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"Tiamin berfungsi sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme karbohidrat. Mengingat pentingnya peran tiamin, maka dilakukan pengembangan teknik pengukuran tiamin yang analog dengan enzyme linked immunosorbent assay (ELISA), dimana antibodi diganti dengan protein pengikat spesifik yaitu protein ikat tiamin kacang hijau (PITKH). Teknik pengukuran ini dilakukan secara kompetitif, kompetitor akan dikompetisikan dengan tiamin bebas yang akan diukur. Kompetitor tersebut berupa konjugat antara tiamin-biotin. Tiamin murni diikatkan dengan biotin menggunakan senyawa perangkai yaitu glutaraldehid. Pada analisis LC-MS ditemukan 3 puncak, . Puncak ke 3 merupakan konjugat tiamin-biotin. Dibuat kurva standar dan diperoleh persamaan garis lurus dengan nilai R2= 0,9986. Uji validasi menggunakan konjugat tiamin-biotin menunjukan nilai coefficient of variation (CV) = 3,81%, nilai ini lebih kecil dari CV Horwitz = 8,12%, akurasi dengan nilai Recovery (R) =94 %-98%. Hasil ini menunjukan syarat pengukuran dengan teknik ELPLA sudah terpenuhi, dengan presisi dan akurasi yang baik. Aplikasi pengukuran kadar tiamin pada serum normal sebanyak 23 sampel didapatkan kadar tiamin berkisar 2,62-9,76 µg/ml. Dengan demikian, teknik ELPLA dengan konjugat tiamin-biotin sebagai kompetitor dapat digunakan pada pengukuran kadar tiamin dalam serum.

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Thiamine has a coenzyme function in several enzymes involved in carbohydrate metabolism. Considering the important role of thiamine, a thiamine measurement technique analogous to the enzyme linked immunosorbent assay (ELISA) was developed, that the antibody was replaced by a specific binding protein named mung bean thiamine binding protein (MBTBP). The measurement technique was carried out competitively in which competitors would be competed with free thiamine to be measured. The competitor is a thiamine-biotin bond. Pure thiamine was bound to biotin using a coupling compound called glutaraldehyde. In the LC-MS analysis we found 3 peaks. The third peak was the thiamine-biotin conjugate. A standard curve was made and the value of its straight line equation was obtained R2= 0,9986. The validation test using thiamine-biotin conjugate showed coefficient of variation (CV) value = 3,81% which was smaller than Horwitz CV = 8,12%, with the accuracy of the Recovery (R) value = 94% – 98%. These results indicated that the measurement requirements for the ELPLA technique had been met with good precision and accuracy. The application of the serum measurements to 23 samples showed thiamine levels ranging from 2,62- 9,76 µg/ml. Thus, the ELPLA technique with thiamine-biotin conjugate as a competitor could be used in the measurement of serum thiamine levels.

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"Tiamin berfungsi sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme karbohidrat. Mengingat pentingnya peran tiamin, maka dilakukan pengembangan teknik pengukuran tiamin yang analog dengan enzyme linked immunosorbent assay ( ELISA), dimana antibodi diganti dengan protein pengikat spesifik yaitu protein ikat tiamin kacang hijau (PITKH). Teknik pengukuran ini dilakukan secara kompetitif, kompetitor akan dikompetisikan dengan tiamin bebas yang akan diukur. Kompetitor tersebut berupa konjugat antara tiamin-biotin. Tiamin murni diikatkan dengan biotin menggunakan senyawa perangkai yaitu glutaraldehid. Pada analisis LC-MS ditemukan 3 puncak, . Puncak ke 3 merupakan konjugat tiamin-biotin. Dibuat kurva standar dan diperoleh persamaan garis lurus dengan nilai R2= 0,9986. Uji validasi menggunakan konjugat tiamin-biotin menunjukan nilai coefficient of variation (CV) = 3,81%, nilai ini lebih kecil dari CV Horwitz = 8,12%, akurasi dengan nilai Recovery (R) =94 %-98%. Hasil ini menunjukan syarat pengukuran dengan teknik ELPLA sudah terpenuhi, dengan presisi dan akurasi yang baik. Aplikasi pengukuran kadar tiamin pada serum normal sebanyak 23 sampel didapatkan kadar tiamin berkisar 2,62-9,76 μg/ml. Dengan demikian, teknik ELPLA dengan konjugat tiamin-biotin sebagai kompetitor dapat digunakan pada pengukuran kadar tiamin dalam serum

