Dentin is a mineralized tissue formed by odontoblasts that are differentiated from ectomesenchymal cells , The molecul ar mech anism of odontoblast diffe rentiation remains unclear, Amino acid transporters play an important role in s up plying nutri tion to normal a nd ca ncer cells including odntoblasts, and for cell proliferation , Amino acid transport system L is a maj or nutrient t ransport system responsible for the Na+' -independent transport o[ neutral amino acids incJuding several essentiaJ amino acids , The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2) , In this study, the expression pattern and role of amino acid transport system L were, therefore, investigated in the differentiation of MDPC-23 cells derived from mouse dental papilla celJs , To determi ne the expression Jevel o[ amino acid transport system L participating in intracelJ ular transport of amino acids in the differentiat ion 0 1' MDPC-23 cells, it was examined by RT-PCR, observation of cell morphoJogy‘ A1izaline red-S staining ancl uptake analysis after inclucing experimental differentiation in MDPC-23 cells The res ults were as follows , The LAT1 mRNA was expressed in the early stage of MDPC-23 cell differentiation , The expression leveJ was gradually increased by time course and it was decreased after the late stage, The LAT2 mRNA was not observed in the earJy stage of MDPC-23 cell differentiation, The LAT2 mRNA was expressed at the 11 days 0 1' MDPC-23 cell differentiation and the expression level was gradually decreased by time course, There was no changes in the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during the differentiation of MDPC-23 cells , The expression of ON mRNA was graduaJJy decreased but the expression of ALP mRNA was increased during differentiation of MDPC-23 cells , The L-Ieucine uptake was increased by time cour se from the early stage to the 9 days in MDPC-23 cell differentiation , The amount of L-Ieucine uptake was maintained to the 11 and 14 days of MDPC-23 cell differentiation As the resul ts‘ it is considered that among neutral amino acid transport system L in differentiation of MDPC-23 cells , the LATl has a key role in cell proliferation in the early stage and middle stage of cell differentiation and the LAT2 has an important roJe in ceJJ differenti ation and mineralization in the Jate stage of cell differentiation for providing cells with neutral a mino acids incJuding several essentiaJ amino acids

System L amino acid transporter is a major route for providing living cells with neutral amino acids including several essential amino acids. To elucidate the expression pattern of L-type amino acid transporter 1 (LAT1) in the bone formation process, the expressions of LAT1 and its subunit 4F2 heavy chain (4F2hc) were investigated in the healing process after the implantation of bone graft materials in the calvarial osseous defected rats. Circular calvarial defects (1 cm in diameter) were made midparietally. The rats were divided into 4 groups of 1 control group and 3 experimental groups. In the control group, the defect was only covered with soft tissue flap. In the experimental groups, they were filled with human particulate dentin (particulate dentin group), with plaster of Paris (plaster of Paris group) and with the mixture of human particulate dentin and plaster of Paris with ratio of 2 : 1 by weight (mixture group). The rats were sacrificed at the 1, 2, 4 and 8 weeks after operation and the RT-PCR analysis and immunohistochemical analysis were performed. In the RT-PCR analysis, the mRNAs of LAT1 and 4F2hc were strongly detected in all 4 groups. In the immunohistochemical analysis, at 1 week after operation, the LAT1 protein and its subunit 4F2hc protein were mainly expressed in the osteoblasts, osteocytes and interstitial tissues of the around the defect and inner part of newly forming bone in all 4 groups. The expressions of LAT1 and 4F2hc proteins were decreased at 2 and 4 weeks after operation. The LAT1 and 4F2hc proteins were scarcely expressed at 8 weeks after operation in all 4 groups. These results suggest that the LAT1 and its subunit 4F2hc highly expressed at the early stage of new bone formation and may have an important role in providing cells with neutral amino acids including several essential amino acids at that stage.

