Bombyx mori
Silkworm Pupae Protein Isolate and Its Application
on High Protein Powdered Milk
A.U. Abdullah*, Ribka, H.T. Utomo, A. Febriani, M.T. Syaputra, & R.R.A. Maheswari
Department of Animal Production and Technology, Faculty of Animal Science, Bogor Agricultural University,
Bogor 16680, Indonesia *email: [email protected]
Abstract
High protein food consumption is a new trend in society with busy life to fulfill the balance of daily protein need. A product with high protein content is high protein powdered milk (HPPM) for instance which commonly use high protein fortificant added through its processing. Bombyx mori silkworm pupae (BSP), by-product of silk spinning industry, has potential to be developed as an alternative for cheaper high protein fortificant. Previous research had isolated BSP protein to obtain protein isolate then fortified it into powdered milk. The continuing study is needed to produce pure protein isolate in order to produce HPPM with better characteristics. The two inventions applied in this protein isolation method were defatting and modifying pH on protein extraction and precipitation process. Hexane was used to extract powdered pupae fat. Protein extraction by NaOH 2N until pH 11 and precipitation by HCl 2N until pH 4.1 were used in this research to make sure all the protein fractions of BSP were dissolved in alkaline condition and precipitated as below its isoelectric pH. The objective of this research was to study the properties of Bombyx mori silkworm pupae protein isolate (BSPPI) and its application on HPPM. The results showed that defatting process could decrease 60.87% fat content of powdered pupae. The modified protein isolation method was able to produce pure BSPPI with 81.84% protein content. Fortification of 20% BSPPI showed very high significant difference (P<0.01) on protein content and protein digestibility of powdered milk, 40.44% and 95.15% respectively.
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Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
Introducton
Proten s a second majorty compound of human body component after waterneeded forhuman growth process. Therefore, the adequacy of daly proten s very mportant.Hgh proten food consumpton s a new trend n socety wth busy lfe to fulfll the balance of daly proten need. A product wth hgh proten content s hgh proten powdered mlk (PM) for nstance whch commonly use hgh proten fortfcant added through ts processng.Fortfcant sources commonly used are whey and casen. Both are very expensve fortfcant and stll depend onmport supply.
Bombyx mori slkworm pupae s by-product of slk spnnng ndustry whch have been used as foods n some countres to provde nutrtonal benefts because t contanshgh and balanced nutrents. Pupae s also known as a better proten
content than soy, fsh, and beef proten (Trvedy et al., 2007) as well as essental
amno acdssuch as BCAA (branched chan amno acds) .e lysne, leucne, and
valne(Tomotake et al.,2010). BCCA are wdely used as food supplement because
of ts effectveness to buld muscle and mprove ts endurance. However, slkworm pupae s generally used as fsh feed ngredents and broler chckens n Indonesa
(Rangacharyulu et al., 2003).
Prevous research had utlzed slkworm pupae as flour for soups and cream cracker ngredent. Artant (2009) and Myatan (2008) stated that slkworm
pupae-based product contans hgh proten and hgh proten dgestblty. Khan et al.(2011)
carred out an nventon to mprove Bombyx mori slkworm pupae-based product
acceptablty by solatng Bombyx mori slkworm pupae protenpror tofortfcaton
to mlk. However, further research s needed to fnd more precse method of proten
solaton to produce pure Bombyx mori slkworm pupae proten solate (BSPPI)n
order to produce powdered mlkwth better characterstcs.The objectve of ths research wasto study the propertes of BSPPI and ts applcaton on hgh proten powdered mlk.
Materals and Methods
The man ngredents used were fresh cow’s mlk (from dary farm of
An-mal Scences Faculty, Bogor Agrcultural Unversty), Bombyx morislkworm
pu-pae (from Rumah Sutera Alam, Capus–Bogor, West Java Provnce), NaOH 2N, HCl 2N, dstlled water, and Whatman flter paper. The man equpments used were refrgerator, centrfuger, pH meter, dgtal scales, spray drer, evaporator, homog-enzer, and blender.
Manufacturing of BSPPI (modified Wang et al.,2010) and Fortified Powdered Milk
powdered pupae, defattng of powdered pupae, solatng ofBSPPI, and dryng of BSPPI. Fresh pupae was pealed and washed by submergng nto clean water. The water was changed repeatedly untl t was clear. After the pupae were clean, they were boled n 15 mnutes and dred by 60 °C oven for 24 hours. Once the pupae were dred, t was powdered usng blender. After obtanng the powdered pupae, the fat was removed by hexane. The defatted powdered pupae proten was extracted usng NaOH 2N untl pH 11 and left for one hour to dssolve the proten completely. As soon as the proten was dssolved, the resdue was removed by centrfuge on 6,000 g-force. The dssolved proten was then precptated by decreasng the pH untl 4.1 usng HCl 2N untl the proten reached ts soelectrc pont and was left for 15 hours to precptate proten completely. The proten precptate was teh obtaned by centrfuge on 6,000 g-force. The last step was cleanng by dstlled water and centrfuge on 6,000 g-force for 3 tmes. BSPPI was then dred by spray drer (nlet
180 oC, outlet 80 oC) to produce powdered BSPPI.Powdered BSPPIwas then added
nto powdered mlk (PM)wth 5 treatments (0%, 5%, 10%, 15%, and 20%).The fortfed powdered mlk was analysed based on chemcal propertes and proten dgestblty.
