Development (NPD) is responsible for developing new products, either new drug formulations or developing generic drug formulations, supplements, herbal

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medicine and other products that are not tested for UDT and BE. The last R&D assistant manager for Existing products, Transfer Formulation, and Stability is in charge of existing products including improving the quality of existing products by complying with the current regulations, surveillance and troubleshooting any problems that occur during the production process. The R&D pharmacist for documentation managed the document such as PPI (Prosedur Pengolahan dan Pengemasan Induk) as well as organizing the document needed for the state-owned enterprise such as Badan Pengawas Obat dan Makanan (BPOM).

The R&D NPD works closely with the BusDev department, who is responsible for collecting data and market analysis, doctors, drug sales, etc. From the data collected, BusDev will request which products will be developed that have good prospects in the market, along with the desired parameters, such as dosage forms, dose, taste, color, etc.

The NPD will then make a pre-formulation and formula development in laboratory scale trials, where the MoA division will develop methods to analyze the product simultaneously. When the ideal formulation is achieved, the stability of the product will be evaluated by the stability division under Existing Product and Transfer Formulation.

Once the data from the analysis is obtained, it will be transferred to the Production and Quality Control department. At the same time, scale up from lab scale to pilot scale (10%

from the commercial batch) is also done, along with optimization for the production process (pilot stability test with a minimum of 6 months drug data stability). Following that, the registration for the product will be made by the R&D pharmacist for documentation for commercialization.

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Figure 1.1. Organizational Structure of PT. Meprofarm

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Figure 1.2. Organizational Structure of R&D Department PT. Meprofarm

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II. INTERNSHIP ACTIVITIES

2.1. Internship Working Conditions

The internship was carried out in the research and development department starting from 4^thof July until 22^ndof December. The first week of the internship was an orientation week, exploring the company and its different departments from manufacturing departments such as QC, QA, R&D, production planning and inventory control (PPIC), production 1 (non-sterile), production 2 (sterile), and engineering. The person in charge of each department took turns to explain a brief description of each department, what kind of job each department did, and the production flow. In this chance, a company tour was also done where the production, starting from mixing to packaging could be observed.

The schedule for the internship was from Monday to Friday, starting from 8.30 AM to 4 PM. There was a lunch break everyday from 12 PM to 12.30 PM. In the first month of internship, the tasks involve literature review, including obtaining as much information regarding Peraturan Badan Pengawas Obat dan Makanan (PerBPOM) and Peraturan Kementrian Kesehatan (Permenkes) that are pertaining to the biscuits development and production. Then, the formulations by theoretical calculations were done based on the PerBPOM that regulate the biscuits development, where the macronutrient and micronutrient content in the products should achieve the target. When the initial formula developed, the ingredients were immediately purchased through the purchasing team. While waiting for the ingredients and utensils to arrive, the field supervisor, Bu Hera, offered to learn about new product formulations with the analyst.

2.2. Internship Task and Experiences

a. Development of fortified biscuits for children aged 6-59 months old

The initial task for this internship was to develop vitamins and minerals fortified biscuits for both children aged 6-59 months and pregnant women. The project commenced with collecting all of the regulations necessary for the development of the biscuits, such as PerBPOM and Permenkes. After that, the competitor's product was analyzed, from its nutritional facts and the composition. The composition of the biscuits was then taken as a benchmark to develop the biscuits.

After acquiring all of the information from the BPOM and the competitor, purchasing of the ingredients and utensils needed was made through the purchasing department. While waiting for the purchased items, the amount of vitamins and

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minerals needed for the fortifications were calculated based on the Peraturan Kementrian Kesehatan (Permenkes) no. 51 tahun 2016 tentang Standar Produk Suplementasi Gizi. In that document, there are specific amounts of both macronutrient and micronutrient standards that need to be fulfilled when producing biscuits. During that time also, the formulation for the biscuits was done by theoretical measurements based on Permenkes no. 51 tahun 2016.

When all of the ingredients and utensils needed arrived, the trial for the fortified biscuits started. Since the formulation was made only based on the competitor’s ingredients with rough measurements, the first trial did not go as expected. The formula was developed until it produced a better product by literature review. However, the project was discontinued. Trial for biscuits for pregnant women was also canceled, thus, the work done for the pregnant women biscuits was only until literature review and analysis of competitors.

b. Development of soy flour substituted sugar-free biscuits

Following that, the sugar-free biscuits were developed using soy flour. The development of the sugar-free biscuits started with the idea of healthy biscuits for diabetics. The idea was to replace the sugar with artificial sweeteners and to improve the nutritional content of the biscuits. Thus, sucralose and soy flour were chosen as the ingredients to replace sugar and to substitute the wheat flour respectively. After that, literature review was done to obtain the formulation of the sugar-free biscuits, along with studying the PerBPOM to understand the requirements to develop the sugar-free biscuits.

Soybean is a good protein source with minimal saturated fat content and excellent profile of amino acids content, making soybean superior to other plant protein.

