The Effect of Acid Solubilization Condition on The Yield of Calcium Extracted from Pacific White Shrimp (Litopenaeus vannamei) Shell PRACTICAL TRAINING REPORT This practical training report is submitted for the partial requirement for Bachelor Degree

  

The Effect of Acid Solubilization Condition on The Yield of

Calcium Extracted from Pacific White Shrimp (Litopenaeus

vannamei ) Shell

  

PRACTICAL TRAINING REPORT

This practical training report is submitted for the partial requirement for

Bachelor Degree

By :

Angela Irena Wibawa 12.70.0034

  

DEPARTMENT OF FOOD TECHNOLOGY

FACULTY OF AGRICULTURAL TECHNOLOGY

SOEGIJAPRANATA CATHOLIC UNIVERSITY

SEMARANG

2015

  

The Effect of Acid Solubilization Condition on The Yield of

Calcium Extracted from Pacific White Shrimp (Litopenaeus

vannamei ) Shell

  BY: ANGELA IRENA WIBAWA

  Student ID : 12.70.0034 Faculty : Agricultural Technology

  This practical training report has been approved and supported by examiner in Practical Training Exam on

  Departement of Food Technology Faculty of Agricultural Technology

  Soegijapranata Catholic University

  th

  Semarang, July 12 2015 Technical Advisor I Technical Advisor II Assoc. Dr. Nattapol Tangsuphoom Dr. Ch.Retnaningsih, MP.

  Dean First of all, the writer wants to say Thank God for His grace and His blessing so this practical training report entitled “The Effect of Acid Solubilization Condition on The Yield of Calcium Extracted from Pacific White Shrimp (Litopenaeus vannamei) Shell could be completed. This practical training report is submitted to fulfill one of the requirements to gain bachelor degree of Agricultural Technology Faculty, Food Technology Department, Soegijapranata Catholic University.

  During the making of this report, the writer wants to say thank you to :

  1. Assoc. Dr. Nattapol Tangsuphoom as the first advisor who has given the best support and guidance for this practical training, from conducting the experiment until the finishing paper.

  2. Dr. Ch.Retnaningsih, MP. as the second advisor who has given the best support and guidance during the internship and the making of this paper.

  3. Saranya Dechapinan as the lab partner who helped the experiment and the making of this paper.

  4. Assoc. Prof. Dr. Visith Chavasit as Dean of Institute of Nutrition Mahidol University, who has given the opportunity to do the practical training in his faculty.

  5. Dr. V. Kristina Ananingsih, ST., MSc. as Dean of Faculty of Agricultural Technology, Soegijapranata Catholic University, for giving the information and chance to join the practical training abroad.

  6. Kartika Puspa Dwiana, S.TP., Practical Training Coordinator of Faculty of Agricultural Technology, Soegijapranata Catholic University, for her understanding and tolerance.

  7. My parents, Triwibawa Mulja Widarsa and Liana Mahdalena Imantono as my parents, my little sister, Anita Angelina Wibawa who allowed to get practical training abroad and supported during the practical training.

  8. Ivana, Naomi, and Vina, who has giving support, help, and being been best practical training mates during the process.

  9. All of people who has directly and indirectly helped and supported during the

  Finally, the writer wants to apologize if there are accidentally errors during the making of this report and hopes that this report can be useful for the others. The readers also can give suggestions to the writer to improve the content in order to be made as one of the good examples for the next practical training.

  Semarang, Writer

  CONTENTS

  TITLE ........................................................................................................................... i APPROVAL PAGE ...................................................................................................... ii PREFACE ..................................................................................................................... iii CONTENTS ................................................................................................................. iv LIST OF TABLES ....................................................................................................... v LIST OF FIGURES ...................................................................................................... vi 1. INTRODUCTION .................................................................................................

  1 1.1. Practical Training ............................................................................................

  1 1.1.1. Institution Profile ..................................................................................

  1 1.1.1.1. Mahidol University ......................................................................

  1 1.1.1.2. Institute of Nutrition Mahidol University ....................................

  1 1.1.1.3. Vision ..........................................................................................

  2 1.1.1.4. Mission ........................................................................................

  2 1.1.1.5. Faculty Member ...........................................................................

  2 1.1.1.5.1. Administrators ......................................................................

