Preparative Reversed-phase High-performance Liquid Research Articles

Unlocking the Potential of Isopropanol as an Eco-Friendly Eluent for Large-Scale Fractionation of Fulvic Acids via Preparative Reversed-Phase High-Performance Liquid Chromatography and Multidimensional RP-HPLC: Evaluation of Molecular Diversity and Element Composition.

Humic substances are organic mixtures of extreme complexity, which significantly complicate their analysis by any method. Fractionation into more homogeneous mixtures seems to be almost the only way to overcome these difficulties. Preparative high-performance liquid chromatography (HPLC) provides almost any amounts of substances required for both further fractionation and studies by other methods. For the first time, isopropyl alcohol (IPA) is proposed for these purposes; its advantages are shown by the example of groundwater fulvic acids (FA). IPA is much safer than conventional solvents and elutes the most nonpolar compounds that are nonelutable by methanol and acetonitrile. The isolated fractions differ significantly in their molecular composition, which is confirmed by ultrahigh-resolution mass spectrometry and molecular spectroscopy. Stepwise IPA-gradient HPLC-UV analysis of each preparative fraction demonstrates the possibilities of multidimensional HPLC for FA. The isolated fractions were studied for contents of a broad range of elements, and the relationships between the molecular and trace-element compositions of the fractions were revealed. Our data reveals the existence of numerous and different organometal compounds in FA composition; thus, the proposed approach can be used for a more in-depth study of the mechanisms of element migration in the environment.

Cytotoxic Flavonoids from Lannea egregia Engl. & K. Krause

Background: Lannea egregia Engl. & K. Krause (family: Anacardiaceae) is a well-known medicinal plant in Nigeria whose various parts have been shown to elicit several biological activities. This study specifically explored the leaf of L. egregia for potential cytotoxic compounds. Methods: n-Hexane, dichloromethane and methanolic extracts of the leaf were prepared using the Soxhlet apparatus and concentrated using the rotary evaporator. Compounds were isolated by reversed-phase preparative high-performance liquid chromatography, and the structures were determined by spectroscopic means. The methanolic extract and the isolated compounds were screened for cytotoxicity against HeLa and MCF-7 cancer cell lines, using the MTT assay. Results: Three flavonoids, myricetin (1), myricetin 3-O-α-L-rhamnoside (2) and quercetin 3-O-α-L-rhamnoside (3), were isolated from the methanolic extract of the leaf of L. egregia. The methanolic extract and compound 3 showed the most potent inhibition profiles against the cells, with IC50 values (Mean ± SEM) of 45.3 ± 1.5 µg/mL and 57.5 ± 0.4 µg/mL for the methanolic extract, and 36.5 ± 2.0 µM and 57.9 ± 10.1 µM for compound 3, against HeLa and MCF-7 cells, respectively. Conclusion: Conclusion: This work shows that L. egregia leaf is moderately cytotoxic, and rich in flavonoids, and the cytotoxicity of the extract is, at least partially, due to the presence of cytotoxic flavonoids. This is the first report on characterized isolated compounds from the leaf of L. egregia and the occurrence of flavonols in the leaf.

Modulation of Brain-Derived Neurotrophic Factor (BDNF) Signaling Pathway by Culinary Sage (Salvia officinalis L.).

Culinary sage (Salvia officinalis L.) is a common spice plant in the mint family (Lamiaceae) well known for its distinctive culinary and traditional medicinal uses. Sage tea has been used traditionally as a brain-enhancing tonic and extracts from sage have been reported to have both cognitive and memory enhancing effects. Brain-derived neurotrophic factor (BDNF) is an endogenous signaling molecule involved in cognition and memory function. In this study, activity-guided fractionation employing preparative reverse-phase high performance liquid chromatography (RP-HPLC) of culinary sage extracts led to the discovery of benzyl 6-O-β-D-apiofuranosyl-β-D-glucoside (B6AG) as a natural product that upregulates transcription of neurotrophic factors in C6 glioma cells. Purified B6AG showed a moderate dose response, with upregulation of BDNF and with EC50 at 6.46 μM. To better understand the natural variation in culinary sage, B6AG was quantitated in the leaves of several commercial varieties by liquid chromatography-mass spectrometry (LC-MS). The level of B6AG in dried culinary sage was found to range from 334 ± 14 to 698 ± 65 μg/g. This study provided a foundation for future investigations, including quantitative inquiries on the distribution of B6AG within the different plant organs, explorations in optimizing post-harvest practices, and aid in the development of sage varieties with elevated levels of B6AG.