Since biscuits are high in caloric but low in fiber content, soy flour made an ideal substitution of wheat flour to improve the nutrient profile of the biscuits. Several trials were done until the desired biscuits were obtained. The formulations were then produced for moisture content analysis and for the sensory acceptance using a 9-point hedonic scale for both fortified and sugar-free biscuits.

c. Other tasks given throughout the internship program

In between trials, the field supervisor gave me several tasks to do including : 1. Calculation of the nutrition facts for supplement products

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2. Competitor’s analysis

3. Literature review for biscuits products and functional ingredients 4. Literature review on analysis method for biscuits

5. Literature review about perBPOM in relation to biscuit development, diabetes, nutrition facts, etc.

Aside from that, when waiting for the ingredients for the trial to arrive, Bu Hera, the field supervisor from Mepro offered to learn more about drug development. Around 3-4 weeks, Ka Yani, Bu Hera’s analyst, taught a lot about drugs and supplements, from lab scale trials to production of certain products, analysis and testing done for products, etc.

In the last week of internship, Bu Hera offered to learn about HPLC. There was one analyst that was analyzing the thiamine and riboflavin content in the supplement product. Since there was leftover premix from the fortified biscuits trial, thiamine and riboflavin contained in the premix was analyzed. Only 2 vitamins were analyzed due to the limited standard used.

2.3. Challenges During Internship

The biggest challenge faced during this internship is that there were no people specialized in the food science field. Almost everyone in PT. Meprofarm’s R&D department came from either apothecary or chemistry analyst backgrounds. Since no one specialized in the food field, all of the work should be done independently, from collecting all of the regulations until the sensory evaluation. Nonetheless, after doing literature review, the problems faced were able to be solved one at a time.

Since PT. Meprofarm is a pharmaceutical company, all of the ingredients needed for the cookie were not available. Thus, the formulation should be done as soon as possible to obtain the ingredients needed. After having the list of ingredients, the purchasing team will be contacted to purchase the items needed. However, the purchasement took a relatively long time, about 1 month until all of the ingredients arrived. It affects the timeline that has been agreed upon the Term of Internship agreement as the time for the trial was shorter than initially planned. Although it affects the initial timeline, the trials were able to be done before the internship period ended.

In formulating the biscuits, many aspects should be fulfilled such as the minimum and maximum dietary components and nutrients. The calculations for the dietary components were challenging, as it affects one another. Hence, the formulations need to be

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done thoroughly to perceive a good result for biscuits as well as fulfilling the macronutrient and micronutrient requirements. During the formulation, the knowledge gained from the human nutrition course came in useful. Since there is a minimum amount of each nutrient that should be fulfilled, it was possible to be calculated beforehand by looking at the nutrition facts.

Upon doing the sensory evaluation for the sugar-free biscuits, the sensory condition is not very suitable. For instance, the success of the sensory evaluation will increase when it is done in an uninterrupted and quiet manner. It is important to keep panelists away from influencing each other. The serving size of the samples should be identical between each sample. Along with that, a palate cleanser should be used in order to remove the residual matter from the previous sample (Heymann and Lawless, 2013). However, in this case, the sensory evaluation was done when the panelists were still working. It is difficult to set up ideal sensory evaluation conditions with this situation.

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III. PROJECT

3.1. Project Description a. Introduction

Health problems are currently faced by the world, which is denoted by an increase in sedentary behavior along with a nutritionally poorer diet (Duarte et al., 2021). A global public health problem that is found worldwide is triple burden malnutrition, which include micronutrient deficiency, underweight (wasting and stunting) and overnutrition (obesity and overweight) (K, 2021). Hence, the significance of different eating habits and the importance of more nutritionally-adjusted food choice has been accentuated, both in academic research and media, specifically in factors of food choice. Coupled with advanced technology, companies could develop a wide range of new concepts of nutritionally-adjusted diets concepts, namely functional products, fortified products, and sweeteners (Amin et al., 2016).

The growing and increasingly demanding market targeted popular foods for incorporation of nutrients as the effective vehicle. One of the potential foods is biscuits, which shows the capability to meet nutritional needs and diet-related illness prevention.

Biscuits have long shelf lives, broadly consumed as snacks or as a complementary to other foods, and are available in many forms and pleasant flavors. Consequently, biscuits are considerably increasing in both production and consumption worldwide (Goubgou et al., 2021).

Biscuits and similar products have been consumed for decades (Chavan et al., 2016). It is considered as the most preferred snack for young and elderly individuals as it has long shelf life, low manufacturing cost, convenience, and the ability to act as a vehicle for important nutrients (Amin et al., 2016). According to the Agricultural Data Center and Information System (2021), the consumption of biscuits in Indonesia in 2021 per week reached 196.194 kg. The main ingredient of biscuits is wheat flour, which are commonly used in bakery products due to its rheological characteristics given to the products (Aleem Zaker et al., 2012) Biscuits are considered unhealthy due to their high content of fat and sugar, but low in protein, fibers, vitamins, and minerals (Ishera et al, 2021).

Biscuits make an ideal product for fortification and has been used as a nutritional strategy to tackle several chronic and nutrition-related diseases, including diabetes, nutrient deficiencies, and obesity (Goubgou et al., 2021). Food fortification has been

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carried out for years to prevent micronutrient malnutrition due to its cost effectiveness (Chadare et al., 2019). As defined by the WHO, food fortification, or is similar to food restoration and enrichment, is the addition of one or more ingredients to the food, whether it is already present in the food or not, that aim to inhibit or enhance a well-studied deficiency related to one or more ingredients in certain groups or in the population. However, micronutrient fortification could affect the sensory attributes of the product, such as taste and mouthfeel properties (Tsikritzi et al., 2014).

