Precipitation Of Casein Research Articles

Inhibition of dipeptidyl peptidase-IV by hydrolysates of beta-lactoglobulin isolated from Gir cow milk

The current study attempts to assess the dipeptidyl peptidase – IV (DPP-IV) inhibitory potential of β-lactoglobulin (β-Lg) isolated from milk of Gir cow. Whey was separated from skim milk by precipitation of casein followed by ultrafiltration (UF) to remove lactose and salts as permeate. β-Lg was separated from the UF retentate using salting out technique. Its purity was confirmed by SDS-PAGE and RP-HPLC. Hydrolysates of β-Lg were prepared using three different enzymes viz., pepsin, trypsin and Flavourzyme at different enzyme to substrate (E:S) ratios (1:25, 1:50, 1:100) and treated for 2-12 h durations. Pepsin-treated hydrolysate showed maximum DPP-IV inhibition of 77.62 ± 0.98 % (IC50 =3.78 mg/mL). Hydrolysate was passed serially through UF filters of 10 and 3 kDa. The permeate of 3 kDa, which exhibited 52.10 ± 0.72 % DPP-IV inhibition was fractionated through RP-HPLC and time based fractions were collected. All 41 fractions were again analysed for DPP-IV inhibition activity and 3 fractions which showed maximum DPP-IV inhibition activity were chosen for LC-MS/MS analysis. One novel peptide was identified through LC-MS/MS, which showed adequate DPP-IV inhibition (IC50 = 6.64 mmolL-1).

Valorization of cheese-making residues in biorefineries using different combinations of dark fermentation, hydrothermal carbonization and anaerobic digestion

Dark fermentation (DF), hydrothermal carbonization (HTC) and anaerobic digestion (AD) are applied, in different combinations, to cheese whey (CW), which is the liquid effluent from the precipitation and removal of milk casein during the cheese-making process. The aim and novelty of this research is to investigate the production of various biofuels (H2-rich gas, hydrochar and biogas) in cascade, according to the waste biorefinery concept. The simplest case is the direct AD of CW. The second investigated possibility is the preliminary HTC of CW, producing hydrochar, followed by the AD of the process water from which hydrochar is separated by filtration. The third possibility is based on DF of CW, followed by the AD of the fermentate (F) from DF. The final possibility is based on DF of CW, followed by HTC of the F, and then AD of the process water. Accordingly, the physical and chemical properties of CW, F, resulting hydrochar and process water (PW), and biomethane potentials of CW, F, and process waters are studied to determine the energy and carbon balances of all variants. In brief, the first variant, direct AD of CW, is believed to be the most energy efficient method.

Neonatal Human Enteroids Take Up Human Milk Extracellular Vesicles That Survive Neonatal Human Digestion

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. HM contains thousands of bioactive components, including extracellular vesicles (HMEVs). HMEVs are lipid bilayer-encased nanoparticles released from mammary epithelial cells that carry biological cargo to the infant. Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aims to develop a neonatal enteroid model and EV isolation pipeline to study HMEV-intestinal epithelial cell (IEC) interactions with the ultimate goal of therapy development. Hypothesis: HMEVs survive in vivo neonatal digestion to be taken up by infant IECs. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from post-pyloric tubes. HMEVs were isolated from raw and pasteurized human milk and digesta (n=3 each) by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. HMEV sub-populations were examined by super-resolution microscopy. HMEV uptake was tested using apical-out neonatal human enteroids isolated from a 2-month-old patient undergoing surgery for ileal atresia and validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid uptake was assessed with CMTPX dye-labeled-HMEVs, dynamin inhibitor, and confocal microscopy. Results: Our HMEV isolation pipeline reliably isolates HMEVs exhibiting classic EV morphology and enriched in extracellular vesicle markers CD81 and CD9, but depleted of b-casein and lactalbumin from HM and digesta. NTA data indicates that only 17.8 ± 2.9% (p>0.0001) of input HMEVs survive to reach the neonatal intestine. Human mammary gland-derived protein BTN1A1 is present in digesta EVs. Neonatal apical-out human enteroids represent a less proliferative and more differentiated epithelium, e.g. down-regulated KI67 (0.05 ± 0.03-fold relative to basal-out, p<0.0001, n=4-6) and elevated CHGA (14.22 ± 9.30-fold relative to basal-out, p=0.0072, n=4-6) as measured by qRT-PCR and normalized to housekeeping gene RPLP0. Neonatal enteroids take up digested HMEVs largely via dynamin-mediated endocytosis, as measured by CMTPX dye intensity normalized to enteroid area using Fiji (2.05 x 105 ± 2.95 x 104 vs 5.38 x 104 ± 6.08 x 103, p<0.0001, n=62-115 enteroids, HMEV-treatment alone and HMEVs + dynamin inhibitor, respectively).Conclusion: Our data suggest that HMEVs survive to the neonatal intestine and can be absorbed by neonatal enteroids largely via dynamin-mediated endocytosis. These data are an important first step to leveraging HMEVs and their cargo to treat neonatal intestinal inflammation. Funding: NIH K01DK129401, NIH R01HD097367, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