Characterization and Relative Quantitation of Wheat, Rye, and Barley Gluten Protein Types by Liquid Chromatography-Tandem Mass Spectrometry.

The consumption of wheat, rye, and barley may cause adverse reactions to wheat such as celiac disease, non-celiac gluten/wheat sensitivity, or wheat allergy. The storage proteins (gluten) are known as major triggers, but also other functional protein groups such as α-amylase/trypsin-inhibitors or enzymes are possibly harmful for people suffering of adverse reactions to wheat. Gluten is widely used as a collective term for the complex protein mixture of wheat, rye or barley and can be subdivided into the following gluten protein types (GPTs): α-gliadins, γ-gliadins, ω5-gliadins, ω1,2-gliadins, high- and low-molecular-weight glutenin subunits of wheat, ω-secalins, high-molecular-weight secalins, γ-75k-secalins and γ-40k-secalins of rye, and C-hordeins, γ-hordeins, B-hordeins, and D-hordeins of barley. GPTs isolated from the flours are useful as reference materials for clinical studies, diagnostics or in food analyses and to elucidate disease mechanisms. A combined strategy of protein separation according to solubility followed by preparative reversed-phase high-performance liquid chromatography was employed to purify the GPTs according to hydrophobicity. Due to the heterogeneity of gluten proteins and their partly polymeric nature, it is a challenge to obtain highly purified GPTs with only one protein group. Therefore, it is essential to characterize and identify the proteins and their proportions in each GPT. In this study, the complexity of gluten from wheat, rye, and barley was demonstrated by identification of the individual proteins employing an undirected proteomics strategy involving liquid chromatography–tandem mass spectrometry of tryptic and chymotryptic hydrolysates of the GPTs. Different protein groups were obtained and the relative composition of the GPTs was revealed. Multiple reaction monitoring liquid chromatography–tandem mass spectrometry was used for the relative quantitation of the most abundant gluten proteins. These analyses also allowed the identification of known wheat allergens and celiac disease-active peptides. Combined with functional assays, these findings may shed light on the mechanisms of gluten/wheat-related disorders and may be useful to characterize reference materials for analytical or diagnostic assays more precisely.

Structure Elucidation of a New Lignan Glycoside from Artemisia ordosica

The EtOAc extract of Artemisia ordosica was separated by preparative reversed-phase high performance liquid chromatography and afforded a new lignan glycoside, artemordolignan glycoside A (1). The structure elucidation of the new compound was carried out by 1D (1H and 13C NMR) and 2D NMR (COSY, HSQC, and NOESY) spectral analysis.

Expression, purification, and characterization of the Degludec precursor DesB30

Diabetes is a chronic metabolic disease, for which recombinant human insulin is the most effective and mainstream treatment. DesB30 is an insulin analogue in which the B chain lacks amino acid 30 (Thr) compared with human insulin. This analogue can be used to produce the long-acting insulin Degludec or Detemir. In this study, a spacer peptide was added before the sequence of DesB30 and the C-peptide was replaced with AAK, a short connecting peptide. The target gene was cloned under the control of AOX1 and expressed as a secretory protein in Pichia pastoris. A high-yield recombination strain was selected by screening for resistance to G418. The basal salts medium was optimized and a lower salt concentration medium was found to show the best effects. Both media were used to compare the yield of high-density fermentation. The maximum yield reached 4.51 g/L in 1/2 basal salt medium, which is the highest reported yield to date. The insulin precursor, which is single-stranded, was purified by weak cation exchange chromatograph and preparative reversed-phase high-performance liquid chromatography (RP-HPLC), from which 73.39% of product was recovered at over 95% purity. The double-stranded protein (DesB30) was obtained by digesting the insulin precursor with trypsin. Using preparative RP-HPLC, the product was obtained with over 95% purity. Finally, the structure of DesB30 was confirmed.

