Histamine Levels in Repeated Thawing Beef | Nurliana | Proceedings of The Annual International Conference, Syiah Kuala University - Life Sciences & Engineering Chapter 5990 12368 1 SM
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
H ist a m ine Le ve ls in Re pe a t e d Tha w ing Be e f
1*
Nurliana, 1 Borgo Mauly Nasut ion, 1 Darm awi, and 1 Sugit o
1
Facult y of Vet erinary Medicine, Syiah Kuala Universit y, Darussalam , Banda Aceh 23111,
I ndonesia
* Corresponding Aut hor: [email protected]
Abst ra ct
The aim of t his resear ch was t o det erm ine t he hist am ine lev els in repeat ed t hawing beef. This
research were using longisim u s dorsi part ( sirloin) of beef. Repeat ed t hawing t reat m ent s of beef
consist ed of P1 as cont rol wit hout freezing, P2 as first t hawing aft er one week freezing, P3 as
second t hawing wit h out freezing t wo week s and P4 wit h freezing t hree week s. The slices of beef
was st orage in freezer at t em perat ure of - 20º C ev ery week s for t hree week s. The t hawing
process was done by st orage in refrigerat or at t em perat ur e of 6º C for 2 70 m inut es aft er being
rem ov ed from t he st orage in freezer ev ery week s for t hree week s. PI C ( Prot ease I nhibit ing
Cock t ail) m et hode were used t o ex t ract ion of enzy m e in beef. Enzy m e Link I m unosorbent Assay
( ELI SA) were used t o det ect ed t he hist am ine lev els wit h 4 50 nm wav e lengt h. The quant it at iv e
dat a from param et ers were analy zed by descript iv e. The result s of t he st udy con cluded t hat
repeat ed t hawing beef in refrigerat or at t em perat ure of 6º C could decrease t he hist am ine lev els.
Ke yw o rds : hist am ine, beef, t hawing
I nt r oduct ion
Meat is one of t he t ypes of foodst uffs of anim al origin, including beef which can hardly be separated
from hum an life ( Nurwant oro et al., 2012) . Beef has enough role in nat ional food cont ext because it is
one com m odit y with a com plete nut rient ( Em har et al., 2014) . Chem ical com posit ion of beef consists
of 56- 72 % water , 15- 22 % protein , 5- 34 % fat and 3.5 % protein not dissolved, include
carbohydrates, organic salts, subst ances dissolved nitrogen, m inerals and vit am ins ( Afiat i, 2009) .
Beef has a lot of benefit s as nut rit ional value, especially protein. Fut herm ore it could being lost if
handled inappropriately because beef is an excellent m edium for t he growt h of m icrobes, especially
bact eria. The existence of bacteria in beef t hat oft en cause dam age and can even lead t o t he source of
t he disease in hum ans. Bact erial contam inated beef t rhroughout cat t le life and since slaught ered, t he
process of preparing carcass unt il t he beef will be consum ed ( Boediono et al., 2012) .
High dem and for beef will increase at cert ain t im es, for exam ple on religious holidays. The price of
m eat will increase cert ainly. I t was caused by high dem and but availabilit y low for beef. People usually
provide m eat one day or t wo days before t he celebrat ion day, t hen st ored in freezer (Maaniaan ,
2009) . Generally, upper and m idle class of beef consum er, catering facilit ies or sellers of m eat buy
beef wit h cheap price and put it int o freezer as soon as posible, t hen beef will be t hawing before it was
sold and cooked ( Suryaningsih, 2010) .
Beef storage by freezing can affect the condit ions of m icrobial cont am inat ion. Som e of bact eria
contam inated beef can produce t he enzym e L - Hist idine Decarboxylase (HDC) which is a form er
precursor of t he biogenic am ines hist am ine. This com pounds can produced by Raoultella terrigena,
Ent erobact er spp., Microbacterium test aceum , Brevibacterium m cbrellneri, Diversus Micrococcus,
St aphylococcus spp ( Heruwat i et al., 2008) .