Protein Content Assay
Proten content was tested by Kjeldahl method (AOAC, 2007). Ths method conssts of three stages .e dgeston, dstllaton, and ttraton.
Protein Digestibility by in vitro Assay (Anderson et al., 1969)
The dgestblty of proten n fortfed powdered mlk was determned usng the multenzyme method. A number of 250 mg powdered mlk added wth 15 ml
HCl 0.1 N contaned 1.5 mg pepsn and were ncubated at 37 oC for 3 hours. The
so-luton was neutralzed usng NaOH 0.5 N and added wth 4 mg pancreatne soso-luton n phosphate buffer 0.2 M pH 8.0 whch contaned 0.005 M sodum azyde. Soluton
of the enzyme mxture were ncubated at 37 oC for 24 hours. The resduewas
ob-taned by centrfuge on 2,500 g-force for 5 mnutes. The proten n resdue ndcated total number of undgestble proten. Total number of proten before and after ths treatment were measured by Kjeldahl method (AOAC, 2007). Apparent Proten D-gestblty (APD) was calculated by equaton below:
APD (%)= (Total proten-undgestble proten)/(Total Proten) ×100%
NaCl Content Assay (Modified Volhard’s Method)
Ash obtaned from 5000 mg sample (W
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Proceeding of the 2nd International Seminar on Animal Industry | Jakarta, 5-6 July 2012
ttrated wth potassum thocyanate 0.1006N untl permanent orange of ts color. NaCl content was calculated based on formula below :
Explanaton :
Coeffcent Factor (CF)= 10, Volume of Blanko (Vblanko)= 5.05 ml
Statistical Analysis
Ths research used a completely randomzed desgn wth5treatments of BSPPI fortfcaton (0%, 5%, 10%, 15%, and 20%) to powdered mlk. All experments and measurements were performed n trplcate and duplcate respectvely and the means reported. The results were expressed as mean values ± standard devaton (n = 3). Analyss of varance (ANOVA), followed by Tukey or Kruskal Walls test, were used for statstcal comparsons among treatments, wth a value of P < 0.05 ndcatng sgnfcant dfference. All data were analyzed usng Statstx 8.0 statstcal software package.
Results and Dscusson
Characteristic of BSPPI
Basc materal usedas aproten fortfcant n ths research was BSPPI. BSPPIwas obtaned through modfedmethodtoncrease thepurty of protensolatecomparedwth prevous researchmethod. The nventonsof ths research are defattng mplemented to powdered pror to proten solaton, pHvaratons usagen protensolaton step,an dwashngforremovng any chemcal resdue n solate.
Table 1. Physcal and Chemcal Profle of BSPPI
Table 2. Powdered Pupae Fat Content Be-fore and After Defattng Process
Parameters Content (%) Sampels Content (%) Proten 81.84 ± 0.62 Powdered pupae 26.43 Proten dgestblty 94.59 ± 0.43 Defatted powdered pupae 10.34 Fat 6.22 ± 0.10
Mosture 7.16 ± 0.03 Ash 3.68 ± 0.69
Yeld 29.94
Defattng s a very mportant step n ths solaton processbecause fats thesec-ondlargestcomponent ofpowdered pupae (n dry bass) after proten.
Protenand-fatbeforedefattng were 60.75% and26.43%respectvely (Table 2)(Khan etal.,
646 nd
60.87±0.19%. The nventons of ths research .edefattng and pHvaratons usage were able to producepure BSPPIwth hgh purty that contans proten about 81.84% and wth less fat content, 6.22% (Tabel 1).It means that proten solaton process used n ths researchcould separate the fat remaned n the sampel.Washng process also successfully washed NaCl remanng after chemcal reacton from NaOH and HCl durng solaton process. Based on the result of NaCl assay, powdered mlk wth or wthout BSPPI were not sgnfcantly dfferent (P>0.05) (Fgure 1a).
Fgure 1. Effect of BSPPI fortfcaton to NaCl content (a), proten content (b), and proten dgestblty (c)of powdered mlk. Explanaton : Dfferent superscrpt (a, b) shows very hgh sgnfcant dfference(P<0.01)
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fortfed by 20% of BSPPI because t had fulflled 20% ofNRV (nutrton reference value) per 100 g of proten (CAC, 2001)
Conclusons
Defattng process decreased 60.87% fat content of powdered pupae. The mod-fed proten solaton method was able to produce pure BSPPI wth 81.84%, 94.59%, 7.16%, 3.68% of proten content, proten dgestblty, water, and ash respectvely. Fortfcaton of 20% BSPPI showed very hgh sgnfcant ncrease (P<0.01) on pro-ten conpro-tent and propro-ten dgestblty of powdered mlk, 40.44% and 95.15% respec-tvely.
Acknowledgement
The authors would lke to gve an acknowledgement to Rumah Sutera Alam
at Capus Bogor-West Java provnce for facltatng us wth Bombyx moripupaeand
Drectorate of Hgher Educaton of Natonal Educaton and Culture, Mnstry of Republc of Indonesa for provdng fund to establsh ths research.
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