  2 1.1.2. Purpose of Practical Training ................................................................

  3 1.1.3. Time and Place of Practical Training ....................................................

  3 1.2. Research ..........................................................................................................

  4 1.2.1. Background of Research .......................................................................

  4 1.2.2. Objectives .............................................................................................

  5 2. RESEARCH METHODOLOGY ..........................................................................

  6 2.1. Materials ...........................................................................................................

  6 2.2. Methods ............................................................................................................

  6 2.2.1. Deproteinization of Shrimp Shell ...............................................................

  6 2.2.2. Calcium Extraction from Shrimp Shell ......................................................

  6 2.2.3. Analyses ....................................................................................................

  7

  2.2.3.1. Ash Content ........................................................................................... 7

  2.2.3.2. Calcium Content ..................................................................................... 8 2.2.3.3. Calcium Extraction Yield .......................................................................

  8 2.2.4. Statistical Analysis ....................................................................................

  8 3. RESULT AND DISCUSSION ..............................................................................

  9 4. CONCLUSION .....................................................................................................

  13 5. REFERENCES ......................................................................................................

  14

  6. APPENDIX........................................................................................................... 15

  Table 1. Chemical Composition of The Deproteinized Shrimp Shell ..........................

  10 Table 2. Ash Content of The Remaining Shell from Different Extraction Conditions .....................................................................................

  10 Table 3. Calcium Content of The Remaining Shell from Different Extraction Conditions .......................................................................................................

  11 Table 4. Calcium Content of The Calcium Solution Obtained from Different Extractions Conditions ....................................................................................

  12 Table 5. Calcium Extraction Yield Obtained from Different Extraction Conditions .......................................................................................................

  12

  Figure 1. Logo of Mahidol University of Thailand ................................................. 1 Figure 2. Map of Mahidol University, Salaya ......................................................... 3

INTRODUCTION 1.1. Practical Training 1.1.1. Institution Profile

  1.1.1.1. Mahidol University

  Mahidol University has its origins in the establishment of Siriraj Hospital in 1888 by His Majesty King Chulalongkorn (Rama V), and the hospital's medical school is the oldest institution of higher learning in Thailand, granting its first medical degree in 1893. Later becoming the University of Medical Sciences in 1943, Mahidol University was renamed with great honor in 1969 by H.M. King Bhumibol Adulyadej , after his Royal Father, H.R.H Prince Mahidol of Songkla , who is widely known as the 'Father of Modern Medicine and Public Health in Thailand'. Mahidol University has since developed into one of the most prestigious universities in Thailand, internationally known and recognized for the high caliber of research and teaching by its faculty, and its outstanding achievements in teaching, research, international academic collaboration and professional services. This diversified institution now offers top quality programs in numerous social and cultural disciplines, including the most doctoral programs of any institution in Thailand, yet has maintained its traditional excellence in medicine and the sciences.

  Figure 1. Logo of Mahidol University of Thailand

  1.1.1.2. Institute of Nutrition Mahidol University

  The Institute of Nutrition, Mahidol University (INMU) was established in 1977 as a national planning and implementation body of the Thai government, with the mission of strengthening the National Food and Nutrition Plan. Mahidol University was selected among other nutrition and nutrition-related institutions due to its dedication to health

  2 fulfilled this mission by conducting research at community and laboratory levels, by providing national and international training and education programs, and by providing technical services in food and nutrition development. The goal has been, and continues to be, the attainment of the highest possible quality of life for individuals, communities, Thai society, and for people living in other countries within and outside the Southeast Asian Region.

  1.1.1.3.Vision

  Institute of Nutrition Mahidol University strives to become a recognized world leader in food and nutrition by 2015 through innovative research, capacity building and the generation and transfer of advanced knowledge and technologies to effectively address critical food and nutrition challenges at individual, community, national and global levels.

  1.1.1.4. Mission

  Institute of Nutrition Mahidol University is committed to ensuring the optimal health and nutrition of all people by conducting timely basic and applied food and nutrition research into the ever-changing health, social and economic conditions that affect individuals, communities, nations and the world; by producing high caliber food and nutrition professionals who are committed to ethically conducting high quality multidisciplinary food and nutrition research studies that recognize, identify and tackle prominent food and nutrition problems; and by providing advanced academic, technical and administrative services for developing and transferring food and nutrition knowledge and technologies for the benefit of all people within and outside of the country through committed professionals, positive leadership and standards of excellence.