On the other hand, sugar-free biscuits were also developed as an alternative snack for diabetes and obese adults. Biscuit products are mainly made with wheat flour as its major ingredient, along with sugar and fat (Nadian et al., 2021). Most biscuits products are high in calories and low in fiber content (Petrović et al., 2016). Excessive consumption of sugar can lead to several diseases such as diabetes and obesity.

Therefore, an increase of functional properties of the biscuits could be done by replacing sugar with artificial sweeteners (Nadian et al., 2021) and soy flour to substitute the wheat flour (Mishra & Chandra, 2012). Currently, there are a number of artificial sweeteners to replace sugar. One of them is sucralose, which is approximately 600 times sweeter than sucrose and it is non caloric. Utilization of sucralose has been approved by the Food and Agriculture (FAO) and World Health Organization (WHO) (Savitha et al., 2018).

Soy flour has a great profile of protein, vitamins, minerals, and fibers that could increase the nutritive aspects to the final product. Research has shown that the addition of soy flour to bakery products increases the nutritional value as well as the sensorial properties of the final product, such as improved color and pleasant taste (Petrovic et al., 2016). According to Ghosal & Kaushik (2020), protein source for cookies should not affect the rheological properties of the cookies, have pleasant or bland flavor, and should not negatively affect the texture of cookies. Study from Aleem Zaker (2012) also observed that the addition of soy flour up to 20% enhances the physical properties of the products. Thus, soy flour makes a great substitute for the biscuits to improve the properties of the biscuits and the nutritional value. The objective of this study is to evaluate the physical properties including diameter, thickness, and spread ratio along with the sensory acceptance of the fortified biscuits and the sugar-free biscuits.

b. Objectives

The objective of this project are :

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- To evaluate the physical properties (diameter, thickness, and spread ratio) for the fortified biscuits for children aged 6-59 months old.

- To evaluate the physical properties (diameter, thickness, and spread ratio) for the sugar-free biscuits enriched with soy flour for diabetics.

- To evaluate the sensory acceptability for the fortified biscuits for children aged 6-59 months old.

- To evaluate the sensory acceptability for the sugar-free biscuits enriched with soy flour for diabetics.

c. Scope of Work

i. Fortified biscuits

- Literature study in biscuits development along with vitamins and minerals fortification in food.

- Gathering PerBPOM and Permenkes needed related to biscuits development, additive ingredients, food fortifications, macronutrient and micronutrient regulations.

- Analyzing competitor’s product on the nutrition facts and the composition of the product.

- Calculating the amount of vitamins and minerals needed for the fortification of the biscuits according to the Permenkes no. 51 tahun 2016.

- Formulation of the biscuits according to the standards that have been set in Permenkes.

- Optimization of the formula.

- Physical properties analysis and sensory acceptance evaluation of the biscuits.

ii. Sugar-free biscuits

- Literature study in sugar free biscuits, diabetes, and glycemic index, and soy flour.

- Gathering PerBPOM needed related to biscuits development, additive ingredients, sugar-free labeling for food products, and macronutrient regulations.

- Analyzing competitor’s product on the nutrition facts and the composition of the product.

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- Formulation of the biscuits according to standards that have been set in PerBPOM.

- Optimization of the formula.

- Physical properties analysis and sensory acceptance evaluation of the biscuits.

d. Hypothesis

The hypothesis of this study are as follows :

i. The H0is the addition of premix to the biscuits will not affect the physical properties (diameter, thickness, and spread factor) of the fortified biscuits, while the H1is the addition of premix to the biscuits will affect the physical properties of the fortified biscuits.

ii. The H0 is the addition of premix to the biscuits will not affect the sensory acceptance of the fortified biscuits, while the H1 is the addition of premix to the biscuits will affect the sensory acceptance of the fortified biscuits.

iii. The H0 is the addition of soy flour to the biscuits will not affect the physical properties (diameter, thickness, and spread factor) of the sugar-free biscuits, while the H1is the addition of soy flour to the biscuits will affect the physical properties of the sugar-free biscuits.

iv. The H0 is the addition of soy flour to the biscuits will not affect the sensory acceptance of the sugar-free biscuits, while the H1is the addition of soy flour to the biscuits will affect the sensory acceptance of the sugar-free biscuits.

e. Problem Formulation

PT. Meprofarm wants to develop healthy food products, including fortified biscuits for children aged 6-59 months old and sugar-free biscuits for adults. There are several factors that need to be considered in developing the fortified biscuits : 1) Macronutrient and micronutrient standards from Permenkes no. 51 tahun 2016, 2) Amount of vitamins and minerals premix needed to be added to the fortified biscuits, 3) Selected ingredients that will be utilized, 4) Composition of the ingredients used.