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Thiamine has a coenzyme function in several enzymes involved in carbohydrate metabolism. Considering the important role of thiamine, a thiamine measurement technique analogous to the enzyme linked immunosorbent assay (ELISA) was developed, that the antibody was replaced by a specific binding protein named mung bean thiamine binding protein (MBTBP). The measurement technique was carried out competitively in which competitors would be competed with free thiamine to be measured. The competitor is a thiamine-biotin bond. Pure thiamine was bound to biotin using a coupling compound called glutaraldehyde. In the LC-MS analysis we found 3 peaks. The third peak was the thiamine-biotin conjugate. A standard curve was made and the value of its straight line equation was obtained R2= 0,9986. The validation test using thiamine-biotin conjugate showed coefficient of variation (CV) value = 3,81% which was smaller than Horwitz CV = 8,12%, with the accuracy of the Recovery (R) value = 94% – 98%. These results indicated that the measurement requirements for the ELPLA technique had been met with good precision and accuracy. The application of the serum measurements to 23 samples showed thiamine levels ranging from 2,62- 9,76 μg/ml. Thus, the ELPLA technique with thiamine-biotin conjugate as a competitor could be used in the measurement of serum thiamine levels"

"Tiamin (Vitamin B1) adalah vitamin B yang pertama kali diidentifikasi. Tiamin berperan sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme energi.Uji laboratorium terhadap kekurangan tiamin dapat dilakukan dengan mengukur kadar tiamin dalam darah. Penelitian ini bertujuan untuk mengetahui kadar tiamin serum pada alkoholic dan penderita DM dengan teknik ELISA, HPLC, dan menggunakan protein ikat tiamin kacang hijau (PITKH) dengan teknik enzyme-labeled protein ligand assay (ELPLA). Untuk melihat faktor-faktor yang mempengaruhi ikatan antara PITKH dengan tiamin digunakan teknik dialisis kesetimbangan. Validitas teknik ELPLSBA dilakukan dengan uji presisi dan akurasi. Teknik ELISA dan HPLC digunakan sebagai pembanding pada pengukuran tiamin serum. Konsentrasi PITKH pasca kromatografi afinitas hasil pengenceran liofilisat stabil selama 30 hari pada suhu -20°C dan 3 hari pada suhu 4°C. Aktifitas pengikatan PITKH dengan tiamin optimum pada pH 7,5. Aktifitas pengikatan ini juga dipengaruhi oleh senyawa alkilasi, oksidator, dan reduktor, tetapi kurang dipengaruhi oleh ion kalsium dan logam-logam berat. Kemampuan PITKH dalam mengukur kadar tiamin serum dengan teknik ELPLA memiliki presisi dengan CV 4,1% dan akurasi dengan nilai R 96-98%. Pengukuran dengan ELISA memberikan hasil yang lebih rendah dari teknik ELPLA, sedangkan uji banding dengan HPLC diperoleh p = 0,102 (p > 0,05) ; artinya tidak terdapat perbedaan yang signifikan antara metode ELPLA dan HPLC. Pengukuran tiamin serum dengan teknik ELISA, HPLC dan ELPLA pada alkoholic dan penderita DM, lebih rendah dari serum normal.