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the Na+-independent transport of neutral amino acids including several essential amino acids. The L-type amino acid transporter 1 (LAT1) is over-expressed to support cell growth in malignant tumors. The double stranded RNA-mediated RNA interference (RNAi) analysis can be in a wide variety of eukaryotes to induce the sequence-specific inhibition of gene expression. In this study, we examined the effect of LAT1 short interfering RNA (siRNA) on cell growth using siRNA of LAT1 in the KB human oral squamous cell carcinoma. In the RT-PCR analysis and western blot analysis, the siRNA of LAT1 inhibited expressions of LAT1 mRNA and protein. The uptake of [14C]L-leucine was inhibited by siRNA of LAT1. In the MTT assay, the siRNA of LAT1 inhibited the growth of the KB cells in the time-dependent manner, indicating that the growth inhibition of KB cell by the siRNA of LAT1 is induced by the blocking of neutral amino acid transport mediated by LAT1. These results suggest that the transport of neutral amino acids including several essential amino acids into the KB human oral squamous cell carcinoma is mediated mainly by LAT1. Further, the LAT1 would be a new target for the inhibition of cancer cell growth.

Amino acids a re required fo1' protein synthesis and energy sources in all living cell s. System L amino acid trans porters neutral amino aci cls i n a Na +' - independent manner. In ma 1ignan t tumors the L-type amino acid t ranspor te r 1(LA1'1), the first isoform 0 1' system L. is highly expressed to suppor t tumo1' cell growth. ln the present study, the expression 0 1' the system L amino acid transporter and BCH cytotoxicity were examined and compa red in both rat gli a l and C6 g lioma cel ls 1'he rat glia l cells expressed the L-type arnino acid t ra nsporter 2(LA1'2). the second isoform 이 system L, with its subu ni t 4F2hc. whereas the LA1'1 was not expressed. 1'he C6 glioma cells expressed LA1'1 with 4F2hc but not LA1'2 1'he [14C]L-leucine uptakes by the glial and C6 glioma cells were inhibi ted by BCH in a co ncent ration-d e pendant man ner wi th the lC50 values of 270.0 :t 13.7 μ M and 73.1 :t 4.5 μ M, respectively. 1'he cell g rowth was inh ibi ted by BCH in the time, concen tration-dependant manner in the gli al and C6 glioma cell s . 1'he rate of cell growth inhi bit ion induced by BCH in the C6 glioma cells was hi gh er than that in the gli al cells 1'hese results s uggesL that the tra ns ports of neutra l amino acids inc1uding several essenti al amino acids into rat g lial and C6 glioma cel ls a re for the most part mediated by LA1'2 and LA1'1, respectively. Therefore‘ the rat glia1 and C6 gJioma cells are excell ent tools for examing the proper ties of LA1'2 and LA1'1, respectively. Moreover, the spec너 i c inh ibi tion 0 1' LATl in cancel cells might be a new rationa le f'or anti -cancer therapy

본 연구는 아미노산의 첨가가 돼지 수정란의 체외 발달율에 미치는 영향을 구명하고자 PEF가 함유된 NCSU-23을 기본배지로 체외성숙 및 체외배양액을 조성한 후 EA(Essential amino acid), NA(Non-essential amino acid) 및 EANA(EA+ NA)를 첨가하여 체외성숙, 체외수정 및 체외발달에 미치는 영향을 조사하였다. 체외성숙 배지에 아미노산을 첨가한 결과 MH 단계까지의 체외성숙율은 NA 첨가군이 83.3%로 대조구 70.0%에 비하여 유의적으로(p<0.05) 높았다. 그러나 체외 수정 이후의 배 발달율과 수정율에서는 아미노산 첨가군과 무첨가군 사이에 유의적인 차이는 없었다. 체외배양액에 아미노산을 첨가한 후 배반포의 내부세포괴(ICM) 세포와 영양배엽(TE) 세포의 발달에 미치는 영향을 조사한 결과, ICM에서는 유의차를 발견할 수 없었으나 TE 세포는 EANA 처리구가 18.0±0.5개로 대조구 16.09±0.56개에 비해 유의적(p<0.05)으로 많았다. 총세포수에서도 EANA 처리구가 50.0±1.0개로 대조구 44.2±1.1개보다 유의적(p<0.05)으로 많았다. 이상의 결과를 종합할 때, 돼지의 체외수정란 생상에 있어서 아미노산의 첨가는 배반포로의 발달율에는 영향을 미치지 못하였으나 체외성숙율을 높이고 배반포의 세포수 향상에 도움을 주는 것으로 판단된다. 특히, 영양배엽(W) 세포의 발달율이 높은 것으로 보아 아미노산의 첨가는 돼지수정란의 착상에 도움을 줄 것이라 기대된다.