As for the sugar-free biscuits, several things that need to be taken into consideration include : 1) Macronutrient composition of biscuits product 2) Sugar-free labeling for food products as mentioned in PerBPOM no. 1 Tahun 2022, 3) Selected

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ingredients that will be used in the biscuit production, 4) Composition of the ingredients used.

f. Experimental Design

Fortified biscuits for children aged 6-59 months old

The formula for the biscuits were developed in August. The experimental design for this study is shown in Figure 3.1. There were 2 different formulations for the biscuits, biscuits dough without premix (F1) and with premix (F2).The different formulations of the dough were then baked at 175^oC for 13 minutes. The biscuits were then analyzed for the spread factor, moisture content and sensory acceptance.

Figure 3.1. Experimental design of biscuits for children aged 6-59 months old project

Sugar-free biscuits

The experimental design for sugar-free biscuits begins with preparation of the dough, where the wheat flour was replaced with different amounts of soy flour, that is 0% (T1), 25% (T2), and 50% (T3). The biscuits were then tested for sensory evaluation using a 9-point hedonic scale.

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Figure 3.3. Experimental design of sugar-free biscuits project

i. Biscuit preparation

- Fortified biscuits for children aged 6-59 months old

The formulation of the biscuits was obtained from Davidson (2016) with modifications. There were two formulations of biscuits, where Formula 1 (F1) contains no vitamins and minerals premixes, while F2 contains vitamins and minerals premixes. Table 3.1. shows the amount of the ingredients needed for the biscuits formulation. The ingredients were weighed accurately using an analytical balance. Premix 1 contains d-alpha-Tocopherol 1000 IU/g, phytomenadione, selenium SE 2000, cyanocobalamin, potassium iodide, and calcium carbonate, where premix 2 contains vitamin A palmitate 1 MIU/gram, dry vitamin D3 100 CWS, thiamine hydrochloride, riboflavin, niacinamide, pyridoxine hydrochloride, and folic acid.

The amount of vitamins and minerals contained in premix 1 and 2 are listed in Appendix 1. and Appendix 2. The premixes were made by weighing the ingredients accurately using analytical balance. It was mixed using a mortar and pestle until it produced a homogenous mixture. The mixture was then meshed using mesh 40 and transferred to a sealed plastic bag. The premixes were stored in a desiccator.

Table 3.1. Composition of F1 and F2 for fortified biscuits

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Ingredients

F1 F2

gram %w/w gram %w/w

Wheat Flour 55 g 55.50% 55 g 54.67%

Sugar 17 g 17.15% 17 g 16.90%

Palm oil 14 g 14.10%% 14 g 13.14%

Skimmed milk powder

11 g 11.10% 11 g 10.93%

Corn starch 0.55 g 0.55% 0.55 g 0.55%

Sodium bicarbonate

1 g 1% 1 g 1%

Calcium carbonate

0 g 0% 0.234 g 0.234%

Dibasic potassium phosphate

0 g 0% 1.260 g 1.26%

Ferrous fumarate 0 g 0% 0.002 g 0.002%

Zinc sulfate 0 g 0% 0.009 g 0.009%

Lecithin 0.55 g 0.55% 0.55 g 0.55%

Premix 1 0 g 0% 0.001 g 0.001%

Premix 2 0 g 0% 0.016 g 0.016%

The biscuits production starts with mixing all of the ingredients using a hand mixer for 2 minutes except for flour and sodium bicarbonate. The flour and sodium bicarbonate were then added to the mixture and then mixed for 1 minute. The dough was rolled into 3 mm thickness and molded with a circle cookie cutter with a diameter of 6 cm. The production flow of the fortified biscuits production could be seen in Figure 3.2.

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Figure 3.2. Production flow of fortified biscuits

- Soy flour substituted sugar-free biscuits

The formulation for the biscuits was obtained from Amin et al.

(2006) with modification. T1 contained 0% soy flour while in T2 and T3, the wheat flour was substituted with 25% and 50% soy flour respectively. The composition of the ingredients used is presented in Table 3.2. All of the ingredients were weighed accurately using an analytical balance.

Table 3.2. Composition T1 for sugar-free biscuits

Ingredients T1 % w/w

Wheat Flour 13.15 g 54.33%

Soy Flour 0 g 0%

Shortening 9 g 37.18%

Skimmed milk powder 1.94 g 8.01%

Sucralose 0.015 g 0.06%

Salt 0.1 g 0.41%

Wheat Flour 9.86 g 40.76%

Soy Flour 3.29 g 13.60%

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Salt 0.1 g 0.41%

Wheat Flour 6.575 g 27.17

Soy Flour 6.575 g 27.17

Salt 0.1 g 0.41%

Figure 3.4. describes the process for the biscuits making. In the first mixing, creaming of shortening and sucralose was done with a hand mixer for 3 minutes. Flour, skimmed milk powder, and salts were added to the creamy mass and mixed for 3 minutes until homogenous. The dough was then rolled into 3 mm thickness with a rolling pin and molded with a circle cookie cutter with a diameter of 6 cm. The molded dough was then placed in a baking tray and baked in a preheated oven at 180^oC for 6 minutes. The biscuits were rested for 30 minutes until they reached room temperature and stored in a sealed plastic bag.