Histam ine is a biogenic am ine com pound produced from decarboxylation of free histidine (α- am ine- βpropionic acid inidosal) ( Lehane and Olley, 1999). This com pound is an im port ant m ediator of
im m ediate t ype allergic react ions ( im m ediate) . Allergic react io by hist am ine has different effect s based
on receptor binding sit es. I n the skin, histam ine causes dilat ion and perm eabilit y of capillaries which
cause redness, burning sensat ion, it ching and swelling. I n the heart , hist am ine will increase heart
rat e. I n t he respiratory syst em , histam ine causes bronchocont ricsion ( increase of bronchus
cont ract ion) , so it is very significant in pat ient s wit h bronchial ast hm a. I n t he digest ive system
hist am ine will increase gast ric acid secret ion and int est inal peristalt ic which causes vom it ing and
diarrhea, ot her effect s caused by hist am ine are increase secret ion gland of saliva, pancreas, bronchus
and tear but generally t hese effect are weak and not fixed ( Syarif et al., 2011) .
Histam ine form at ion process was influenced by presence of L - Hist idine Decarboxylase (HDC)
( Bennour et al., 1991) . Hum an body has effect ive system t o det oxify t oxins in t he digest ive t ract.
Monoam ine oxidase, diam ine oxidase and hist am ine N - m et hylt ransferase are digest ive enzym e which
256
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
m etabolize hist am ine int o t he digest ive t ract becom e non- t oxic com pounds. However, possibilit y of
hist am ine poisoning st ill need t o warn because hist am ine det oxificat ion system only works on
condit ion of t he daily norm al int ake ( I ndriat i et al., 2006) . At very high int ake of histam ine, t he
system will not able to detoxify of t his com pounds. 15 ppm levels of hist am ine consum pt ion can cause
allergic sym pt om s and 100 ppm consum pt ion m ay cause poisoning (Viciana et al., 1995)
Based on t he results from t he previous studies, the authors would like t o explain t he effect of
repeat ed t hawing in refrigerat or t o t he levels of histam ine in beef.
M a t e r ia le and m e t hods
Pr ocedur e
Be e f Fr e ez ing a nd Tha w ing
250 gram s beef were divided int o 4 t reatm ent s, a t reat m ent consisted of six repet it ions wit h different
sam ples and each sam ple weight of 10 gram s, the first t reat m ent ( P1) was not done freezing
( preparated and ext racted direct ly) , ot hers 3 t reatm ent s ( P2, P3, P4) were frozen in a freezer at - 20º C
t em perature with t hawing every week. St orage of beef ( P2, P3 and P4) were done for one, t wo and
t hree weeks, respect ively. Thawing conducted at t em perat ure of 6º C in refrigerat or unt il t he ent ire ice
m elt for 270 m inutes with a perm anent packaging.
Pr epa r a t ion of Be e f Ex t r a ct
10 gram s of t hawed beef was washed wit h cold PBS once, t hen it cut int o sm all pieces and taken 10
m g t o incorporated int o the m icro t ube, protease inhibit or 500μL added and RIFA buffer (1:100),
cooled for 30 m inutes in t he freezer, t hen vort ex using m ult i volt exer for 15 m inutes, and cent rifuged
using a cent rifuge cool on speed 10,000 rpm at 4°C for 20 m inutes, t hen t he supernat ant was tested.
H ist a m ine Assays Using ELI SA Test
Som e steps were used t o test t he levels of histam ine in beef using ELI SA t echniques. 50 m L sam ple
was inserted in t he ELI SA plate wells, t hen added 50 m L Biot inylat ed Detect ion Ab, incubated for 45
m inutes at 37º C tem perat ure, then it was washed wit h ELI SA washer for t hree t im es, then it was
added 100 uL HRP Conjugate for each ELI SA plate wells and incubated again for 30 m inutes at 37º C
t em perature, t hen it was washed wit h ELI SA Washer five t im es, then added 90 μL Substrate Reagent
pit t ing each ELI SA plat e and incubated for 15 m inutes at a tem perat ure of 37º C, t hen added 50 uL
St op Solut ion and read it s absorbance wit h ELI SA reader at 450 nm wave lengt h.
D a t a Ana lysis
Result datas will be analyzed by descript ive analysis.
Results a nd D iscussion
The result of ELI SA test of beef which repeated t hawing was shown in Table 1 .