  3

  The main administrators of the faculty are in follow: a. M.Sc. and Ph.D. Degree Program in Nutrition: Dr. Gene Charoonruk.

  b. M.Sc. Degree Program in Food and Nutritional Toxicology: Asst. Prof. Chaniphun Butryee

  c. M.Sc. Degree Program in Food and Nutrition for Development (International program): Asst. Prof. Sitima Jittinandana d. M.Sc. Degree Program in Nutrition and Dietetics (International program): Asst. Prof. Chanida Pachotikarn 1.1.2.

Purpose of Practical Training

  a. To give an experience to explore food in the research, so the student can implement the knowledge that they learned in the scientist world or real food industry.

  b. To give an opportunity to adapt with new circumstances and neighborhood.

  c. To broaden the knowledge and experience of international exposure.

  The practical training is conducted in the Institute of Nutrition, Mahidol University,

  th th Salaya Thailand from 16 January to 26 March 2015.

  4 located in999 Phutthamonthon 4 Rd., Salaya, Phutthamonthon, Nakhon Pathom 73170, Thailand. Tel. 0-2800-2380 Fax. 0-2441-9344).

Research 1.2.1. Background of Research

  Calcium is one of the macro mineral which is needed for human body for the skeletal system. Houtkooper (2004) stated that calcium is needed to build and maintain teeth and bones, it helps muscles and nerves to work properly, and also helps to prevent high blood pressure. Inadequate consumption of calcium can cause diseases such as osteoporosis, osteomalacia, and bone fracture. The calcium needed per day for every individual is different and it depends on their ages. Females need more calcium than males since their mass bone more decrease rapidly, especially those who are menopause which reduce the amount of estrogen hormone. Fikawati (2005) also reported that female students need more calcium intake for replenishing than do male students. Thus the risk of osteoporosis is also higher in women than men (Agustin 2009). A study conducted in Bandung City suggested that calcium intake of adolescent students does not meet the requirement yet which is under 75% RDA.

  Shell of the crustaceans, which is the waste from seafood processing can cause environmental problem because some of flesh left in the shells is the ideal growth media for pathogenic bacteria (Sini et al., 2007). Crustaceans shell consists mainly of chitin, a polymer of N-acetylglucosamine, which naturally exists in complex structure with minerals, especially calcium, and protein. Ravichandran, et al (2009) reported that shrimp shell contains 32.5% protein, 26.6% minerals which mostly is calcium, 9.8% lipid, and 12.3% moisture. Therefore the shell waste is usually utilized as the source for chitin extraction, from which minerals and protein are removed by using strong acid and alkali solutions to solubilize the minerals and protein from the shell. The obtained protein and calcium are frequently discarded as by products.

  Nowadays, calcium is added into several products such as fruit juice and dairy products. It is also fortified in dairy-substitute, cereal based beverages like soy milk. Therefore, calcium extracted from shrimp shell can be utilized for food fortification with very low cost. Different types of calcium, which have different solubility and bioavailability, can

  6 calcium extracted from shrimp shell.

  The main objectives of this study are determine the effect of the condition used for acid solubilization on the yield of the calcium extracted from shrimp shell.

RESEARCH METHODOLOGY

  2.1. Materials

  Frozen shell of Pacific white shrimp (Litopenaeus vannamei) was obtained from a shrimp processing plant of Charoen Pokphand Food (Public) Co., Ltd. Serine endoprotease (Alcalase 2.4L) was donated by Brenntag (Thailand) Co., Ltd. All other chemicals unless otherwise stated were obtained from Sigma Aldrich.

  2.2. Methods 2.2.1. Deproteinization of Shrimp Shell

  First of all, shrimp shell was thawed and then the thawed shell was mixed with de- ionized water at the weight to volume ratio of 1:10. After that, the enzyme was added into them at the concentration of 0.1% (v/w) of the shell and the mixture was heated in a

  o

  temperature-controlled water bath for 2 hours at pH 9 and 50

  C, which is the optimal condition of the enzyme, to hydrolyze and remove the protein from the shell. After that,

  o

  the mixture was heated at 90 C for 10 minutes in the water bath to inactivate the enzyme and after that it was put immediately into the ice bath to cool down the room temperature (28°C). The deproteinized shell was separated from protein solution by

  o

  wire screen and dried in the hot air oven at 50 C for 6 hours. The dried deproteinized shrimp shell was packed and sealed in LDPE bags and kept in the desiccator for further analysis. Chemical composition of the deproteinized shell was analyzed according to AOAC Official Method.

