IntroductionDietary supplementation

Introduction
Dietary supplementation is an attractive, non-invasive means of enhancing and optimizing important physiological functions, including the immune system [1]. The ability of dietary supplementation to optimize immune function is seen as particularly important among those groups of individuals who may have an under-developed or poorly functioning immune system, such as infants, young children, immunocompromised subjects and the elderly [2,3]. Recent advances in research have greatly increased understanding of the importance of the gut microbiota. Bacterial colonization of the intestine in a gut-microbiome system is critical to the normal development of many aspects of physiology. Further understanding BINUS University, Alam Sutra Campus, Tangerang, Indonesia. of the mechanisms underlying the gut-microbiome will provide new insight into the symbiotic relationship between gut microbiota and their mammalian hosts, and help to identify the potential for microbial-based preventive strategies to aid in health promotion. Probiotics are defined as living microorganisms that when administered in adequate amounts, confer a health benefit to human health [4]. Lactobacillus plantarum IS-10506 is a novel
probiotic strain isolated from a yogurt-like product, dadih, an Indonesian traditional buffalo milk of West Sumatera origin [5]. In vitro probiotic properties have been proven, such as acid and bile tolerance, adhesion properties, and competitiveness against pathogens [6,7]; in vivo [8,9] and human studies have been conducted in serial studies [10–12]. The primary functions of the gastrointestinal tract are digestion and absorption of nutrients, and electrolyte and water homeostasis; hence, the integrity of the intestinal brush border is a key element in preventing systemic absorption of enteric toxins and bacteria. Gastrointestinal lymphoid tissue plays an important role in controlling transepithelial passage of bacteria across the intestinal mucosa by synthesizing more antibody molecules than any other lymphoid tissue. Gastrointestinal antibodies are produced in the form of immunoglobulin and are key players in efficient humoral mucosal immunity, in particular the maintenance of the integrity of the epithelial barrier.
Zinc is an essential trace element of exceptional biologic and public health importance. Zinc is an essential component of a large number of enzymes, and plays a central role in cellular growth and differentiation of tissues with rapid turnover, including the immune system and the gastrointestinal tract [13,14]. Zinc deficiency involves impaired immune function, sub normal growth and development, and in children causes growth retardation, delayed sexual maturation, and infection susceptibility. Intervention trials have demonstrated that the supplementation with zinc can also enhance the immune response [15]. There is no evidence that zinc
causes harm when given to non-septic, immunocompetent children [16].

Materials and methods
Preparation of diets The tested powder contained maltodextrin as placebo and probiotic L. plantarum IS-10506 of dadih origin was identified by 16S rRNA gene sequencing as L. plantarum (Gene Bank accession no. DQ860148). For experimental purposes, microencapsulated bacteria were incorporated at a dose of 2.3 × 10 11 CFUs/g powder, and has been shown to exhibit neither significant loss of viability nor contamination because of storage during the entire study period.
Zinc was supplemented as zinc sulfate powder. Placebo, probiotic or zinc were given in microencapsulated form and were identical in appearance.

Subjects and trial criteria
Forty-eight apparently healthy children within the age range of 12–24 months (median 16.8 months) were selected for this study. Prior to commencement of the trial, the selection criteria were generated from the records of participating health care providers. Inclusion criteria were apparently healthy children, between the ages of 12 and 24 months and agreement to conform to the trial guidelines or provide notification of non-compliance. Exclusion criteria were congenital abnormality or disease, gastrointestinal disease, regular use of products with probiotic bacteria, receiving antibiotic therapy within two weeks prior to the intervention study and non-agreement to avoid potentially conflicting nutritional or trace element supplements during the 90 days of the trial. Compliance with supplementation was confirmed by the subjects by direct report to the health care provider. Protocol The trial protocol was approved by the Faculty of Medicine, University of Indonesia Ethics Committee. Participation in the study was voluntary, and written informed consent was obtained before the start of the study from the parents or guardian of children for their child’s participation in the study. The study period was from August 2009 to March 2010 in Larangan, Ciledug districts, Tangerang, Banten Province, Indonesia. The study was a doubleblind, randomized, placebo-controlled pre-post intervention trial. Material Transfer Agreement was obtained from Ministry of Health for analyses of blood samples in Japan.