The study of cornified cell envelope has been used to investigate the differentiation factors and to advance oral carcinogenesis, CE of human oral keratinocytes are in wet condition as saliva containing many proteases, growth factors, and many kinds of bacteria, The analysis of CE in Immortalized human oral keratinocyte(IHOK) derived from normal human oral keratinocyte(NHOK) will be used to study the pathogenesis of oral squamous cell carcinoma, The purpose of this study was to analyze the amino acid component derived from CE of cultured NHOK and IHOK, It will be helpful to study the role of transfected E6/E7 gene in forming CE, and to examine the pathogenesis of oral squamous cell carcinoma, After primry culture of NHOK, IHOK were cultured in KBM bullet kit at 370C under 95% C02 incubator, Growth curve according to calcium concentration, cornified cell envelope measurement(CEM), and protein chemistry for amino acid component of CE were done(Mena :f::SD) , respectively. The obtained results were as follows, lHOK showed small areas of stratification, more compact, with irregular border and tightly apposed cells in 1,2 mM Ca++, Cornified cell envelope exhibited an aggregated group of empty space surrounded by the remained cell membrane, During the terminal differentiation in cultured NHOK and IHOK, insoluble cornified cell envelope formation was increased, CEM of NHOK was about 4 folds than that of lHOK under high calcium, Amino acid component of both groups showed Pro/Glu(SPR) , Gln/Glu(lnvolucrin) , and Gly(Loricrin) in descending order, From the aboving results, ít was suggested that when the terminal dífferentiation in cultured NHOK and IHOK, major amino acid component of CE in cultured lHOK was the same to that of cultured NHOK, It was thought that E6 and E7 gene should be involved in preventing the differentiation and proliferation of IHOK from making CE,

This study was conducted to examine contents of total acid and free amino acids in the Demi-glace with added quantity of Omija extracts. Firstly, The level of Total acid content of Demi-glace sauces was 1.08~1.89% and Omija extracts was 2.77~7.24%. The more Omija extracts added, there was the higher total acid contents. Sauces and extracts of 5% Omija was the highest. Secondly, Total free amino acids contents of control was 2518.52mg%, and Omija sauces was 2261.52~2894.14mg%. 2% Omija sauces was the highest among them. Hydroxyproline of total 34 free amino acids was the highest, and Glutamic acid 158.42mg%, Proline 78.90mg% was next in order. Arginine was the highest with 27.40~34.40mg% among 9 essential amino acids contents. Glutamic acid was the highest contents with 123.18~158.42mg%. Compared to control's(0.41mg%), Omija added group was 20.63~27.82mg% and it was the highest increase. While other 15 amino acid was analyzed, Hydroxyproline was the highest contents with 1,737.22~2,205.80mg%. Compared to control group(15.63mg%), proline was 57.01~78.90mg% Omija added group and it was increased with the highest contents. In essential amino acid, flavor enhancing amino acid and other amino acid were increased and the highest contents with 2% added Omija sauce. Thirdly, sensory characteristics of Demi-glace sauces based on overall preference, It was find that 2% added Omija was the best. 2% added was the best for color, flavor, taste, texture, overall acceptability(P〈.001). In terms of Demi-glace sauces' gender preference, male and female people liked 2% added Omija color, flavor, taste, texture, overall acceptability. It was find that there was no significant differences between male and female.