Ta ble 1 . Histam ine levels ( ppm ) in beef after repeated t hawing
Repet it ions
No t hawing
1 st t hawing
( one week in freezer)
1
2
3
4
5
6
avarage
6.108x10 - 6
7.642 x10 - 6
0.856 x10 - 6
0.004 x10 - 6
0.15 x10 - 6
4.416 x10 - 6
3.20 x10 - 6
4.312 x10 - 6
9.514 x10 - 6
0.456 x10 - 6
0.204 x10 - 6
0.048 x10 - 6
16.66 x10 - 6
5.20 x10 - 6
2 nd t hawing
( t wo weeks in freezer)
0.006 x10 - 6
2.636 x10 - 6
0.026 x10 - 6
0.054 x10 - 6
0.516 x10 - 6
7.21 x10 - 6
1.74 x10 - 6
3 r d t hawing
( t hree weeks in
freezer)
0.002 x10 - 6
0.686 x10 - 6
4.144 x10 - 6
1.046 x10 - 6
0.292 x10 - 6
1.208 x10 - 6
1.23 x10 - 6
The results showed that the histam ine levels lowest in t he second week ( P3) and t hen t hird week ( P4)
along durat ion of freezing and frequent t hawing t reatm ent , t here were 1.74 x10 - 6 ppm and 1.23 x10 - 6
ppm ( Table 1 and figure 1) . Hist am ine levels increased during t he first week of t hawing t reatm ent
( P1) was 5.20 x10 - 6. it was m ay be caused by freezing storage at - 20°C , which t he HDC enzym e ( LHist idine Decarboxylase) inact ivat ed t o produce t he hist am ine, so it could reduced or st opped.
According t o Mat ulessy et al., ( 2010) , t he inhibit ion of cellular m et abolism in t he body t issues of
anim als t o produce HDC enzym e will t ake place at - 18°C t em perat ure. HDC enzym e act ivit y was as
precursor of hist am ine form at ion influenced by pH, t em perat ure and low oxygen availabilit y, t he
highest enzym e act ivit y at 37°C and t he opt im um pH was 6.0 ( Eitenm iller et al., 1982 ; Kusum awat i
and I ndriat i, 2008) . Thawing process was done over - 10°C t em perat ure, bacterial act ivit y will ret urn
257
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
norm ally, alt hough som e of t hem died. Sublet hal bact eria will also die over t im e in freezing st orage. it
caused increase t he level of histam ine in P2. Maybe due t o the presence of HDC enzym e as bacteria
product ion. I t was act ive again after sublethal phase for one week in freezing st orage, t hen it
decreased at P3 and P4 caused of cont inuous freezing t reat m ent, t hen sublet hal bact eria died and
act ivit y of HDC enzym e also halt ed because tem perat ure was t oo ext rem e. Som e of t he HDC enzym e
was produced by bact eria in sublet hal phase at - 20° C ( Mat ulessye et al., 2010) .
The growt h of
bact eria, hist am ine form ing and act ivit y of HDC enzym e can be inhibited at 5°C or lower. Exam ple
Ent erobact er, one of a fam ily of Gram negat ive psychrot rophic.
6
Histamine levels
5
4
3
2
1
0
P1
P2
P3
P4
Treatments
Figur e 1 . Graph hist am ine levels aft er test ed ELI SA
Beef freezing was done by freezing t he beef below t he froze point of t he liquid at t em perat ur of - 20º C
t o - 45º C could inhibit t he growt h of m icrobes, proteolyt ic processes, hydrolysis processes, lipolyt ic
processes and slight oxidat ive processes ( Safit ra and Put ra, 2013) . This stat em ent was support ed by
Lawrie ( 1991), clot t ing speed will increase in line wit h t he decline of t em perature at - 20°C. Alm ost
98% of t he water cont ained in beef will freeze quickly and produce soft ice cryst als, whereas t he slow
freezing will form large ice crystals could dam age cells and caused denat urat ion of proteins. I n such
t his st uat ions bacteria will grow faster in denat urated protein m edium .
Possibilit y t he lowest histam ine levels in t he second and t hird week of freezig storage not causing
allergy and poisoning if consum ed. Thawing t reat m ent on m eat at t em perat ure of 6°C for 270 m inutes
in t he refrigerat or can reduce hist am ine levels. I t could happen due t o no m icrobial cont am inat ion in
m eat , so it safe for consum pt ion. According t o Suryaningsih ( 2010) beef thawing process is done by
put t ing t he frozen beef
at t em perat ure 5- 10°C and let at least 1 hour to inhibit m icrobial
recont am inat ion.
Conclusion
St orage of beef in the freezer at t em perat ure of -20 °C and repeated t hawing in refrigerat or at
t em perature of 6 °C could decrease t he level of his tam ine.