  Extraction of calcium was performed by mixing the dried deproteinized shrimp shell with HCl solution at the weight to volume ratio of 1:40. The concentration of acid was calculated to obtain the stoichiometric equivalence ratio of the extracted calcium compound, i.e., 2:1 for CaCl

  2 in this study. Extractions were performed at room temperature, 50 and 70°C for 1, 2, and 4 hours on a hot plate with magnetic stirrer.

  After that, the mixture was cooled immediately to the room temperature and the remaining shell was separated from the calcium solution by wire screen before being

  8

  2.2.3.1. Ash Content

  The ash contents in the shells before and after calcium extraction were analyzed according to AOAC Official Method. The crucible and lid were put in the furnace prior

  o

  to heating at 550 C for overnight. After that the crucible was cooled in the desiccator for 30 minutes. Then, the crucible and the lid were weighed. One gram of the sample was put in the crucible. After that, the crucible was heated over flow above Bunsen flame with lid half covered until the fume was no longer produced. Then, crucible with lid and

  o

  sample was put in the furnace and heated at 550 C for overnight. During heating, lid was not covered to prevent loss of fluffy ash but it was put on after the heating was complete, which was noticed as the sample turned to grey. The crucible was then cooled down in the desiccator. The weight of crucible with lid and the remaining ash was recorded and the ash content was calculated based on the following equation:

  2.2.3.2. Calcium Content

  The calcium contents both in the solution and in the remaining shell from each extraction condition were analyzed by using an atomic absorption spectrometry (AAS) equipped with flame and graphite furnace. The remaining ash from the shell was added 5 ml of 4 N nitric acid solution and diluted with distilled water to obtain the ash solution of the final volume of 50 ml. For calcium content analysis by AAS, aliquots (125 µl) of the ash solution from the remaining shell and the calcium solution were added with 2.5 ml of 4 N nitric acid solution and 5 ml of 5% lanthanum oxide and diluted with distilled water to the final volume of 25 ml prior to AAS analysis.

  2.2.3.3. Calcium Extraction Yield

  The calcium extraction yield was calculated from the calcium contents in the shell before and after extraction based on the following equation:

  9

Statistical Analysis

  All experiment was performed in triplicates. The results were presented as means ± standart deviations. Statistical software (SPSS 18.0, SPSS, Chicago, Illinois, U.S.A.) was used to analyze the data.

RESULTS AND DISCUSSION

  Hartini et al. (2003) stated that the source of calcium is widely-available in broccoli, cabbage, legumes, milk, dairy products and also fortified products such as soy milk. Houtkooper (2004) also added that the calcium needed for individual ages between 9-18 years old is 1300 miligram each day. The adults between 19-50 years old need 1000 miligram calcium per day and for people

  ≥ 50 years old need 1200 miligram calcium per day. Fikawati (2005) reported that 76.2% from 1254 students consume the calcium less than 75% of RDA and the RDA for Indonesian is 1200 miligram/ day for the teenagers.

  Extracted Calcium from the shrimp shell can be utilized for the fortification because it is cheap but there are several factors that can influence the percent yield of shrimp shell such as pH, acid, temperature and time and this study used two parameters to find the suitable condition for the % extraction yield. Before it obtained % extraction yield, it used deproteinization to remove the protein and get the calcium from the Pacific White shrimp shell. Then, calcium extracted with HCl to get the mixture of calcium with acid from the shrimp shell. Percon et al (2003) reported that HCl didn’t give the good effect to the molecular weight in the demineralization of chitin but Poeloengasihhernawan (2009) stated it has high ability to remove ash and protein than acetic acid. After that, AAS was used to analyze the extracted calcium.

  The result of chemical compostion of the deproteinized shrimp shell is shown in the table 1.