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Thiamine (vitamin B1) was the first B vitamin to have been identified. It serves as a coenzyme for several enzymes involved in energy metabolism. The laboratory test against thiamine deficiency can be done by measuring thiamine levels in the blood. The aim of this study was to determine the serum thiamine levels in alcoholics and DM by ELISA, HPLC, and using mung bean thiamine binding protein (MBTBP) with the development of enzyme-labeled protein ligand assay (ELPLA) method. The equilibrium dialysis technique was used to see the factors affecting the bond between TBP and thiamine. The ELPLA method validity was performed with precision and accuracy tests. ELISA and HPLC methods were used as comparators for measurements of serum thiamine. The MBTBP concentration of post-chromatographic affinity resulted from dilution of lyophilisate was stable for 30 days at -20°C and 3 days at 4°C. The optimal pH for binding MBTBP to thiamine was 7,5. This binding activity was also affected by alkylation, oxidizing, and reducing agents, but it was less affected by calcium ions and heavy metals. MBTBP ability to measure serum thiamine levels with the ELPLA technique has precision with CV 4,2% and accuracy with R 96-98%. Measurements by ELISA has lower result than ELPLA. The comparison test with HPLC method obtained p = 0,102 (p > 0,05); meaning no significant difference between ELPSLBA and HPLC methods. Serum thiamine level by ELISA, HPLC and ELPLSBA techniques in alcoholic and DM patients were lower than normal serum."

"Latar BelakangTiamin merupakan mikronutrien yang dibutuhkan dalam proses metabolisme energi. Defisiensi tiamin dapat menyebabkan berbagai kelainan jangka pendek dan jangka panjang, terutama pada populasi khusus, seperti pasien dengan diabetes atau stunting. Meskipun pentingnya kecukupan tiamin, belum terdapat standar tiamin serum bagi populasi Indonesia. Hal ini disebabkan oleh metode pengujian yang belum optimal untuk dilakukan pada sampel besar. Karena itu dikembangkan metode pengukuran yang lebih ekonomis dan praktis menggunakan prinsip ELISA namun menggunakan protein ikat tiamin. Protein ini telah ditemukan dalam berbagai spesies kacang-kacangan. Penelitian ini mengeksplorasi keberadaannya pada spesies kacang yang masih kurang diteliti, yaitu kacang lima (Phaseolus lunatus)MetodeKacang lima dihaluskan dan dibuat tiga sampel kacang, masing-masing sebanyak 5 gram. Tiap sampel dilarutkan menggunakan dapar fosfat. Larutan tersebut disentrifugasi dan supernatan diambil. Dilakukan proses salting out dengan ammonium sulfat, lalu larutan kembali disentrifugasi. Presipitat diisolasi dan dilarutkan dengan dapar fosfat lalu menjalani proses dialisis. Kadar protein diukur menggunakan spektrofotometer. Ditambahkan larutan tiamin pada sampel hasil salting out, kemudian dilakukan dialisis kesetimbangan untuk mengukur aktivitas pengikatan tiamin. Absorbansi dialisat dibaca untuk menghitung jumlah tiamin terikat.HasilPenelitian ini menunjukkan bahwa kacang lima mengandung rata-rata 7,61 gram protein/100 gram kacang. Ditemukan aktivitas pengikatan tiamin yang bervariasi antara 573,16 dan 878,09 μg/10 gram kacang dengan rasio tiamin terikat per jumlah protein 0,95μg/mg protein.KesimpulanDitemukan protein ikat tiamin pada kacang lima dengan aktivitas yang tinggi dibandingkan dengan sumber lainnya.

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Introduction

Thiamine is a micronutrient essential for energy metabolism, and its deficiency may lead to various disorders, especially on certain populations, such as diabetic and stunting patients. In Indonesia, there is no established serum thiamine standard, partly due to the unoptimal testing methods available. Therefore, a more economical and practical measurement method was developed using the ELISA principle but using thiamine binding protein found in various species of beans. This research explores its presence in a lesser-studied lima bean (Phaseolus lunatus)

Method

Lima beans were ground and three samples are prepared, each containing 5 grams of bean. Each sample is dissolved using phosphate buffer. The solution was centrifuged and the supernatant was collected. Ammonium sulfate was added to precipitate protein, then the solution was centrifuged again. The precipitate was isolated and dissolved in phosphate buffer, then dialysed. Protein levels were measured using a spectrophotometer. Thiamine solution was added to the salted out sample, then equilibrium dialysis was carried out to measure the thiamine binding activity. Absorbance reading of the dialysate were used to quantify bound thiamine.