본 연구는 콩, 메주, 그리고 된장을 제조하여 장기간(12개월) 숙성시키는 과정 주에 아미노산성질소, 아미노산, 유리아미노산 및 색도의 변화를 관찰하고자 수행되었다. 된장의 제조는 한국식품개발원의 지침에 따라 수행하였으며 제조 직후, 6개월 숙성 및 12개월 숙성후에 시료를 분석하였다. 된장의 아미노산성질소는 제조 직후에는 콩과 메주에 비하여 높았으나(p<0.05) 숙성기간이 경과함에 따라 감소하는 경향이었다. 된장의 아미노산 총량은 콩보다 매우 낮았으나 유리아미노산 총량은 매우 높았다(p<0.05). 된장의 아미노산 총량은 숙성중 뚜렷한 차이를 보이지 않았으나 유리아미노산 총량은 숙성기간이 경과함에 따라 감소하는 경향을 보였다. 아미노산에 대한 유리아미노산의 비율(유리율)은 총량으로 콩의 경우 0.8%, 메주의 경우 17.3%, 그리고 된장의 경우 숙성중 20.4~32.9%이었다. 된장의 숙성중 아미노산이나 유리아미노산의 조성은 변화되었으나 어느 시점에서나 지미성분인 glutamic acid가 가장 많이 검출되었으며, 그 유리율은 21.1~41.5%이었다. 된장의 색도(명도, 적색도 및 황색도)는 숙성기간이 경과함에 따라 낮아졌다. 이로부터 된장의 아미노산 함량은 비록 콩에 비하여 매우 낮지만, 아미노산 유리율은 메주의 제조와 발효로부터 증가되고 된장의 숙성과정에서 더욱 증가되는 것을 알 수 있다. 된장의 아미노산성질소, 아미노산 및 색도에 의한 관능적 품질은 숙성 12개월 후에는 떨어지는 것으로 보인다.

Arrùno acid transpoπers play an important role in supplying nutrition to cells and for cell proliferation. System L is a major nutrient transport system responsible for the Na+-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LATl) is upregulated to support tumor cell growth. In the present study, we have examined the expression and function of system L amino acid transporter in FaDu human pharyngeal squamous carcinoma cells. RT-PCR, real-time quantitative RT-PCR and westem blot analysis have revealed that the FaDu cells express LATl together with its associaω19 protem 4F2hc, whereas the FaDu cells do not express the other system L isoform L-type amino acid transporter 2 (LAT2). 까le uptake of L-(14Clleucine by FaDu cells is Na+-independent and almost completely inhibited by system L selective inhibitor 2-aminobicyclo-(2,2,1)-heptane-2- carboxylic acid (BCH). The profiles of the inhibition of L-[I4Cllellcine uptake by variolls amino acids in the FaDu cells are comparable with those for the LA T1 expressed in Xenopus 。()(찌es. π1e majority of L-[I4Clleucine uptake is, therefore, mediated by LAT1 in the FaDu cells. These results suggest that the transport of neutral amino acids including several essential amino acids in the FaDu human pharyngeal squamous carcinoma cells mediated by LAT1. In addition, specific inhibition of LAT1 by such agents as BCH in pharyngeal squamous cell carcinomas will be a new rationale for anti-cancer therapy.

본 연구는 체외에서 한우 난포란의 핵성숙과 그 후의 초기 배발달에 있어서 체외성숙 배지에 아미노산의 첨가가 난포란의 제1극체(PB) 출현율, 배발달율 그리고 배반포의 세포수에 미치는 영향을 검토하였다. 첨가하는 아미노산의 종류와 농도는 각각 MEM 배지의 non-essential amino acids (NEAA)와 BME 배지의 essential amino acids(EAA)는 1, 2, 4배 및 유청중의 lactalbumine hydrolysate(LAH)는 1, 5, 10 ㎎/㎖이었다. 그 결과 NEAA와 EAA의 경우 PB 출현율은 1배 첨가군이 미첨가군보다 유의하게 높았지만(p＜0.05), 첨가량이 증가할수록 오히려 감소하였다. 그러나 배반포로의 배발달율은 모든 군에서 비슷한 경향이었다. 그리고 배반포의 총 세포수와 총 세포수중 TE 세포수는 2배 처리군이 가장 높았으며, ICM 세포수는 아미노산 첨가량이 증가할수록 많아졌다. 한편 LAH의 경우 PB 출현율은 5㎎ 첨가군이 가장높았으며, 배반포로의 발달율은 미첨가군과 1 ㎎ 첨가군이 5 ㎎ 첨가군과 10 ㎎ 첨가군보다 각각 유의하게 높았다(p＜0.05). 그리고 배반포의 세포수는 NEAA와 EAA를 이용하였을 경우와 비슷한 경향이었다. 이상의 결과로부터 체외성숙용 배지에 아미노산의 첨가는 생산된 배반포의 품질을 향상시킬 수 있기 때문에 배반포의 체외생산에 이용할 수 있는 새로운 아미노산의 종류 및 농도를 탐색할 필요가 있다고 사료된다.