Re fe re n ce s
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Bennour, M., Marrak chi, A.E. , Bouch rit i,N. Ham am a, A., Ouadaa.M.E. ( 19 91) . Chem ical and m icrobiological
assessm ent of m ack erel ( Scom ber scom bru s) st ored in ice. J. Food Prot ect ion . 5: 789 –792.
Budiono, H., Harlis, Ret n i, Budiart i, S. ( 2012) . Threshold an aly sis as an indicat or of E. Coli cont am inat ion of beef at
t he slaught er house, Jam bi Dist r ict . Biospecies. 5: 14- 21
Eit enm iller, R.R., Orr, J.H., Wallis, W.W. ( 1982) . Hist am ine form at ion in fish: m icrobiological and biochem ical
condit ion. I n R.E. Mart in, G.J. Flick ,. C.E. Hebard, and D.R. Ward. ( eds.) . Chem ist ry and Biochem ist ry
of Marine Food Product s. AVI Publ. Connect icut . 1: 39- 50.
Em har, A., Aj i,J. M. M., Agust ina, T. ( 2014) . Beef supply chain analy sis in Jem ber dist rict . Ber kala I lm iah Pert anian.
1: 53- 61.
Heruwat i, E.S., Sophia, R.A., Mangunwardoy o, W. ( 2008) . I nhibit ion of L- Hist idin decarbox y lase produced by
hist am ine form ing bact eria using benzoic acid. Jurnal Pasca Panen Dan Biot ekn ologi Kelaut an Dan
Perikanan. 3: 97- 107.
I ndriat i, N., Rispay eni, Heruwat i. E.S. ( 2006) . St udies of hist am ine form ing bact eria on peda m ack erel during
processing. Jur nal Penelit ian Perikanan I ndonesia. 1: 117– 123.
Kusm arwat i. A., I ndriat i. N.( 20 08) . I nhibit ion of Pangium edule Reinw seed ex t ract act iv e t o t he growt h of
hist am ine for m ing bact eria. Jurnal Pascapanen dan Biot eknologi Kelaut an dan Perikanan. 3: 29- 3 6.
Lawrie, R. A. 1991. Meat Scien ce. 3rd ed. Pergam on Press, Ox ford.
Lehane, L., Olley . J. ( 199 9) . Nat ional Office of Anim al and Plant Healt h. Canberra.
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Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
Maaniaan. 2009. The qualit y of frozen m eat . Balai Penelit ian Ternak , Bogor.
Mat ulessy D.N. , Sury ant o, E., Rusm an. ( 201 0) . Ev aluat ion of phy sical charact erist ic, chem ical com posit ion and
m icrobial qualit y of broiler carcass frozen in t he t radit ional m ark et nort h Halm ahera dist rict , Nort h
Maluk u. Bulet in Pet ernakan. 34: 1 78- 18 5.
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wat er, Escherich ia coli assesm en t of beef m arinat ed in garlic j uice. Jurnal Aplikasi Teknologi Pangan.
1: 20- 22.
Safit ra A.G., Put ra. A.B.K. ( 2013) . St udies of v ariat ion in ev aporat or cooling load low st age cascade refrigerat ion
sy st em using a con cent ric t ube t y pe heat ex changer wit h t he refrigerant work ing fluid in t he high
st age of m usicool- 22 and low st age of r- 404a. Jurnal Teknik Pom it s. 2: 95 - 100.
Sury aningsih, L. ( 2010) . St udies of v arous t hawing m et h ods t o t enderness, wat er holding capacit y and cook ing lost
of shrink part of beef.. 2 nd Nat ional Sem inar on Anim al Hunbandry Facult y "Pr oduct ion Sy st em s
based on local Ecosy st em . Bandung.
Sy arif, A., Est uningt y as, A., Set iawat i, A., Mu cht ar, Ar if,A., Bahry ,A., Suy at na, B., Dewot o, F.D., Ut am a,H.R. ,
Darm an sj ah ,H., Wiria, I ., Nafrialdi,M.S.S., Wilm ana, Ascobat P.F., Set iabudy , P., Sunary o,R. ,
Wardhini, R., Suher m an, S. , ,S.K. Ganiswarna,S.G., Arozal, V.H.S., Mar iana, W., I st iant or o, Y. ,
Sadik in, Y.H. , Louisa, Z.D., Ely sabet h. M. ( 20 11) . Far m acology and dan Therapy . FKUI press,
Jak art a.