Results

Lima beans contain an average of 7.61 grams of protein/100 grams. Thiamine binding activity ranged from 573.16 to 878.09 μg per 10 grams of beans with a ratio of bound thiamine per amount of protein of 0.95 μg/mg protein.

Conclusion

Thiamine binding protein was found in Lima beans with high activity compared to other sources."

"ABSTRAKLatar Belakang: Tiamin merupakan kelompok vitamin B yang dibutuhkan manusia namun harus diperoleh dari sumber luar. Tiamin memiliki banyak fungsi penting dalam tubuh, sehingga kekurangan tiamin dapat menyebabkan masalah serius. Pemeriksaan kadar tiamin dalam tubuh dengan metode yang tersedia saat ini membutuhkan biaya yang mahal. Berdasarkan hal tersebut, lahirlah ide untuk menggunakan prinsip ELISA dengan memanfaatkan protein pengikat tiamin dari sumber yang mudah diperoleh dan murah untuk skrining. Penelitian ini bertujuan untuk mengetahui adanya protein pengikat tiamin pada ketan hitam (Oryza sativa L.).Metode: Isolasi protein pengikat tiamin dari ketan hitam (Oryza sativa L.) dilakukan dengan metode salting out, dialisis, dan dialisis kesetimbangan. Salting out menggunakan konsentrasi garam amonium sulfat 90%.Hasil: Dalam penelitian ini, konsentrasi protein total adalah 5.190,48 g/mL setelah tahap salting out. Adanya protein pengikat tiamin dalam protein total dapat dibuktikan dengan dialisis ekuilibrium. Protein pengikat tiamin dari ketan hitam (Oryza sativa L.) dapat mengikat tiamin pada dialisis kesetimbangan 0,479 g/gram tepung.Kesimpulan: Ketan hitam (Oryza sativa L.) mengandung protein pengikat tiamin.ABSTRACTBackground: Thiamine is a group of B vitamins that humans need but must be obtained from external sources. Thiamine has many important functions in the body, so thiamine deficiency can cause serious problems. Examination of thiamine levels in the body with currently available methods is expensive. Based on this, the idea was born to use the ELISA principle by utilizing thiamine binding proteins from sources that are easily obtained and inexpensive for screening. This study aims to determine the presence of thiamine binding protein in black sticky rice (Oryza sativa L.).Methods: Isolation of thiamine binding protein from black sticky rice (Oryza sativa L.) was carried out by salting out, dialysis, and equilibrium dialysis methods. Salting out using a 90% concentration of ammonium sulfate salt.Results: In this study, the total protein concentration was 5,190.48 g/mL after the salting out stage. The presence of thiamine-binding protein in total protein can be demonstrated by equilibrium dialysis. Thiamine binding protein from black sticky rice (Oryza sativa L.) can bind thiamine at equilibrium dialysis of 0.479 g/gram flour.Conclusion: Black sticky rice (Oryza sativa L.) contains thiamine binding protein."

"Latar Belakang. Penelitian ini bertujuan untuk membakukan teknik pengukuran asam folat serum menggunakan protein ikat folat PIF yang diisolasi dan dimurnikan secara utuh dari susu sapi segar dengan teknik enzyme labeled protein ligand binding assay ELPLBA . Metode. Metode yang digunakan adalah metode eksperimen yang menguji validitas dan perbandingan teknik ELPLBA untuk pembakuan teknik pengukuran folat serum.Hasil. Hasil isolasi dan pemurnian PIF menghasilkan kadar 3 tiga mg/mL. Identifikasi SDS-PAGE dan Western blot menunjukkan 3 tiga pita protein yang diperiksa adalah protein ikat folat. Validitas teknik uji keterulangan menunjukkan nilai yang dapat diterima CV.