Subjects were randomly assigned consecutively into four groups: placebo, probiotic, zinc, and combination of probiotic and zinc. At enrollment, children were randomized using sealed
envelopes containing a note assigning the subject to one of the four study arms. There were 36 children (19 boys) in the study groups (supplemented), and 12 (7 boys) in the control group.
The probiotic L. plantarum IS-10506 was supplemented at a dose of 2.3 × 10 10 CFUs per day; zinc was supplemented at a dose of 20 mg/day as zinc sulfate. Supplementation was conducted for 90 days. Age, sex, weight, adverse events were recorded for each child, and a physical examination was conducted by a physician every one month during the intervention study. Two children in the zinc group and one child in the combination probiotic and zinc group were dropped from the study because they moved out of town and the blood sample was not sufficient. Stool and blood sample collection Peripheral blood samples were withdrawn from subjects by venipuncture and stool samples were collected at baseline and at the end of the study period. Fecal secretory IgA (sIgA) was assessed by ELISA kit at the Institute of Human Virology and Cancer Biology (IHVCB) laboratory, Faculty of Medicine, University of Indonesia,
and serum zinc was assessed by ICP-MS in the Trace Element Laboratory, Department of Public Health, Gunma University Graduate School of Medicine, Japan.

Analysis of fecal sIgA
Fresh samples of stool were collected in the morning and transported immediately to the laboratory and stored at −80◦ C. Measurements of sIgA were carried out using ELISA kits (K8870 and K6500, Immundiagnostik, Bensheim, Germany) according to the manufacturer’s instructions. Stool samples weighing 80–120 mg were diluted with appropriate amounts of buffer provided in the kit to give constant dilutions. The stool were suspended in diluent buffer, vortexed, and centrifuged at 13,000 rpm (=13,000 × g) for 5 min in 1.5 mL tubes. Supernatants were diluted 1:250 in wash buffer. Standards, controls and stool samples were simultaneously transferred to microplates coated with antibodies specific for sIgA. Anti-sIgA antibody conjugated with peroxidase was used for development. For each well, the optical density was measured at 450 nm on a microplate ELISA reader (Dynex, Heidleberg, Germany). The results of the test samples were calculated from the standard curve and expressed as concentration ( g/g) by wet weight of stool. Serum blood preparation and zinc analysis Venous blood samples were collected from children and centrifuged at 3000 rpm for 10 min to obtain serum and kept at −80◦C until analysis. Each serum sample (0.1 mL) was digested with 1 mL of 60% ultrapure nitric acid and 0.1 mL of 30% ultrapure hydrogen peroxide in a perfluoroalkoxy polymer vessel using the following procedure: heating at 90◦C for 0.5 h, heating at 120◦ C for 2.5 h, and finally heating at 150◦ C for 2 h on a hot plate. After cooling, the digested solutions of serum were diluted up to 5 mL with Milli-Q water in polypropylene sample tubes. Blank and certified reference materials (CRMs) were subjected to the same treatment as the samples. To minimize contamination from devices, all containers were soaked in an acid bath (a solution of 6% (V/V) of nitric acid) for a minimum of 24 h, followed by final rinses with Milli-Q water. Serum zinc concentrations were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) with an ELAN DRC II(Perkin Elmer, Waltham, MA, USA). The accuracy of the analysis was checked with CRMs: Seronorm Trace Element Serum Level 2 (No.203105, Billingstad, Norway).