Viciana, N., Jov er, M.T., Caron,M.S.V. ( 1995) . Liquid chrom at ographic m et hod for det erm inat ion of biogenic
am ines in fish and fish product . Journal of AOAC I nt ernat ion al. 78: 1045- 10 50.
259
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
H ist a m ine Le ve ls in Re pe a t e d Tha w ing Be e f
1*
Nurliana, 1 Borgo Mauly Nasut ion, 1 Darm awi, and 1 Sugit o
1
Facult y of Vet erinary Medicine, Syiah Kuala Universit y, Darussalam , Banda Aceh 23111,
I ndonesia
* Corresponding Aut hor: [email protected]
Abst ra ct
The aim of t his resear ch was t o det erm ine t he hist am ine lev els in repeat ed t hawing beef. This
research were using longisim u s dorsi part ( sirloin) of beef. Repeat ed t hawing t reat m ent s of beef
consist ed of P1 as cont rol wit hout freezing, P2 as first t hawing aft er one week freezing, P3 as
second t hawing wit h out freezing t wo week s and P4 wit h freezing t hree week s. The slices of beef
was st orage in freezer at t em perat ure of - 20º C ev ery week s for t hree week s. The t hawing
process was done by st orage in refrigerat or at t em perat ur e of 6º C for 2 70 m inut es aft er being
rem ov ed from t he st orage in freezer ev ery week s for t hree week s. PI C ( Prot ease I nhibit ing
Cock t ail) m et hode were used t o ex t ract ion of enzy m e in beef. Enzy m e Link I m unosorbent Assay
( ELI SA) were used t o det ect ed t he hist am ine lev els wit h 4 50 nm wav e lengt h. The quant it at iv e
dat a from param et ers were analy zed by descript iv e. The result s of t he st udy con cluded t hat
repeat ed t hawing beef in refrigerat or at t em perat ure of 6º C could decrease t he hist am ine lev els.
Ke yw o rds : hist am ine, beef, t hawing
I nt r oduct ion
Meat is one of t he t ypes of foodst uffs of anim al origin, including beef which can hardly be separated
from hum an life ( Nurwant oro et al., 2012) . Beef has enough role in nat ional food cont ext because it is
one com m odit y with a com plete nut rient ( Em har et al., 2014) . Chem ical com posit ion of beef consists
of 56- 72 % water , 15- 22 % protein , 5- 34 % fat and 3.5 % protein not dissolved, include
carbohydrates, organic salts, subst ances dissolved nitrogen, m inerals and vit am ins ( Afiat i, 2009) .
Beef has a lot of benefit s as nut rit ional value, especially protein. Fut herm ore it could being lost if
handled inappropriately because beef is an excellent m edium for t he growt h of m icrobes, especially
bact eria. The existence of bacteria in beef t hat oft en cause dam age and can even lead t o t he source of
t he disease in hum ans. Bact erial contam inated beef t rhroughout cat t le life and since slaught ered, t he
process of preparing carcass unt il t he beef will be consum ed ( Boediono et al., 2012) .
High dem and for beef will increase at cert ain t im es, for exam ple on religious holidays. The price of
m eat will increase cert ainly. I t was caused by high dem and but availabilit y low for beef. People usually
provide m eat one day or t wo days before t he celebrat ion day, t hen st ored in freezer (Maaniaan ,
2009) . Generally, upper and m idle class of beef consum er, catering facilit ies or sellers of m eat buy
beef wit h cheap price and put it int o freezer as soon as posible, t hen beef will be t hawing before it was
sold and cooked ( Suryaningsih, 2010) .
Beef storage by freezing can affect the condit ions of m icrobial cont am inat ion. Som e of bact eria
contam inated beef can produce t he enzym e L - Hist idine Decarboxylase (HDC) which is a form er
precursor of t he biogenic am ines hist am ine. This com pounds can produced by Raoultella terrigena,
Ent erobact er spp., Microbacterium test aceum , Brevibacterium m cbrellneri, Diversus Micrococcus,
St aphylococcus spp ( Heruwat i et al., 2008) .