Figure 3.4. Procedure for the sugar-free biscuits production

ii. Physical properties measurement

Physical properties of the biscuits include diameter, thickness, and spread

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thickness of the biscuits (Saric et al., 2014). The diameter and the thickness of the biscuits was measured according to Mudgil et al. (2017). Six biscuits were laid down edge to edge and the length were measured with a ruler to obtain the average value of the diameter. For the thickness, six biscuits were stacked and their thickness was measured using a caliper. The samples were reorder and restacked three times to obtain the average diameter thickness value respectively.

iii. Moisture content analysis

Moisture content analysis was conducted with a rapid moisture analyzer (Mettler Toledo HB43 Halogen Moisture Analyzer). The sample was grinded and homogenized using mortar and pestle then weighed into 1 gram. The samples were analyzed with a temperature of 105^oC. The moisture content was recorded every 5 minutes and was taken as the final moisture content when the triplicate number of the same moisture content appears.

iv. Sensory acceptance evaluation

The sensory acceptance was conducted to evaluate the organoleptic properties of the biscuits, including color, using a 9-point hedonic scale. There were 10 untrained panelists from the R&D division. The inclusion criteria are young adults aged 17-35 years old who regularly consume biscuits and have no gluten and soy allergies.

Prior to the sensory evaluation, the biscuits were freshly baked, cooled down to room temperature, and served in a sealed plastic bag. The biscuits were coded with a random 3-digit number and simultaneously served to the panelist.

Panelists should rinse their mouth in between samples. The panelists were asked to rate the color, taste, texture, and overall liking of the biscuits, ranging from 1 (dislike extremely) to 9 (like extremely).

v. Data analysis

The data presented were the mean and standard deviation. The data obtained from the moisture content analysis could not be statistically analyzed because there were no replications. On the other hand, the data obtained from diameter, thickness, and spread factor were analyzed using the Shapiro-Wilk test to determine if the data is normally distributed or not. The result showed that the data

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was normally distributed, both in fortified biscuits and sugar-free biscuits. Thus it was analyzed using independent t-test and one-way ANOVA respectively.

The sensory acceptance for the fortified biscuits was done through a 9-point hedonic scale. Normality test was conducted using the Shapiro-Wilk normality test and the result showed that the data were normally distributed for aroma and overall liking, not normally distributed for taste and texture. The data were then analyzed using Mann-Whitney U test for the normally distributed data and independent t-test for the not normally distributed data to obtain the p-value for each attribute.

For the sugar-free biscuits, the sensory acceptance was also conducted with a 9-point hedonic scale. Results from the Shapiro-Wilk normality test showed that the data were normally distributed. The data were then further analyzed using one-way ANOVA.

vi. Analysis of thiamine and riboflavin in the premix 2

Premix 2 for the fortified biscuits was stored in a sealed plastic bag and has been put on the desiccator for 5 months. Vitamin B1 and B2 were analyzed using HPLC with a UV-VIS detector at UV 267 nm. The sample weighed and was put in a 50 mL volumetric flask. Then NaOH 0.1 N and acetonitrile was added to the flask.

The mixture was degassed for 10 minutes using a sonic water bath. Afterwards, formic acid 0.1% was added to the flask and degassed once again using a sonic water bath for 15 minutes. After the degassing process, the flask was added with more formic acid 0.1% until 50 mL. The solution was then filtered using Whatman filter paper and the solution was diluted by transferring the solution to a 20 mL volumetric flask with addition of formic acid 0.1% until 20 mL. The solution was filtered using 0.45 μM Millipore membrane into the HPLC vial.

The HPLC with C18 column and gradient elution was applied using a mobile phase consisting of acetonitrile and dapar format pH 2.6. The injection volume was 2 μL, the flowrate was 0.8 mL/min, and the temperature of the column was 40^oC.

The signal (peak area) of the vitamins were recorded at 267 nm.

The peak area obtained from the analysis was calculated using the following formula to obtain the concentration :

𝐶𝑜𝑛𝑐. = ^𝑎_𝑏 ✕ ^𝑐

𝑑✕ ^𝑒

𝑓 ✕ ^𝑔

ℎ ✕𝑖

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Where :

a : area of the sample b : area of the standard c : weight of the standard d : weight of the premix weighed e : dilution factor the sample f : dilution factor of the standard g : weight of the premix

h : weight of the sample in the premix i : concentration of the standard

3.2. Result and Discussion

a. Fortified biscuits for children aged 6-59 months old i. Physical properties measurement

The result from the diameter, thickness, and spread ratio value could be seen in Table 3.3. The p-value for all physical properties are >0.05, meaning no significant difference is observed between F1 and F2. Diameter and spread ratio in F2 were insignificantly higher with the value of 62.93 ± 0.40 mm and 8.412 ± 0.03 mm respectively, while F1 has higher thickness value of 7.5 ± 0.1 mm.

Spread factor. Study from Shahid (2009) reported that size of the biscuits and spread potential are affected by the particle size of flour and moisture content. Result presented in Table 3.3. Is aligned with the research from Shahid (2009), where the increase of diameter and spread factor along with the decrease in thickness is observed in the increasing concentration of vitamin A in the form of retinyl acetate. Addition of zinc sulfate decreases the diameter and thickness of the cookies, caused by the reduction of starch network and strength of gluten (Usman et al., 2021).