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Background. This study was aimed to standarized technique of folic acid level serum measurment using folate binding protein FBP that isolated and purified from fresh bovine milk with enzyme labeled protein ligand binding assay ELPLBA technique. Method in this study, we performed an experiment research validated dan compared measurement techniques of ELPLBA with competitive ELISA for the standardization of the serum folic acid measurement technique. Results. FBP concentration yielded from isolated and purification was resulted 3 mg mL. SDS PAGE and western blot result showed 3 three protein bands that was confirmed to be FBP. Validity test repeatability indicate an acceptable CV 10 , whereas reproducible test showed poor results over a 5 day period. The results of the accuracy test of the enzyme labeled protein ligand binding assay technique showed good accuration. Linearity test of two samples showed quite linear results. Comparison of folic acid level measurement in serum between ELPLBA and ELISA technique showed there is no difference between both technique based on independent test T test at 95 confidence level. Conclusion. the enzyme labeled protein ligand binding assay technique on serum folic acid measurements were quite valid dan equivalent to the results obtained by competitive ELISA techniques."

"Pangan fungsional adalah pangan yang dapat memenuhi asupan harian dan memberikan manfaat kesehatan. Probiotik merupakan salah satu komponen pangan fungsional berupa sel bakteri hidup yang dalam jumlah tertentu dapat memberikan manfaat kesehatan. Selain pada fermentasi makanan, bakteri asam laktat juga dimanfaatkan dalam fermentasi minuman seperti fermentasi sari kacang hijau. Isolat bakteri asam laktat TM2 adalah bakteri asam laktat yang diisolasi dari tapai ketan hitam dan memiliki aktivitas antibakteri yang tinggi. Penelitian ini bertujuan untuk mengetahui aktivitas antibakteri filtrat hasil fermentasi terhadap Bacillus cereus dan Klebsiella oxytoca. Medium kacang hijau dibuat dengan cara merendam kacang hijau selama 12 jam kemudian direbus dan disaring. Selanjutnya starter isolat BAL TM2 diinokulasikan ke dalam medium kacang hijau dan difermentasi selama 1, 2, dan 3 hari. Pertumbuhan bakteri dihitung dengan metode Total Plate Count (TPC). Pengukuran pH dilakukan dengan menggunakan pH meter sedangkan total asam diukur dengan metode titrasi. Pengujian aktivitas antibakteri dilakukan dengan metode cylinder plate assay. Hasil penelitian menunjukkan bahwa terjadi pertumbuhan populasi bakteri asam laktat dari starter hingga hasil fermentasi hari ke-3. Medium kacang hijau mengalami penurunan pH dan kenaikan konsentrasi asam selama fermentasi. Hasil pengujian aktivitas antibakteri menunjukkan aktivitas penghambatan tertinggi pada filtrat fermentasi 2 hari terhadap Bacillus cereus (19-20 mm) dan Klebsiella oxytoca (14 mm). Kesimpulan penelitian ini adalah filtrat medium kacang hijau menunjukkan aktivitas antibakteri terhadap Bacillus cereus dan Klebsiella oxytoca. Aktivitas tertinggi didapatkan pada filtrat fermentasi 2 hari dan mengalami penurunan efektivitas pada fermentasi 3 hari. Filtrat lebih efektif menghambat pertumbuhan Bacillus cereus dibanding Klebsiella oxytoca.

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Functional food is food that meets daily intake requirements and provides health benefits. Probiotics are a component of functional food consisting of live bacterial cells that can offer health advantages. Besides food fermentation, lactic acid bacteria are also utilized in beverage fermentation, such as mung bean juice. The lactic acid bacteria isolate TM2, derived from black sticky rice tapai, exhibits high antibacterial activity. This study aims to evaluate the antibacterial activity of the fermentation filtrate against Bacillus cereus and Klebsiella oxytoca. The mung bean medium was prepared by soaking the beans for 12 hours, then boiling and filtering them. Next, the LAB TM2 isolate was inoculated into the mung bean medium and fermented for 1, 2, and 3 days. Bacterial growth was measured using the Total Plate Count (TPC) method. pH was measured with a pH meter, while total acidity was determined through titration. Antibacterial activity was tested using the cylinder plate assay method. The results showed growth of lactic acid bacteria from the starter culture to the third day of fermentation. The mung bean medium exhibited a decrease in pH and an increase in acid concentration during fermentation. The highest inhibitory activity was observed in the 2-day fermentation filtrate against Bacillus cereus (19-20 mm) and Klebsiella oxytoca (14 mm). In conclusion, the mung bean medium filtrate demonstrates antibacterial activity, peaking at 2 days and decreasing thereafter, with greater effectiveness against Bacillus cereus compared to Klebsiella oxytoca."