  Table 1. Chemical Composition of The Deproteinized Shrimp Shell

  Composition Content (/100 g)* Wet weight basis Dry weight basis

  Moisture 9.96 g 90.05 g Protein (N x 6.25) 41.79 g 46.41 g Ash 25.48 g 28.30 g Calcium 8365.805 mg 9290.280 mg Dietary fiber 30.38 g 33.74 g

  11 deproteinized shrimp shell based on the wet weight basis which is 8365.805 mg/100 g and the dry weight basis which is 9290.280 mg/100 g. On the previous study by Ravichandran (2009), it mentioned that the calcium content in the shrimp shell in Indian White Shrimp (Penaeus indicus) is higher than its flesh so shrimp shell is the good source of calcium. It is also supported by Okoye (2005) that shrimp waste is good source for calcium but the bioavailability for the protein is low for the broiler chicken. The result of ash content of the remaining shell from different extraction conditions is shown in the table 2.

  

Table 2. Ash Content of The Remaining Shell from Different Extraction

Conditions o

  Temperature (

  C) Ash content (mg/100g)* 1 hour 2 hours 4 hours 28 3.90 ± 0.24 3.81 ± 0.20 5.00 ± 0.35 50 3.00 ± 0.34 3.12 ± 0.09 2.94 ± 0.21 70 3.17 ± 0.11 3.14 ± 0.60 2.68 ± 0.47

• Mean ± standard deviation of triplicate samples From the table 2, it showed that the higher temperature, the lower ash content and the

  o

  longer time, the lower ash content too but the data in the temperature 28

  C, 4 hours showed that the longer time, the higher ash content. Poeloengasihhernawan (2009) stated that the ash of the shrimp shell are higher than those of heads because of the different chemical composition of the raw material but preconditioned shell has lower ash content than head and after the demineralization process, shell has higher ash content than head. Fall (2012) reported that the highest ash was obtained from 100% shrimp shell meal for hybrid tilapia and it could substitute the soybean meal up to 60% for hybrid tilapia’s meal. Trung (2012) also reported that The purified chitosan showed the higher quality than chitosan especially lower ash and protein content. Then shrimp shell powder also has the higher ash than raw chitin (Khorrami, 2012).

  12 is shown in table 3.

  

Table 3. Calcium Content of The Remaining Shell from Different Extraction

Conditions o

  Temperature (

  C) Calcium content (mg/100g)* 1 hour 2 hours 4 hours 28 1052.92 ± 89.03 1001.29 ± 47.89 1010.66 ± 188.64 50 971.05 ± 112.71 941.47 ± 76.15 955.44 ± 149.02 70 954.58 ± 183.90 769.50 ± 51.60 803.10 ± 33.00

• Mean ± standard deviation of triplicate samples From the data above, it showed that the higher temperature, the lower calcium content from the remaining shell and the longer time, the lower calcium content too but the calcium content increased slightly at 4 hours for each temperature. The result of calcium content of the calcium solution obtained from different extractions conditions is shown in the table 4.

  

Table 4. Calcium Content of The Calcium Solution Obtained from Different

Extraction Conditions o

  Temperature (

  C) Calcium content (mg/100g) 1 hour 2 hours 4 hours 28 214.12 ± 3.00 216.15 ± 15.60 221.18 ±7.48 50 224.06 ± 7.67 227.1 ± 12.61 226.89 ± 3.10 70 224.33 ± 21.00 227.1 ± 8.62 239.38 ± 6.00

• Mean ± standard deviation of triplicate samples Based on the data above, it showed that the higher temperature, the higher calcium content of the calcium solution and the longer time, the higher calcium content of the solution too.

  13 shown in the table 5.

  

Table 5. Calcium Extraction Yield Obtained from Different Extraction Conditions

o

  Temperature (

  C) Calcium extraction yield (%)* 1 hour 2 hours 4 hours

  aA bA aA

  28 87.41±1.06 87.91±0.43 87.92±2.25

  aA bA aA

  50 88.39±1.35 88.75±0.91 88.58±1.78

  aA aA aA

  70 88.59±2.20 90.80±0.62 90.40±0.40

• Mean ± standard deviation of triplicate samples As we can see from the table, there was no difference of heating temperature in 1 hour and 4 hours in the extraction yield but there was difference of temperature in 2 hour and

  o

  70 C was different than the other temperatures in the extraction yield. For the time, we

  o

  can see that there was no difference of time in the same temperature so 70

  C, 2 hours is the suitable condition to be utilized when it used HCL as the acid.