It has been said that amino acid transporters play an important role in supplying nutrition to normal and cancer cells and for cell proliferation. System L is a major nutrient transport system responsible for the Na+-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1(LAT1) is up-regulated to support tumor cell growth. In the present study, we have examined the function of LAT1 and its expression in the KB human oral epidermoid carcinoma cells. RT-PCR, western blot analysis and immunohistochemical analysis have revealed that KB cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas KB cells do not express the other system L isoform LAT2. The uptake of L-[14C]leucine by KB cells is Na+-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of L-[14C]leucine uptake by amino acids in the KB cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of L-[14C]leucine uptake is, therefore, mediated by LAT1 in the KB cells. These results suggest that the uptakes of neutral amino acids including several essential amino acids in the KB oral epidermoid carcinoma cells mediated by LAT1. In addition, specific inhibition of LAT1 by such agents as BCH in human oral squamous cell carcinomas will be a new rationale for anti-cancer therapy.

본 연구는 체외에서 소 난포란유래의 배반포 생산에 있어서 배지 내에 첨가하는 외인성 고정질소 원으로써 아미노산과 FBS의 첨가효과를 검토하였다. 소 난포란의 체외성숙은 TCM-l99용액, 체외 수정은 Fer-TALP용액으로 행하였으며, 체외수정후 24시간째(day 1)의 수정난자를 체외배양에 제공하였다. 체외배양용 기초배지는 YS용액, 기초 배양법은 25개 난자/10 배지의 단순.미소적배양법을 이용하였다. 본 연구의 결과를 요약하면 다음과 같다. 1.

세리신 잠의 발육, 실샘 성장, 세리신 량, 혈액 아미노산 조성 등 세리신 잠의 특성을 알아보기 위하여 잠 120 과 같은 조건으로 사육하여 얻은 결과는 다음과 같다. 1. 부화율에 있어서 세리신 잠은 85%이며 잠 120 은 95%이었다. 특히 세리신 잠은 부화가 고르지 않았다. 2. 유충 기간에 있어서 세리신은 20 일 23 시간이었고, 잠 120 은 21 일 22시간이었다. 3. 실샘에 있어서 세리신 잠은 중부실샘이 대부분이며 후부 실샘의 길이는 약 2~3cm 이었고 굴곡은 없었다. 4. 세리신 잠에 있어서 상족후 나방까지의 기간은 약 12 일이었으며, 화용후 화아까지는 7 일로 대단히 짧은 특성을 보였다. 5. 고치에 있어서 세리신 잠은 이주 얇고 가벼웠으며 견충중은 2.7cg 이며 견충 비율은 3.65%이였다. 6. 세리신 (sericin protein)양에 있어서, 세리신 잠의 견충이 sericin 만 으로 구성되어 있고 잠 120 은 28%가 sericin 이었다. 세리신 잠의 견충 당 sericin 율은 잠 120 의 34.6%정도였다. 7. 혈액 아미노산의 양에 있어서 세리신 잠은 histidine, lysine, glutamic acid 가 많았으며 5 령 발육에 따른 조성의 변화에 있어서 대부분의 혈액 아미노산들은 증가하였다.

In order to establish the processing conditions for salt-fermented liquefaction of anchovy(Engrulis japonica), changes in the amino acid composition from oligopeptides during fermentation periods were analyzed. Experimental sample A: chopped whole anchovy, adding 20% water, heating at 50℃ for 9 hrs and then adding 10% NaCl. Sample B: chopped whole anchovy, adding 20% water, heating at 50℃ for 9 hrs and then adding 13% NaCl. Sample C: chopped whole anchovy adding 13% NaCl. Sample D: whole anchovy adding 17% NaCl. The total amino acids from oligopeptides in fermented liquefaction of anchovy increased in early fermentation period and reached highest level, and then they declined irregularly during fermentation. Their maximum amounts were just after heating at 50℃ for 9 hrs in sample A, after 15 days in sample B, and after 60 days in samples C and D. The fermented liquefaction of anchovy extracts were rich in glutamic acid, aspartic acid, proline, glycine, alanine, lysine and valine. However, the contents of most amino acids fluctuated by the experimental specimens and fermenting periods. Among them glutamic acid was the most abundant amino acid which was occupied 0.6~27.7%(average 24.0%) in the content of total amino acids from oligopeptides. The contribution of the amino acid composition from oligopeptides to extractive nitrogen was occupying average 20.8 and 17.5% in rapid- and low salt-fermented liquefaction(sample A, B and C) and traditional fermented liquefaction(sample D), respectively.