"Terdapat banyak metode untuk mendeteksi kontaminasi melamin dalam makanan atau produk pakan termasuk immunoassay. Dalam penelitian ini, hapten melamin disintesis dengan mereaksikan 2-kloro-4,6-diamino-1,3,5-triazina (CAAT) dengan asam 6-aminokaproat. Karakterisasi hapten dengan spektrometer Fourier Transform Infrared (FTIR) menunjukkan absorpsi ikatan N-H dari gugus amina sekunder alifatik pada frekuensi 3350-3310 cm-1 dan vibrasi ulur ikatan C-N pada daerah sidik jari 1250-1020 cm-1. Karakterisasi dengan 1H-NMR menunjukkan pergeseran kimia pada 4,20 ppm (t, 1H), sedangkan dengan 13C-NMR pada pergeseran kimia 139 ppm. Karakterisasi dengan spektrometer massa menunjukkan M+. m/z 240,4 untuk hapten (C9H16N6O2). Imunogen disintesis dengan cara menkonjugasikan hapten dengan bovine serum albumin (BSA) dengan menggunakan metode ester aktif. Konjugat hapten-BSA menyerap panjang gelombang maksimum UV pada 224 nm. Data menunjukkan bahwa hapten dan imunogen berhasil disintesis. Hapten-BSA kemudian disuntikkan ke kelinci untuk menghasilkan antibodi poliklonal. Setelah tiga minggu imunisasi, serum menunjukkan adanya antibodi melalui Gel Agarose Precipitation Test (AGPT). Dari hasil optimasi awal indirect ELISA diketahui konsentrasi optimum coating antigen hapten-OVA adalah 1 μg/mL dan konjugat HRP-anti rabbit IgG adalah 1:10.000. Dari hasil indirect competitive ELISA diperoleh bahwa pengenceran optimum antibodi yang masih memberikan respon positif berupa absorbansi yang tinggi adalah 1:125 (6 μg/mL). Antiserum yang dihasilkan memiliki sensitivitas yang cukup baik terhadap melamin dengan nilai IC50 sebesar 2,83 ppm dan limit deteksi (IC15) sebesar 0,22 ppm melalui indirect competitive ELISA.

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There are many method to detect the melamine contamination in food or feed product including immunoassay. In this study, hapten of melamine was synthesized by reacting 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) and 6-aminocaproic acid. Characterization of hapten with fourier transform infrared spectroscopy (FTIR) showed secondary N-H stretching vibrational band at 3350-3310 cm-1 and C-N stretching vibrational band at fingerprint region 1250-1020 cm-1. Characterization of hapten with 1H-NMR showed chemical shift at 4,20 ppm (t, 1H), and 139 ppm by 13C-NMR. Hapten also characterized with high-resolution mass spectrometry (HRMS) that showed M+. m/z 240,4 for hapten (C9H16N6O2). The Immunogen was prepared by coupling hapten to bovine serum albumin (BSA) using the active ester method. Hapten-BSA conjugate showed the maximum wavelength of UV absorpstion at 224 nm. The data pointed out that hapten and immunogen was successfully synthesized. Hapten-BSA conjugate then injected into rabbit to produce the polyclonal antibodies. After three weeks immunization, serum showed the presence of antibodies through Agar Gel Precipitation Test (AGPT). The initial optimization of indirect ELISA showed that the optimum concentration of coating antigen hapten-OVA was 1 mg/mL and HRP-conjugated anti-rabbit IgG was 1:10,000. The optimum antibody dilution that still gave a positive response was 1:125 (6 mg/mL) through indirect competitive ELISA. Antiserum produced has a good sensitivity to melamine with IC50 2.83 ppm and a detection limit (IC15) 0.22 ppm using indirect competitive ELISA."