Histam ine is a biogenic am ine com pound produced from decarboxylation of free histidine (α- am ine- βpropionic acid inidosal) ( Lehane and Olley, 1999). This com pound is an im port ant m ediator of
im m ediate t ype allergic react ions ( im m ediate) . Allergic react io by hist am ine has different effect s based
on receptor binding sit es. I n the skin, histam ine causes dilat ion and perm eabilit y of capillaries which
cause redness, burning sensat ion, it ching and swelling. I n the heart , hist am ine will increase heart
rat e. I n t he respiratory syst em , histam ine causes bronchocont ricsion ( increase of bronchus
cont ract ion) , so it is very significant in pat ient s wit h bronchial ast hm a. I n t he digest ive system
hist am ine will increase gast ric acid secret ion and int est inal peristalt ic which causes vom it ing and
diarrhea, ot her effect s caused by hist am ine are increase secret ion gland of saliva, pancreas, bronchus
and tear but generally t hese effect are weak and not fixed ( Syarif et al., 2011) .
Histam ine form at ion process was influenced by presence of L - Hist idine Decarboxylase (HDC)
( Bennour et al., 1991) . Hum an body has effect ive system t o det oxify t oxins in t he digest ive t ract.
Monoam ine oxidase, diam ine oxidase and hist am ine N - m et hylt ransferase are digest ive enzym e which
256
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
m etabolize hist am ine int o t he digest ive t ract becom e non- t oxic com pounds. However, possibilit y of
hist am ine poisoning st ill need t o warn because hist am ine det oxificat ion system only works on
condit ion of t he daily norm al int ake ( I ndriat i et al., 2006) . At very high int ake of histam ine, t he
system will not able to detoxify of t his com pounds. 15 ppm levels of hist am ine consum pt ion can cause
allergic sym pt om s and 100 ppm consum pt ion m ay cause poisoning (Viciana et al., 1995)
Based on t he results from t he previous studies, the authors would like t o explain t he effect of
repeat ed t hawing in refrigerat or t o t he levels of histam ine in beef.
M a t e r ia le and m e t hods
Pr ocedur e
Be e f Fr e ez ing a nd Tha w ing
250 gram s beef were divided int o 4 t reatm ent s, a t reat m ent consisted of six repet it ions wit h different
sam ples and each sam ple weight of 10 gram s, the first t reat m ent ( P1) was not done freezing
( preparated and ext racted direct ly) , ot hers 3 t reatm ent s ( P2, P3, P4) were frozen in a freezer at - 20º C
t em perature with t hawing every week. St orage of beef ( P2, P3 and P4) were done for one, t wo and
t hree weeks, respect ively. Thawing conducted at t em perat ure of 6º C in refrigerat or unt il t he ent ire ice
m elt for 270 m inutes with a perm anent packaging.
Pr epa r a t ion of Be e f Ex t r a ct
10 gram s of t hawed beef was washed wit h cold PBS once, t hen it cut int o sm all pieces and taken 10
m g t o incorporated int o the m icro t ube, protease inhibit or 500μL added and RIFA buffer (1:100),
cooled for 30 m inutes in t he freezer, t hen vort ex using m ult i volt exer for 15 m inutes, and cent rifuged
using a cent rifuge cool on speed 10,000 rpm at 4°C for 20 m inutes, t hen t he supernat ant was tested.
H ist a m ine Assays Using ELI SA Test
Som e steps were used t o test t he levels of histam ine in beef using ELI SA t echniques. 50 m L sam ple
was inserted in t he ELI SA plate wells, t hen added 50 m L Biot inylat ed Detect ion Ab, incubated for 45
m inutes at 37º C tem perat ure, then it was washed wit h ELI SA washer for t hree t im es, then it was
added 100 uL HRP Conjugate for each ELI SA plate wells and incubated again for 30 m inutes at 37º C
t em perature, t hen it was washed wit h ELI SA Washer five t im es, then added 90 μL Substrate Reagent
pit t ing each ELI SA plat e and incubated for 15 m inutes at a tem perat ure of 37º C, t hen added 50 uL
St op Solut ion and read it s absorbance wit h ELI SA reader at 450 nm wave lengt h.
D a t a Ana lysis
Result datas will be analyzed by descript ive analysis.
Results a nd D iscussion
The result of ELI SA test of beef which repeated t hawing was shown in Table 1 .