CONCLUSION

  Temperature and time can affect the percent extraction yield because the higher

  o

  temperature and time, the higher extraction yield and 70 C is the suitable temperature because it obtained the highest % extraction yield and 2 hours is the suitable time for to obtain the yield.

REFERENCES

  Agustin, R. The Relationship between Status Nutrition, Lifestyle, and Habits of Calcium and Vitamin D with Genesis Osteoporosis and Osteoponia on Citizens

  ≥ 45 years in Taman Wisma Asri North Bekasi. FKMUI; 2009.

  AOAC. 1990. Official methods of analysis of the Association of Official Analytical Chemists. 15th edition. Washington, DC, Association of Official Analytical Chemists. Fall, Jean, Yi-Theng Tseng, Diegane Ndong, Shin-Shyn Sheen. The Effects of Replacement of Soybean Meal by Shrimp Shell Meal on the Growth of Hybrid Tilapia (Oreochromis niloticus x Oreochromis aureus) Reared Under Brackish Water. International Journal of Fisheries and Aquaculture Vol.4 (5), pp. 85-91, April 2012 . Available online a Fikawati, S., Syafiq, A., Puspasari, P. The Factors Associated with Calcium Intake in Young in Bandung. Vol.24 No.1. Universa Medicina; 2005. Hartini TNS, Winkvist A, Lindholm L, Stenlund H, Persson V, Nurdiati DS, and Surjono A & Hakimi M. Nutrient intake and iron status of urban poor and rural poor without access to rice fields are affected by the emerging economic crisis: the case of pregnant Indonesian women. Eur J Clin Nutr. 2003 May; 57(5) : 654. Available fro Houtkooper, L., Farrell, V. A. Calcium supplement guidelines. Arizona Cooperative Extension ; 2004. Khorrami, M, G. D. Najafpour, H. Younesi, M. N. Hosseinpour. Production of Chitin and Chitosan from Shrimp Shell in BatchCulture of Lactobacillus plantarum. 2012.

  Chem. Biochem. Eng. Q. 26 (3) 217–223.

  Okoye, F.C., G.S. Ojewola and K. Njoku-Onu. Evaluation of Shrimp Waste Meal as A Probable Animal Protein Source for Broiler Chicken. 2005. Int. J. Poult Sci., pp: 458- 461.

  Poeloengasihhernawan, Crescentiana D., Satriyo K.Wahono Suharto, M. Kismurtono. Optimization of Chitin Production from Penaeus monodon Shells at Ambient Temperature. Surabaya; 22 Dec. 2009, ISSN 2086-1931.

  Ravichandran, S., Rameshkumar, G. Prince, A. R. Biochemical composition of shell and flesh of the Indian white shrimp (Penaeus indicus). 2009.American-Eurasian

  16 from shrimp shell by using Bacillus subtilis fermentation. Carbohydrate Research. 2007 Nov 26; 342(16): 2423. Available from Trung, Trang Si, Pham Thi Dan Phuong. Bioactive Compounds from By-Products of Shrimp Processing Industry in Vietnam. Journal of Food and Drug Analysis, Vol. 20, Suppl. 1, 2012, Pages 194-197. Available fro Appendix A: Table of Result Table 1. Result of ANOVA for Different Time

  1 hours

  a

ANOVA

  Yield Sum of Mean Squares df Square F Sig.

  Between 2.378 2 1.189 .459 .653 Groups Within Groups 15.560 6 2.593 Total 17.938

  8

  a. time = 1 2 hours

  a

ANOVA

  Yield Sum of

  Squares df Mean Square F Sig. Between 13.304

  2

  6.652 14.274 .005

  Groups Within Groups 2.796

  6

  .466

  Total 16.099

  8

  a. time = 2 4 hours

  a

ANOVA

  Yield Sum of Mean Squares df Square F Sig.

  Between 9.907 2 4.953 1.766 .249 Groups Within Groups 16.826 6 2.804 Total 26.733

  8

  18

  Table 2. Result of ANOVA for Different Temperature o

  Room Temperature (28

  C)

  a ANOVA

  Yield Sum of Mean Squares df Square F Sig.