Hasil (Bahasa Indonesia) 1:

PendahuluanSuplementasi diet adalah cara yang menarik, non-invasif untuk meningkatkan dan mengoptimalkan fungsi fisiologis yang penting, termasuk sistem kekebalan tubuh [1]. Kemampuan suplementasi Diet untuk mengoptimalkan fungsi kekebalan tubuh dipandang sebagai terutama penting di antara kelompok-kelompok individu yang mungkin memiliki memberatkan atau buruknya fungsi sistem kekebalan tubuh, seperti bayi, anak-anak muda, kekebalan subjek dan orang tua [2,3]. Kemajuan dalam penelitian telah sangat meningkatkan pemahaman tentang pentingnya mikrobiota usus. Kolonisasi bakteri usus dalam sistem usus-microbiome sangat penting untuk perkembangan normal banyak aspek fisiologi. Pemahaman lebih lanjut BINUS University, Alam Sutra Campus, Tangerang, Indonesia. mekanisme yang mendasari usus-microbiome akan memberikan wawasan baru ke dalam hubungan simbiotik antara mikrobiota usus dan host mamalia mereka, dan membantu untuk mengidentifikasi potensi mikroba berbasis strategi pencegahan untuk membantu dalam promosi kesehatan. Probiotik didefinisikan sebagai mikroorganisme hidup yang bila diberikan dalam jumlah yang memadai, memberikan manfaat kesehatan untuk kesehatan manusia [4]. Lactobacillus plantarum IS-10506 adalah sebuah novelprobiotik strain terisolasi dari yoghurt-seperti produk, Dadiah, susu kerbau tradisional Indonesia asal Sumatera Barat [5]. Properti in vitro probiotik telah terbukti, seperti asam dan toleransi empedu, sifat adhesi dan daya saing terhadap patogen [6,7]; di vivo [8,9] dan penelitian pada manusia telah dilakukan dalam serial studi [10-12]. Fungsi utama dari saluran pencernaan yang pencernaan dan penyerapan nutrisi dan homeostasis elektrolit dan air; oleh karena itu, integritas perbatasan sikat usus adalah elemen kunci dalam mencegah penyerapan sistemik enterik racun dan bakteri. Jaringan limfoid pencernaan memainkan peran penting dalam mengontrol transepithelial bagian dari bakteri di mukosa usus oleh mensintesis molekul antibodi lebih daripada setiap jaringan limfoid lainnya. Antibodi pencernaan yang dihasilkan dalam bentuk antibodi dan merupakan pemain kunci dalam efisien kekebalan mukosa humoral, khususnya pemeliharaan integritas penghalang epitel.Zinc merupakan trace elemen penting dari luar biasa pentingnya biologis dan kesehatan masyarakat. Seng adalah komponen penting dari sejumlah besar enzim, dan memainkan peran penting dalam pertumbuhan sel dan diferensiasi jaringan dengan pergantian cepat, termasuk sistem kekebalan tubuh dan sistem pencernaan [13,14]. Defisiensi Zinc melibatkan fungsi kekebalan tubuh terganggu, sub normal pertumbuhan dan perkembangan, dan anak-anak menyebabkan kelambatan pertumbuhan, tertunda pematangan seksual, dan infeksi kerentanan. Uji intervensi telah menunjukkan bahwa suplementasi dengan seng dapat juga meningkatkan respons imun [15]. Tidak ada bukti bahwa sengpenyebab membahayakan ketika diberikan kepada non-septic, anak-anak immunocompetent [16].Bahan dan metodePersiapan Diet maltodekstrin diuji bubuk terkandung sebagai plasebo dan probiotik L. plantarum IS-10506 asal Dadiah diidentifikasi oleh 16S sequencing gen rRNA sebagai L. plantarum (gen Bank aksesi no. DQ860148). Untuk tujuan eksperimental, bakteri microencapsulated digabungkan dengan dosis 2.3 × 10 11 CFUs g bubuk, dan telah terbukti menunjukkan baik kerugian yang signifikan viabilitas maupun kontaminasi karena penyimpanan selama masa studi seluruh.Seng dilengkapi sebagai bedak sulfat seng. Plasebo, probiotik, atau seng diberikan dalam bentuk microencapsulated dan sama dalam penampilan.Mata pelajaran dan kriteria percobaanEmpat puluh delapan rupanya sehat anak-anak dalam rentang usia 12-24 bulan (median 16.8 bulan) yang dipilih untuk studi ini. Sebelum dimulainya sidang, kriteria seleksi yang dihasilkan dari catatan penyedia layanan kesehatan yang berpartisipasi. Kriteria inklusi yang tampaknya sehat anak-anak, berusia antara 12 dan 24 bulan dan perjanjian sesuai dengan pedoman percobaan atau memberikan pemberitahuan non-kepatuhan. Kriteria pengecualian adalah kelainan bawaan atau penyakit, penyakit pencernaan, penggunaan rutin produk dengan bakteri probiotik, menerima terapi antibiotik dalam waktu dua minggu sebelum studi intervensi dan perjanjian non-untuk menghindari berpotensi bertentangan gizi atau elemen