Isolation and identification of a novel algicidal peptide from mackerel muscle hydrolysate

To help remedy damage from harmful algal blooms, an attempt was made to isolate an algicidal substance previously observed to be present in mackerel muscle hydrolysate. Crude extract was obtained by cold acetone precipitation, and it dissolved best in water. Through molecular weight cut-off determination and tricine-SDS PAGE, the algicidal substance was determined to be a peptide of <1 kDa. Based on this result, purification was first performed using size exclusion chromatography and preparative reverse phase high-performance liquid chromatography. Then, the active algicidal fraction was applied to an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry system, followed by MS/MS analysis. The algicidal peptide had linear structure consisting of amino acids with sequence NH-KMNF-COOH. Its calculated properties were: molecular weight 538.66 g/mol; isoelectric point 9.91; net charge +1 at pH 7.0; and 50% hydrophobicity. Algicidal ability of the identified peptide was confirmed using synthesized peptide. The LC50 values toward four harmful algal blooming species were 0.69, 0.83, 0.85 and 1.24 mg/ml for Alexandrium fundyense, A. catenella, Heterocapsa triquetra, and Prorocentrum minimum, respectively. There was no coincidence in the sequence of the identified peptide with those of known metabolites in the APD, Norine, CAMP, UniProt and METLIN databases. Consequently, this algicidal substance originating from mackerel protein was deduced to be a novel peptide that can usefully be applied to relieve harmful algal blooms.

Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying unidentified terpene synthase genes from edible plants of the family Araliaceae.

A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2 (ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium), and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition, silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic resonance, [α]D, and gas chromatography-mass spectrometry analyses. Herein, the products of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata "Udo" and a Tps gene (named AeTps1) in Aralia elata "Taranoki" were studied resulting in identifying functionalities of these cryptic Tps genes.

A modern purification method for volatile sesquiterpenes produced by recombinant Escherichia coli carrying terpene synthase genes.

Most volatile sesquiterpenes had been purified from plants using distillation and preparative gas chromatography, which is not applicable to many laboratories that do not possess a needed facility. Thus, this review focuses on a modern purification method for volatile sesquiterpenes using Escherichia coli cells that functionally express terpene synthase (Tps) genes. It was recently developed that recombinant E. coli cells carrying Tps genes were cultured in two-layer media (n-octane/TB medium) without harming the cells, and the volatile hydrophobic compounds trapped in the n-octane were purified by two-phase partition (alkane/alkaline 50% MeOH), silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography (if necessary). Consequently, it was found that the volatile sesquiterpenes are easily purified, the structures of which can then be determined by nuclear magnetic resonance, [α]D and gas chromatography-mass spectrometry analyses. The antioxidant activities of several volatile sesquiterpenes are also presented in this review.

Polyphenols from Euphorbia pekinensis Inhibit AGEs Formation In Vitro and Vessel Dilation in Larval Zebrafish In Vivo.

To identify active compounds in the roots of Euphorbia pekinensis for treatment of diabetic complications, an active column fraction from a 70% EtOH extract of E. pekinensis root was purified by preparative reversed-phase high-performance liquid chromatography, leading to the isolation of a new ellagic acid derivative, 3,3'-di-O-methylellagic acid 4-O-(6"-O-galloyl)-β-D-galactopyranoside (1: ), along with three known compounds, geraniin (2: ), 3,3'-di-O-methylellagic acid 4-O-β-D-xylopyranoside (3: ), and ellagic acid 3,3'-dimethyl ether (4: ). The structure of the new compound was established by extensive spectroscopic studies and chemical evidence. The inhibitory effects of isolated compounds 1: -4: on advanced glycation end-products (AGEs) formation were examined. All compounds exhibited considerable inhibition of AGEs formation and IC50 values of 0.41 - 12.33 µM, compared with those of the positive controls aminoguanidine (IC50 = 1122.34 µM) and quercetin (IC50 = 27.80 µM). In addition, the effects of 2: and 4: on the dilation of hyaloid-retinal vessels induced by high glucose (HG) in larval zebrafish were investigated; both compounds significantly reduced the HG-induced dilation of hyaloid-retinal vessels relative to the HG-treated control group.