  Between .499 2 .250 .117 .892 Groups Within Groups 12.813 6 2.135 Total 13.312

  8

  a. trt = RT

  o

  50 C

  a ANOVA

  Yield Sum of Mean Squares df Square F Sig.

  Between .188 2 .094 .048 .953 Groups Within Groups 11.633 6 1.939 Total 11.821

  8

  a. trt = 50C

  o

  70 C

  a ANOVA

  Yield Sum of Mean Squares df Square F Sig.

  Between 8.335 2 4.167 2.329 .178 Groups Within Groups 10.736 6 1.789 Total 19.071

  8

  a. trt = 70C

  19

  Table 3. Result of Post Hoc Test for Different Time

  1 hour

  b yield a

  Duncan Subset for alpha = 0.05

  Trt N

  1 RT 3 87.4140

  50C 3 88.3926

  70C 3 88.5895 Sig. .420

  Means for groups in homogeneous subsets are displayed.

  a. Uses Harmonic Mean Sample Size = 3.000.

  b. time = 1 2 hours

  b yield a

  Duncan Subset for alpha =

  0.05 Trt N

  1

  2 RT 3 87.9080

  50C 3 88.7463

  70C 3 90.8019 Sig. .183 1.000

  Means for groups in homogeneous subsets are displayed.

  a. Uses Harmonic Mean Sample Size = 3.000.

  b. time = 2

  20 4 hours

  b yield a

  Duncan Subset for alpha = 0.05

  Trt N

  1 RT 3 87.9192

  50C 3 88.5792

  70C 3 90.4002 Sig. .130

  Means for groups in homogeneous subsets are displayed.

  a. Uses Harmonic Mean Sample Size = 3.000.

  b. time = 4

  Table 4. Result of Post Hoc Test for Different Temperature o

  Room Temperature (28

  C)

  b yield a

  Duncan Subset for alpha = 0.05 time N

  1

  1 3 87.4140

  2 3 87.9080

  4 3 87.9192 Sig. .696

  Means for groups in homogeneous subsets are displayed.

  a. Uses Harmonic Mean Sample Size = 3.000.

  b. trt = RT

  21

  o

  50 C

  b yield a

  Duncan Subset for alpha = 0.05 time N

  1

  1 3 88.3926

  4 3 88.5792

  2 3 88.7463 Sig. .773

  Means for groups in homogeneous subsets are displayed.

  a. Uses Harmonic Mean Sample Size = 3.000.

  b. trt = 50C

  o

  70 C

  b yield a

  Duncan Subset for alpha = 0.05 time N

  1

  1 3 88.5895

  4 3 90.4002

  2 3 90.8019 Sig. .098

  Means for groups in homogeneous subsets are displayed.

  a. Uses Harmonic Mean Sample Size = 3.000.