Table 3.5. Measurement of diameter, thickness, and spread factor of fortified biscuits

Physical Properties Formula

p-value F1 (without premix) F2 (with premix)

Diameter (mm) 62.89 ± 0.34^a 62.93 ± 0.40^a 0.895

Thickness (mm) 7.5 ± 0.1^a 7.48 ± 0.03^a 0.722

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Spread Factor 8.39 ± 0.07^a 8.412 ± 0.03^a 0.502 Note : Parametric test with independent t-test was performed to find the statistical differences among samples. Values with different superscript letters in similar row indicate

significant difference (P < 0.05)

ii. Moisture content analysis

The moisture content of F1 and F2 for the biscuits were 5.41% and 4.15%.

Lower moisture content was observed in the fortified biscuit. This result could be caused due to the micronutrient content that increases the solid content (Herawati et al., 2015). Another possible reason for lower moisture content in the fortified biscuits is due to the binding interaction of the vitamins and minerals in the premix with the free water molecules in the biscuits (Mileiva et al., 2017).

iii. Sensory acceptance evaluation

Several organoleptic properties of the biscuits were analyzed, which are color, taste, texture, and overall liking. Figure 3.5. shows the acceptability of the biscuits. The sensory acceptance was analyzed using Mann-Whitney U test due to the data that is not normally distributed. The result shows that the p-value for aroma, taste, and overall liking is p>0.05, meaning that there is no significant difference between F1 and F2.

Table 3.6. Sensory acceptance of fortified biscuits

Sensory Attributes Formula

p-value F1 (without premix) F2 (with premix)

Aroma 6.7 ± 0.88^a 6.8 ± 0.67^a 0.538

Taste 6.7 ± 0.83^a 6.4 ± 0.73^a 0.837

Texture 6.1 ± 1^a 6 ± 0.78^a 0.905

Overall Liking 7.1 ± 0.71^a 7 ± 0.78^a 0.776

Note : Parametric test with independent t-test (taste and texture) and non-parametric test with Mann-Whitney U test (aroma and overall liking) was performed to find the statistical differences among samples. Values with different superscript letters in similar row indicate

The overall liking for both F1 and F2 were 6.5 ± 0.88 and 6.5 ± 0.71 respectively, meaning that biscuits with and without premix were slightly liked by

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the panelists. F1 has the highest acceptance in taste with the value of 6.7 ± 0.83 and F2 has the highest acceptance in aroma with the value of 6.8 ± 0.67, however it was not significantly different (p>0.05). All of the sensory attributes values analyzed were not significantly different where the p-value is >0.05, meaning that the null hypothesis is accepted, thus there is no difference in unfortified and fortified biscuits. This result aligned with the result from Herawati et al. (2015) which shows that the addition of micronutrient premix does not alter both taste and color of the biscuits. Research from Osei et al., (2008) proof that fortification of several micronutrient (vitamin A, vitamin D, vitamin E, vitamin C, thiamine, riboflavin, niacin, pyridoxine, folic acid, cyanocobalamin, iron, zinc, iodine, and copper) to a school meal did not affect the sensory acceptability.

Figure 3.7. Illustration of sensory acceptance of fortified biscuits with and without premix

iv. Analysis of thiamine and riboflavin in the premix 2

Results from HPLC analysis are shown in Table 3.5. and Table 3.6. for thiamine and riboflavin respectively. The premix 2 was stored in a sealed plastic and placed in a desiccator. After 5 months of storage, the concentration of both thiamine and riboflavin was decreasing to 45.08% and 56.69% respectively. These findings could be caused by the sensitivity of thiamine and riboflavin to light exposure (Ahmad et al., 2012; Gonzales, 2022), due to the storage condition of the premix. The premix is stored in a transparent plastic, therefore exposing the premix to light.

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Table 3.7. Concentration of thiamine

Sample Concentration

Thiamine_1 43.29%

Thiamine_2 42.98%

Thiamine_3 48.98%

Average Concentration 45.08%

Table 3.8. Concentration of riboflavin

Sample Concentration

Riboflavin_1 57.77%

Riboflavin_2 57.51%

Riboflavin_3 57.78%

Average Concentration 57.69%

b. Soy flour substituted sugar-free biscuits for diabetics i. Spread factor analysis

Table 3.7. shows the result of diameter, thickness, and spread factor of the sugar-free biscuits. It was found a significant difference in diameter and spread factor of the biscuits, while no significant difference is observed in thickness. The diameter of the biscuits is decreasing with increasing amounts of soy flour substitution, from 62.72 ± 0.92 mm to 60.06 ± 0.10 mm (p=0.022). The spread ratio of the biscuits are also decreasing with the increase of soy flour substitution, ranging from 16.18 ± 0.12 to 15.64 ± 0.23 (p=0.013).