Rapid Identification of Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides from Ruditapes philippinarum Hydrolysate.

Dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides were rapidly identified from Ruditapes philippinarum hydrolysate. The hydrolysate was fractionated by ethanol precipitation and preparative reverse phase high-performance liquid chromatography (RP-HPLC). The fraction which showed the highest DPP-IV inhibitory activity was then analyzed by a high-throughput nano-liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC ESI-MS/MS) method, and the sequences of peptides were identified based on the MS/MS spectra against the Mollusca protein data from the UniProt database. In total, 50 peptides were identified. Furthermore, molecular docking was used to identify potential DPP-IV inhibitors from the identified peptides. Docking results suggested that four peptides: FAGDDAPR, LAPSTM, FAGDDAPRA, and FLMESH, could bind pockets of DPP-IV through hydrogen bonds, π-π bonds, and charge interactions. The four peptides were chemically synthesized and tested for DPP-IV inhibitory activity. The results showed that they possessed DPP-IV inhibitory activity with IC50 values of 168.72 μM, 140.82 μM, 393.30 μM, and >500 μM, respectively. These results indicate that R. philippinarum-derived peptides may have potential as functional food ingredients for the prevention of diabetes.

Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria.

Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.

Purification and Identification of a Novel Angiotensin I-Converting Enzyme Inhibitory Peptide from Sesame Meal

An angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated and identified from sesame meal by successive chromatography. Sesame meal hydrolysates were obtained by treatment with alcalase, papain, neutrase and pepsin. The ACE inhibitory activities of each hydrolysate were investigated and the alcalase produced hydrolysate with the highest ACE inhibitory activity. The protein hydrolysate generated with alcalase was then separated by an ultrafiltration membrane with 10 kDa molecular weight cut-off membranes, Sephadex G-50 and semi preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Biological functions of all fractions were assayed, and Y4, a fraction from semi preparative RP-HPLC, was found to display the highest ACE inhibitory activity. The molecular weight of Y4 was 873.3 Da and the amino acid sequence was Tyr-Ala-His-Tyr-Ser-Tyr-Ala determined by TOF–MS/MS. The IC50 value for ACE inhibitory activity of fraction Y4 was 0.6 mg/mL. These results suggested that the sesame meal hydrolysates may be a beneficial ingredient for nutraceuticals and pharmaceuticals against hypertension and its related diseases.

Chemical Constituents of the Culture Broth of Phellinus linteus and Their Antioxidant Activity

The medicinal fungus Phellinus linteus, in the family Hymenochaetaceae, has been used as a traditional medicine for the treatment of various diseases. In this study, the chemical constituents of the culture broth of P. linteus were investigated. P. linteus was cultured in potato dextrose broth medium, and the culture broth was extracted with ethyl acetate. The ethyl acetatesoluble portion was concentrated and subjected to ODS column chromatography, followed by Sephadex LH-20 column chromatography. Six compounds (1~6) were purified by preparative reversed-phase high-performance liquid chromatography. Spectroscopic methods identified their structures as caffeic acid (1), inotilone (2), 4-(3,4-dihydroxyphenyl)-3-buten-2-one (3), phellilane H (4), (2E,4E)-(+)-4'-hydroxy-γ-ionylideneacetic acid (5), and (2E,4E)-γ-ionylideneacetic acid (6). Compounds 1, 2, and 3 exhibited potent dose-dependent antioxidant activity.

Comparative studies on performance of CCC and preparative RP-HPLC in separation and purification of steroid saponins from Dioscorea zingiberensis C.H.Wright.