utama suplemen selama 90 hari pengadilan. Kepatuhan suplementasi dikukuhkan oleh subyek oleh laporan langsung kepada penyedia perawatan kesehatan. Protokol-protokol percobaan telah disetujui oleh Fakultas Kedokteran, Universitas Indonesia Komite etika. Partisipasi dalam studi adalah sukarela, dan persetujuan tertulis diperoleh sebelum memulai studi dari orang tua atau wali dari anak-anak untuk anak-anak mereka partisipasi dalam studi. Masa studi adalah dari Agustus 2009 sampai Maret 2010 di Larangan, Ciledug Kabupaten, Tangerang, Propinsi Banten, Indonesia. Studi ini adalah doubleblind, acak, plasebo-terkontrol intervensi pra-posting sidang. Perjanjian Transfer material Diperoleh dari Departemen Kesehatan untuk analisis sampel darah di Jepang.Subyek secara acak berturut-turut menjadi empat kelompok: plasebo, probiotik, seng, dan kombinasi probiotik dan seng. Di pendaftaran, anak-anak secara acak menggunakan disegelamplop berisi catatan menugaskan subjek untuk salah satu studi empat lengan. Ada 36 anak (19 anak laki-laki) dalam kelompok belajar (ditambah), dan 12 (7 anak laki-laki) dalam kelompok kontrol.Probiotik L. plantarum IS-10506 dilengkapi dengan dosis 2.3 × 10 10 CFUs setiap hari; Seng dilengkapi dengan dosis 20 mg/hari sebagai sulfat seng. Suplementasi dilaksanakan selama 90 hari. Umur, jenis kelamin, berat badan, efek samping direkam untuk setiap anak, dan pemeriksaan fisik dilakukan oleh seorang dokter setiap satu bulan selama studi intervensi. Dua anak dalam kelompok Seng dan satu anak dalam kelompok probiotik dan seng kombinasi dijatuhkan dari studi karena mereka pindah ke luar kota dan sampel darah tidak cukup. Bangku dan darah sampel koleksi sampel darah perifer yang ditarik dari subyek oleh venapungsi dan tinja sampel dikumpulkan pada awal dan pada akhir masa studi. IgA air tinja (sIgA) dinilai oleh ELISA kit di Institut manusia virologi dan kanker biologi (IHVCB) laboratorium, Fakultas Kedokteran, Universitas Indonesia,dan seng serum dinilai oleh ICP-MS di laboratorium Trace Element, Departemen Kesehatan masyarakat, Gunma Universitas Graduate School of Medicine, Jepang.Analisis sIgA tinjaFresh samples of stool were collected in the morning and transported immediately to the laboratory and stored at −80◦ C. Measurements of sIgA were carried out using ELISA kits (K8870 and K6500, Immundiagnostik, Bensheim, Germany) according to the manufacturer’s instructions. Stool samples weighing 80–120 mg were diluted with appropriate amounts of buffer provided in the kit to give constant dilutions. The stool were suspended in diluent buffer, vortexed, and centrifuged at 13,000 rpm (=13,000 × g) for 5 min in 1.5 mL tubes. Supernatants were diluted 1:250 in wash buffer. Standards, controls and stool samples were simultaneously transferred to microplates coated with antibodies specific for sIgA. Anti-sIgA antibody conjugated with peroxidase was used for development. For each well, the optical density was measured at 450 nm on a microplate ELISA reader (Dynex, Heidleberg, Germany). The results of the test samples were calculated from the standard curve and expressed as concentration ( g/g) by wet weight of stool. Serum blood preparation and zinc analysis Venous blood samples were collected from children and centrifuged at 3000 rpm for 10 min to obtain serum and kept at −80◦C until analysis. Each serum sample (0.1 mL) was digested with 1 mL of 60% ultrapure nitric acid and 0.1 mL of 30% ultrapure hydrogen peroxide in a perfluoroalkoxy polymer vessel using the following procedure: heating at 90◦C for 0.5 h, heating at 120◦ C for 2.5 h, and finally heating at 150◦ C for 2 h on a hot plate. After cooling, the digested solutions of serum were diluted up to 5 mL with Milli-Q water in polypropylene sample tubes. Blank and certified reference materials (CRMs) were subjected to the same treatment as the samples. To minimize contamination from devices, all containers were soaked in an acid bath (a solution of 6% (V/V) of nitric acid) for a minimum of 24 h, followed by final rinses with Milli-Q water. Serum zinc concentrations were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) with an ELAN DRC II(Perkin Elmer, Waltham, MA, USA). The accuracy of the analysis was checked with CRMs: Seronorm Trace Element Serum Level 2 (No.203105, Billingstad, Norway).