아미노산 엽면 시비가 멜론 묘의 생육에 미치는 영향에 대하여 연구하였다. 처리 시기는 본엽 1매 전개시(Ll)와 2매 전개시(L2)로 나누었고, 아미노산 농도는 무처리구, 10, 20, 30 mg . L-1로 설정하였다. Ll에서 생체중과 건물중은 아미노산 처리구에서 높았다. 초장은 3차 조사시에 Ll-30에서 가장 높게 나타났다. L2에서 3차 조사시 생체중은 L2-30처리구에서 높게 나타났다. 초장은 2차와 3차 조사시 L2-30에서 높게 나타났다. 제1본엽의 염장과 엽폭의 경우, Ll의 3차 조사시 처리 효과가 나타났다. L2에서 염장은 처리구간에 유의적인 차이를 보이지 않았고, 엽폭은 3차 조사시에 아미노산 처리구에서 높게 나타났다 제2본엽의 염장과 엽폭은 Ll의 2차와 3차 조사시에 아미노산 처리구들에서 높게 나타났다. L2에서는 처리구간에 유의적인 차이를 보이지는 않았다 제7본엽의 염장과 엽폭은 시비 및 조사 시기에 관계없이 처리간에 유의성을 보이지는 않았다. 이상의 결과, 제1본엽이 전개된 후 아미노산의 농도를 20mg. L-1 이상, 회수를 3회 이상 엽면살포 하거나, 제2본엽이 전개된 후 아미노산을 시비할 경우에는 아미노산 농도를 30 mg . L-1으로 설정하고 회수를 2회 이상 시비한다면 생육이 향상된 묘를 생산할 수 있을 것으로 사료된다.

In order to establish the processing condition of rapid- and low salt-fermented liquefaction of anchovy (Engrulis japonica), effect of temperature on crude enzyme activity of anchovy viscera, pretreatment conditions, and the minimum content of adding NaCl were investigated. The minimum limitation of NaCl content for anchovy liquefaction was 10%. Sample A(water adding, heating, adding 10% NaCl): chopped whole anchovy adding 20% water and then heating for 9 hrs at 50℃ and then adding 10% NaCl and then fermented at room temperature(8-29℃) for 180 days. Sample B(water adding, heating, adding 13% NaCl): chopped whole anchovy adding 20% water and then heating for 9 hrs at 50℃ and then adding 13% NaCl and then fermented at room temperature for 180 days. Sample C(adding 13% NaCl): chopped whole anchovy and then adding 13% NaCl and then fermented at room temperature for 180 days. Sample D(adding 17% NaCl): whole anchovy adding 17% NaCl and then fermented at room temperature for 180 days. The content of free amino acids such as aspartic acid, serine and threonine fluctuated severely according to the pretreatment methods. Possibly they might be recommend quality indices of standardization for salt-fermented liquefaction of anchovy. As for the relation between fermentation period(X) and individual free amino acid(Y), five kinds of free amino acids such as glutamic acid, valine, glycine, lysine, and alanine showed highly significant in their coefficient of determination in most of samples. They might be recommend as quality indices for salt-fermented liquefaction of anchovy during fermentation. The difference of taste between products of the rapid- and low salt-fermented liquefaction and the traditional salt-fermented liquefaction were caused by their composition of the free amino acids ratios, in which were umami, sweet, and bitter taste in the extracts of anchovy during fermentation. The appropriate fermentation period of the sample A was shorten 30 days than the sample B and 60 days than the samples C and 90 days than the sample D in the processing of anchovy.