  b. trt = 70C

  22

  3 5 200 8944 8365.805 106.9

  1 5 200 9309 8365.805 111.3

  2

  70

  1 5 200 9891 8365.805 118.2

  1

  70

  81.0

  1 5 200 6779 8365.805

  1

  70

  4

  2

  50

  3 5 200 9079 8365.805 108.5

  4

  50

  3 5 200 9350 8365.805 111.8

  2

  50

  99.6

  3 5 200 8335 8365.805

  2

  70

  1 5 200 8599 8365.805 102.8

  2 5 200 8599 8365.805 102.8

  4

  1

  70

  98.7

  3 5 200 8260 8365.805

  1

  70

  2 5 200 9553 8365.805 114.2

  4

  70

  2 5 200 9350 8365.805 111.8

  70

  70

  2 5 200 9350 8365.805 111.8

  2

  70

  2 5 200 9079 8365.805 108.5

  2

  70

  1 5 200 8538 8365.805 102.1

  4

  70

  1 5 200 9823 8365.805 117.4

  4

  50

  1

  Table 5. Result of % Calcium Extraction Yield

  95.6 Room

  2 5 200 8768 8365.805 104.8 Room

  4

  2 5 200 8700 8365.805 104.0 Room

  2

  2 5 200 9241 8365.805 110.5 Room

  2

  1 5 200 8741 8365.805 104.5 Room

  4

  1 5 200 9282 8365.805 111.0 Room

  4

  1 5 200 7997 8365.805

  2 5 200 8599 8365.805 102.8 Room

  2

  90.7 Room

  1 5 200 7591 8365.805

  2

  1 5 200 8971 8365.805 107.2 Room

  1

  1 5 200 8633 8365.805 103.2 Room

  1

  Original sample % yield Room

  Condition time rep Wet dried sample Final volume Content

  4

  1

  50

  2

  2 5 200 8903 8365.805 106.4

  1

  50

  1 5 200 9038 8365.805 108.0

  4

  50

  1 5 200 9241 8365.805 110.5

  4

  50

  1 5 200 9411 8365.805 112.5

  50

  3 5 200 8430 8365.805 100.8 Room

  1 5 200 9241 8365.805 110.5

  2

  50

  1 5 200 9343 8365.805 111.7

  1

  50

  1 5 200 9005 8365.805 107.6

  1

  50

  3 5 200 8633 8365.805 103.2

  1

  3 5 200 8768 8365.805 104.8

Appendix B: Documentation

  

Figure 1. Raw Shrimp Shell

Figure 2. Dried Shrimp Shell

Figure 3. Calcium Extraction

  

Appendix : Result of Atomic Absorption

Spectrocophy

  25

  y = 0.02365x + 0.0018 R² = 0.9996

  4 1 0.125 25 0.275 11.55180 2310 231

  50

  4 1 0.125 25 0.269 11.29810 2260 226

  50

  1 2 0.125 25 0.265 11.12896 2226 223

  50

  1 2 0.125 25 0.256 10.74841 2150 215

  50

  2 3 0.0625 25 0.125 5.20930 2084 208

  50

  2 3 0.0625

  25 0.14 5.84355 2337 234

  0.05

  25 0.28 11.76321 2353 235

  0.1

  0.15

  0.2

  0.25

  0.3

  0.35

  2

  4

  6

  8

  10

  12

  50

  2 1 0.125

  The Result of Atomic Absorption Spectrocopy from Calcium content in HCL Solution Concentration Intensity

  4 1 0.0625 25 0.139 5.80127 2321 232 Room

  1 0.025 4 0.097 8 0.197

  10

  0.24

  12

  0.28 Slope 0.0237

  Intercept 0.0018 Condition Time rep Wt Total volume Int. reading Conc. (ppm) Content (ppm) mg/100g

  Room

  1 1 0.125 25 0.257 10.79070 2158 216 Room

  1 1 0.125 25 0.267 11.21353 2243 224 Room

  2 1 0.0625 25 0.114 4.74419 1898 190 Room

  2 1 0.0625

  25 0.12 4.99789 1999 200 Room

  4 1 0.0625 25 0.131 5.46300 2185 219 Room

  50

  2 2 0.125 25 0.275 11.55180 2310 231 Room

  2 2 0.125 25 0.259 10.87526 2175 218 Room

  4 2 0.125 25 0.261 10.95983 2192 219 Room

  4 2 0.125 25 0.256 10.74841 2150 215 Room

  1 3 0.125 25 0.251 10.53700 2107 211 Room

  1 3 0.125 25 0.257 10.79070 2158 216

  50

  1 1 0.125 25 0.268 11.25581 2251 225

  50

  1 1 0.125 25 0.278 11.67865 2336 234

  50

  2 1 0.125 25 0.275 11.55180 2310 231

  14

  26

  2 2 0.0625 25 0.136 5.67442 2270 227

  70

  1 3 0.125 25 0.246 10.32558 2065 207

  70

  4 2 0.0625 25 0.143 5.97040 2388 239

  70

  25 0.14 5.84355 2337 234

  4 2 0.0625

  70

  25 0.14 5.84355 2337 234

  2 2 0.0625

  70

  70

  70

  4 1 0.0625 25 0.128 5.33615 2134 213

  70

  4 1 0.0625 25 0.147 6.13953 2456 246

  70

  2 1 0.125 25 0.256 10.74841 2150 215

  70

  2 1 0.125 25 0.277 11.63636 2327 233

  70

  1 1 0.0625 25 0.148 6.18182 2473 247

  70

  1 1 0.0625 25 0.102 4.23679 1695 169

  1 3 0.125 25 0.261 10.95983 2192 219