Table 3.9. Measurement of diameter, thickness, and spread factor of sugar-free biscuits

Physical Properties Formula

p-value T1 (0%) T2 (25%) T3 (50%)

Diameter (mm) 62.00 ± 0.60â 61.00 ± 0.44âb 60.06 ± 0.10^b 0.022 Thickness (mm) 3.83 ± 0.04â 3.85 ± 0.03â 3.84 ± 0.01â 0.778

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Spread Factor 16.18 ± 0.12^a 15.87 ± 0.22^ab 15.64 ± 0.23^b 0.013 Note : Parametric test with one-way ANOVA was performed to find the statistical differences among samples. Values with different superscript letters in similar row indicate

Spread factor is the most prime quality characteristic of the biscuits, where greater values of spread factor is more desirable (Devi & Khatkar, 2016). The highest spread factor could be observed in T1. Significant difference (p<0.05) is also observed in the spread factor, meaning the substitution of soy flour has an effect on the spread factor of the samples. Decrease in the spread ratio of the biscuits attributable to the limited free water present in the dough caused by the soy flour that is competing for available hydrophilic sites (Kulthe et al., 2014).

Research from Sharma & Devi (2021) also observed a decrease in spread ratio with increased levels of soy flour, which could be caused by the increase in the protein content as soy is a rich source of protein. Protein could restrict the increase in spread ratio by binding with water. Another study from Mudgil et al. (2017) reported that the water absorption capacity of the flour used affects the biscuits’

spread, where higher water absorption will restrict the spreading of the biscuits due to the quicker partitioning of free water to the hydrophilic sites of the flour.

ii. Moisture content analysis

Moisture content of the sugar-free biscuits was analyzed using a rapid moisture analyzer. The result showed that the moisture content of T1, T2, and T3 are 3.50%, 3.96% and 4.17% respectively. Highest moisture content was found in the biscuits with 50% substitution of soy flour and the lowest moisture content was found in the biscuits with no substitution of soy flour. Higher moisture content is related to the shelf life of the biscuits. Higher moisture content induces proliferation of microbes leading to spoilage and shorter shelf life of baked products (Ndife et al., 2014).

Moisture content of the biscuits decreased with increasing amounts of soy flour added to the biscuits. This result is in agreement with the research conducted by Aleem Zaker et al. (2012) that found increasing the amount of soy flour will increase the moisture content. High water binding capacity of soy flour might be responsible for retaining moisture content resulting in higher value in the product.

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Study from Kulthe et al. (2014) also showed a similar result, which is caused by higher moisture retention possessed by soy flour than wheat flour.

iii. Sensory acceptance evaluation

Data for the sensory acceptance evaluation of the sugar-free biscuits are presented in Table 3.8. No significant differences (p>0.05) are found in aroma, texture, and overall liking, where taste showed significant difference (p<0.05). For aroma, both T1 and T2 have the same aroma score of 6.8 ± 1.03 and 6.8 ± 0.97 respectively, where aroma of T3 decreased slightly with the score of 6.7 ± 1.00.

Ndife et al. (2014) reported that incorporation of soy flour to biscuits commonly linked with beany aroma.

Texture is not significantly affected by different levels of soy flour substitution. However, the score decreased from 7.00 ± 1.05 to 6.2 ± 0.93 as the level of soy flour increased. Ghoshal & Kaushik (2020) reported the same result, where higher levels of soy flour incorporated result in decline in texture. One of the problems in preparation of soymeal fortified goods is coarse texture and dry mouthfeel which is attributed to strong flavor profile resulting in unpalatable and presence of fiber.

Taste was significantly affected (p<0.05) by the substitution of soy flour to the biscuits. T2 received the highest score of taste with the score of 77.1 ± 1.

Overall liking score also showed a similar trend, where 25% incorporation of soy flour to the biscuits received the highest score of 7.4 ± 0.97 and score for 50%

incorporation decreased to 6.7 ±0.97. The most optimum substitution in this study was observed in T2 with 25% of soy flour substitution. Study from Sharma & Devi (2021) showed similar results where cookies below 30% incorporation of soy flour have higher taste and overall liking and after 30% level of substitution, the acceptability decreased. Higher levels of incorporation of soy flour results in greater hardness and poor flavor (Ghoshal & Kaushik, 2020). The undesirable taste of soy flavor is caused by several chemical compounds, including furans, aldehydes, alcohols, trihydroxy fatty acid, fatty acid dimers, phenolics, furfurals, and oxidized phosphatidylcholine present in soybean (Lock, 2007).

Table 3.10. Sensory acceptance of fortified biscuits

Sensory Formula

p-value

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Attributes T1 (0%) T2 (25%) T3 (50%)

Aroma 6.8 ± 1.03â 6.8 ± 0.97â 6.7 ± 1.00â 0.965 Taste 7.2 ± 0.87âb 7.7 ± 1â 6.5 ± 0.88^b 0.022 Texture 7.00 ± 1.05â 6.4 ± 1.22â 6.2 ± 0.93â 0.228 Overall Liking 7.1 ± 1â 7.4 ± 0.97â 6.7 ±0.97â 0.312 Note : Parametric test with one-way ANOVA was performed to find the statistical differences among samples. Values with different superscript letters in similar row indicate

Figure 3.6. Illustration of sensory acceptance of sugar-free biscuits enriched with 0%, 25%, and 50% soy flour

3.3. Conclusion and Recommendation a. Conclusion

Biscuits are one of the most popular foods among young and elderly which is consumed worldwide. However, it is nutritionally poor as it is mostly high in calories and low in fiber content. Therefore, it is an ideal vehicle for food fortification.