Ta ble 1 . Histam ine levels ( ppm ) in beef after repeated t hawing
Repet it ions
No t hawing
1 st t hawing
( one week in freezer)
1
2
3
4
5
6
avarage
6.108x10 - 6
7.642 x10 - 6
0.856 x10 - 6
0.004 x10 - 6
0.15 x10 - 6
4.416 x10 - 6
3.20 x10 - 6
4.312 x10 - 6
9.514 x10 - 6
0.456 x10 - 6
0.204 x10 - 6
0.048 x10 - 6
16.66 x10 - 6
5.20 x10 - 6
2 nd t hawing
( t wo weeks in freezer)
0.006 x10 - 6
2.636 x10 - 6
0.026 x10 - 6
0.054 x10 - 6
0.516 x10 - 6
7.21 x10 - 6
1.74 x10 - 6
3 r d t hawing
( t hree weeks in
freezer)
0.002 x10 - 6
0.686 x10 - 6
4.144 x10 - 6
1.046 x10 - 6
0.292 x10 - 6
1.208 x10 - 6
1.23 x10 - 6
The results showed that the histam ine levels lowest in t he second week ( P3) and t hen t hird week ( P4)
along durat ion of freezing and frequent t hawing t reatm ent , t here were 1.74 x10 - 6 ppm and 1.23 x10 - 6
ppm ( Table 1 and figure 1) . Hist am ine levels increased during t he first week of t hawing t reatm ent
( P1) was 5.20 x10 - 6. it was m ay be caused by freezing storage at - 20°C , which t he HDC enzym e ( LHist idine Decarboxylase) inact ivat ed t o produce t he hist am ine, so it could reduced or st opped.
According t o Mat ulessy et al., ( 2010) , t he inhibit ion of cellular m et abolism in t he body t issues of
anim als t o produce HDC enzym e will t ake place at - 18°C t em perat ure. HDC enzym e act ivit y was as
precursor of hist am ine form at ion influenced by pH, t em perat ure and low oxygen availabilit y, t he
highest enzym e act ivit y at 37°C and t he opt im um pH was 6.0 ( Eitenm iller et al., 1982 ; Kusum awat i
and I ndriat i, 2008) . Thawing process was done over - 10°C t em perat ure, bacterial act ivit y will ret urn
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Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
norm ally, alt hough som e of t hem died. Sublet hal bact eria will also die over t im e in freezing st orage. it
caused increase t he level of histam ine in P2. Maybe due t o the presence of HDC enzym e as bacteria
product ion. I t was act ive again after sublethal phase for one week in freezing st orage, t hen it
decreased at P3 and P4 caused of cont inuous freezing t reat m ent, t hen sublet hal bact eria died and
act ivit y of HDC enzym e also halt ed because tem perat ure was t oo ext rem e. Som e of t he HDC enzym e
was produced by bact eria in sublet hal phase at - 20° C ( Mat ulessye et al., 2010) .
The growt h of
bact eria, hist am ine form ing and act ivit y of HDC enzym e can be inhibited at 5°C or lower. Exam ple
Ent erobact er, one of a fam ily of Gram negat ive psychrot rophic.
6
Histamine levels
5
4
3
2
1
0
P1
P2
P3
P4
Treatments
Figur e 1 . Graph hist am ine levels aft er test ed ELI SA
Beef freezing was done by freezing t he beef below t he froze point of t he liquid at t em perat ur of - 20º C
t o - 45º C could inhibit t he growt h of m icrobes, proteolyt ic processes, hydrolysis processes, lipolyt ic
processes and slight oxidat ive processes ( Safit ra and Put ra, 2013) . This stat em ent was support ed by
Lawrie ( 1991), clot t ing speed will increase in line wit h t he decline of t em perature at - 20°C. Alm ost
98% of t he water cont ained in beef will freeze quickly and produce soft ice cryst als, whereas t he slow
freezing will form large ice crystals could dam age cells and caused denat urat ion of proteins. I n such
t his st uat ions bacteria will grow faster in denat urated protein m edium .
Possibilit y t he lowest histam ine levels in t he second and t hird week of freezig storage not causing
allergy and poisoning if consum ed. Thawing t reat m ent on m eat at t em perat ure of 6°C for 270 m inutes
in t he refrigerat or can reduce hist am ine levels. I t could happen due t o no m icrobial cont am inat ion in
m eat , so it safe for consum pt ion. According t o Suryaningsih ( 2010) beef thawing process is done by
put t ing t he frozen beef
at t em perat ure 5- 10°C and let at least 1 hour to inhibit m icrobial
recont am inat ion.
Conclusion
St orage of beef in the freezer at t em perat ure of -20 °C and repeated t hawing in refrigerat or at
t em perature of 6 °C could decrease t he level of his tam ine.
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