Steroid saponins from Dioscorea zingiberensis C.H.Wright were separated for the first time using two chromatographic methods for comparison: counter-current chromatography (CCC) coupled with evaporative light scattering detector (ELSD) and preparative reversed phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet detector. Ethyl acetate-n-butanol-methanol-water (4:1:2:4, v/v) was chosen as the two-phase solvent system for CCC, while the acetonitrile-water (25:75 for the first step and15:85 for the second step, v/v) was used as the mobile phase in the preparative RP-HPLC. The following five steroid saponins were purified by theses two chromatographic methods, in one-step operation by CCC and by two-step operation in preparative RP-HPLC: 1) 26-O-β-D- glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (compound A), 2) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 4) 26-triol-3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (compound B), 3) 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β, 22ζ, 26-triol-3-O-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside (compound C), 4) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-{α-L-rhamnopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)]}-β-D-glucopyranoside (compound D) and 5) 26-O-β-D-glucopyranosyl-(25R)-furost-5, 20(22)-diene-3β, 26-diol-3-O-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosy-(1→2)]-β-D-glucopyranoside (compound E). The purities of these five steroid saponins separated by both methods were over 95%, and structural identification of these compounds was performed by ESI-MS, and 13C NMR. Comparison of these two established approaches revealed that CCC required a longer separation time but with less solvent consumption, whereas preparative RP-HPLC gave a shorter separation time but with higher solvent consumption. These results demonstrated that either of these two methods of different separation mechanism is feasible, economical and efficient for rapid preparative isolation and purification of steroid saponins from Dioscorea zingiberensis C.H.Wright.

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid–liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by 1H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described.

Identification of 4″-isovaleryl-spiramycin III produced by Bacillus sp. fmbJ

The production of secondary metabolites with antibiotic properties is a common characteristic to Bacillus spp. These metabolites not only have diverse chemical structures but also have a wide range of bioactivities with medicinal and agricultural interests such as antibiotic. Bacillus sp. fmbJ has been found to produce lipopeptides fengycin and surfactin in accordance with our previous report. In this study, another antimicrobial substance was separated and purified from the culture supernatant of strain fmbJ using the silica gel column chromatography and preparative reversed-phase high-performance liquid chromatography. By means of electrospray ionization mass spectroscopy, infrared spectroscopy, and nuclear magnetic resonance, the antagonistic compound was determined to be 4″-isovaleryl-spiramycin III with the molecular weight of 982 Da. This report is the first to introduce the finding of spiramycin produced from Bacillus sp. The study provides a novel source for the production of spiramycin in pharmaceutical industries.

Isolation of gallic acid and methyl gallate from folium Toonea Sinensis and validated method for their quantitation using LC-based technologies

Summary Silica-gel column chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC) were sequentially employed for the isolation of two antioxidants including gallic acid and methyl gallate from Folium Toonea Sinensis. An RP-HPLC-UV method was then developed and validated to rapidly determine their content in this herb with ethyl gallate as internal standard. The quantitation was performed on an XBridge Shield RP18 column (150 mm × 4.6 mm, 5 μm) under 40°C. The mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution was driven at 1.0 mL min−1 under gradient elution, and 270 nm was selected to monitor the separation. To evaluate the fitness for purpose of the method and to investigate the difference in the content of analytes among different samples, the leaves collected from five production sites were analyzed. The newly established method is suitable for routine analysis of gallic acid and methyl gallate in the herb and, hence, can assist in its quality assessment. It was also found that not only the content of two antioxidants but also the ratio varied significantly among different geographical origins. In addition, three samples from Yantai, Zumadian, and Zhenjiang were distinguished as they have a much higher content ratio than the other two.

Phytochemistry and bioactivity of Pedicularis sibthorpii growing in Iran

The methanol extract of the aerial parts of the medicinal plant Pedicularis sibthorpii Boiss., Scrophulariaceae, growing in the Azerbaijan province of Iran, was found to be active in the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and the antibacterial agar well diffusion assays, but no general toxicity was observed in the brine shrimp lethality assay. A combination of solid-phase extraction (SPE) and preparative reversed-phase high-performance liquid chromatography (prep-RP-HPLC) analyses of the methanolic extract afforded three phenylethanoids (verbascoside, martynoside and isomartynoside), an iridoid (aucubin), a flavonoid (luteolin 7-O-β-D-glucopyranoside) and mannitol, and the structures of these compounds were elucidated unambiguously by spectroscopic means. The distribution of the isolated compounds within the genus Pedicularis has also been discussed.

A novel bioactive peptide from yoghurts modulates expression of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine β-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of β-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide β-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.