Physical properties of fortified biscuits were not significantly affected (p>0.05) by the addition of the premix. Sensory attributes of the fortified biscuits were not affected significantly (p>0.05) by the addition of the premix. The overall liking of the unfortified and fortified biscuits were lower than the fortified biscuits.

For the sugar-free cookies, significant differences (p<0.05) were observed in diameter and spread ratio, where higher amounts of soy flour replacement into the

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formula, the lower amount of diameter and spread ratio were observed. Additionally, the sensory acceptance result of the sugar-free biscuits revealed that taste was significantly affected by the substitution of soy flour, where aroma, texture, and overall liking not. T2 (25% soy flour substitution) was rated the highest score for the taste, and it's significantly different from T3 (50% soy flour substitution).

b. Recommendations

Further study to investigate the micronutrient content in the biscuits should be conducted to evaluate the degradation and stability of micronutrient during the process.

Moisture content analysis should be done in replicates to obtain the result from the statistical analysis where the significance of the treatment could be analyzed. Other analysis should be measured such as proximate analysis to determine the macronutrient content and color analysis to enhance the food quality and to understand the consumer’s acceptance.

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IV. SELF REFLECTION

Since the 4^thof July, I got the opportunity to do my internship at PT. Meprofarm in the R&D department. I got the opportunity to develop biscuits products for children aged 6-59 months and sugar-free biscuits. This project has expanded my knowledge about the formulation of a biscuits product. I also got the chance to understand the BPOM regulation about food products, labeling and nutrition facts guidelines, food additives used in food products, and other regulations.

This experience has enabled me to understand how the industry works, such as how to develop a product from a laboratory scale, pilot scale, and production scale. Along with that, I can also experience what it feels like to be working in a corporate industry, especially in the R&D department. Although PT. Meprofarm is a pharmaceutical company, but it has similar ecosystem as the food and beverage industry in terms of inventory control, production scale, and product development scheme. Another insightful knowledge gained from this internship is about pharmaceutical products and industry. All of my colleagues were very friendly and sometimes, they even explained things I have never learned before, which is mostly about pharmaceutical product development. I also got the chance to be involved in the trial of new products and several analyses such as dissolution tests, sample preparations, and HPLC analysis. I believe this will be a great insight for me for several job opportunities in my interest in the R&D field, analysis, and product development.

During this internship program, I perceive myself as a hardworking and detail oriented person. Throughout this program, I did most of the things by myself. Starting from collecting the regulations applied for biscuit development, formulations, trials, and sensory evaluation. Most of the decisions were made by myself. The field supervisor gave me directions but the decision maker was always me. Thus, the decision making skills were also sharpened. Furthermore, I am also a fast learner and an expeditious person. I can easily grasp the instructions given by the supervisor.

For instance, there are several sensory evaluation methods. I must identify the needs of doing the sensory evaluation, the purpose of the product, and all other aspects to decide on which sensory evaluation methods should be used. In this situation, both decision making and observing skills were used and sharpened. Another case would be when the results of the biscuits trial were not as expected, I must correctly identify which variables should be changed, added, or removed to achieve the desired result. There are times when both me and the supervisor did not understand one concept. When faced with that kind of situation, I will do more research and literature review to solve the confusion.

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Nonetheless, I still have difficulties in making the same perspective between me and my supervisor. Sometimes, there are concepts that I had learned before but it is not familiar to my supervisor. When I try to explain those concepts, sometimes I too could not explain the underlying mechanism behind it. But, I always try to deepen my understanding by doing as much literature review as possible so that I can explain the concepts clearly.

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V. CONCLUSION & RECOMMENDATION

5.1. Conclusion

The aim of this internship program is to obtain ideal formulation for biscuits products, both for children aged 6-59 years old and sugar-free biscuits. The goal has been partially achieved by obtaining an ideal formulation for the sugar-free biscuits. Due to the company’s circumstances, the trial for the biscuits for children must be discontinued.

In food science and nutrition, there were no courses related to the production of the biscuits. There are courses that support the fundamental principles, such as food chemistry and food additives. The knowledge gained from the courses could be applied to improve the formulation of the biscuits.

5.2. Recommendation

PT Meprofarm has a positive working environment which supports the intern to have a great experience while doing the internship program. The knowledge acquired from these experiences were very insightful, although it is mostly about drug development. Thus, PT Meprofarm could accommodate students who want to learn more about drugs and medicines.

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Development of Fortified Biscuits For Children Aged 6-59 Months Old and Sugar-Free Biscuits Enriched with Soy Flour For Diabetics

INTERNSHIP REPORT

DEVELOPMENT OF FORTIFIED BISCUITS FOR CHILDREN AGED 6-59 MONTHS OLD AND SOY FLOUR

SUBSTITUTED SUGAR-FREE BISCUITS FOR DIABETICS

By

Valerie Valentina Dharmawan 19010142

i3L – Indonesia International Institute for Life Sciences School of Life Sciences

Food Science and Nutrition

CERTIFICATE OF APPROVAL

COPYRIGHT NOTICE

STATEMENT OF ORIGINALITY

ABSTRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

LIST OF ABBREVIATIONS

I. INTRODUCTION

II. INTERNSHIP ACTIVITIES

III. PROJECT

IV. SELF REFLECTION

V. CONCLUSION & RECOMMENDATION

REFERENCES