A combination of Sephadexes for the milk whey protein fractions isolation

Whey proteins perform important biological functions. They can also be a source of numerous natural bioactive peptides that have a positive effect on various physiological systems of the body. For research and usage of this phenomenon, the question of these proteins isolating with saving of their structure and biological functions is relevant. Considering the significant difference in molecular weights, gel filtration is a promising method. The purpose of the study was the fractionation of milk whey proteins using gel filtration with a combination of different dextran gels. We used in the work milk whey proteins after isoelectric precipitation of casein and isolation of low molecular weight peptides by gel filtration on Sephadex G-25. The first gel filtration of the whey protein preparation was performed on a column with Sephadex G-150. For further fractionation were used three sectors from the first two chromatographic peaks obtained by gel filtration on Sephadex G-150. The first peak contains high molecular fractions of proteins eluting with a volume close to the free volume of the chromatographic column. The second asymmetric peak consists mainly of β-lactoglobulin and α-lactalbumin mixture. The third peak includes low molecular weight polypeptides and peptides that are not fixed in a polyacrylamide gel. The combined chromatographic fractions of three sectors from the first two chromatographic peaks were re-fractionated on a G-100 column. Electrophoretic analysis of chromatographic fractions after repeated chromatography of milk whey proteins confirmed the feasibility of choosing a certain sequence of sephadex. At the same time, the yield of β-lactoglobulin fraction increases by 12 ± 1 %, and α-lactalbumin by 9 ± 1 % compared to the results obtained by repeated gel filtration on one Sephadex (G-100). The degree of purification of β-lactoglobulin and α-lactalbumin, according to the densitometry, was 93 ± 5 % and 77 ± 4 %, respectively.

Isolation of casein for stable isotope ratio analysis of butter, cheese, and milk powder.

Stable isotope ratio analysis (SIRA) is commonly used for the authentication of dairy commodities, providing evidence to support the geographical origin and production background of products. We set out to optimise methods for the isolation of a common constituent (casein) from three dairy commodities, which would permit easier inter- and intra-commodity comparisons following SIRA. Three published methods for isolation of protein (from cheese, milk, and butter) were adapted to yield protein (casein) fractions from commercial cheddar cheese, whole milk powder (WMP), and butter samples with a high degree of purity for subsequent SIRA. The casein fractions isolated underwent elemental analysis (H, C, and N), protein determination, and some also underwent SIRA of O and S. Two-way analysis of variance and Tukey post hoc comparisons tested differences between methods. For each product, an optimised casein isolation method was chosen based on the C/N ratio and protein content. An optimum solvent lipid extraction (petroleum spirit-diethyl ether (2:1)) and casein precipitation method was chosen for cheddar cheese casein. A final solvent lipid extraction (heptane-isopropanol (3:2)) was necessary for WMP and butter casein extraction. δ13 C and δ2 H values validated the methods' abilities to remove contaminating lipid and isolate pure casein. Casein of high purity, for subsequent SIRA, can be isolated from cheddar cheese, WMP, and butter following modifications of previously published methods.

Evaluation of the Physical and Chemical Treatment of Wastewater for the Dairy Industry

Dairy wastewater generally contains fats, lactose, whey proteins, and nutrients. Casein precipitation causes the effluent to decompose into a dark, strong-smelling sludge. Fluid waste contains soluble organic matter, suspended solids, and gaseous organic matter, which cause undesirable taste and smell, grant tone and turbidity, and advance eutrophication, which plays an essential role in increasing biological oxygen demand (BOD) in water. It also contains detergents and disinfecting agents from the rinses and washing processes, which increase the need for chemical oxygen (COD). One of the characteristics of dairy effluents is their relatively high temperature, high organic contents, and wide pH range, so the discharge of wastewater into water bodies without treatment leads to deterioration of water quality and ecological imbalance, and therefore treatment is required. To remove or reduce environmental damage. Dairy wastewater treatment includes mechanical, physical, chemical, and biological methods. Organic treatment techniques are reasonable for treating wastewater from the dairy business because of their high biodegradability. Notwithstanding, the long-chain unsaturated fats framed during lipid hydrolysis show an inhibitory impact during anaerobic treatment. Chain block reactors (SBR) and top stream anaerobic slop cover of sludge (UASB) frameworks are the most encouraging advancements for the organic treatment of dairy wastewater. Many papers have applied high-impact exercise and technical methods to the dairy business's anaerobic wastewater treatment of dairy wastewater. However, the two techniques actually have a few disadvantages. The most vital objective of these studies is to track down savvy and naturally manageable ways to deal with and empower the reuse and management of wastewater and waste. Consequently, elective treatments to organic treatment are physical and substance techniques, for example, coagulation, retention, layer cycles, and electrolysis. This section gives a primary survey zeroing in on physical and compound treatment strategies for dairy wastewater treatment. It is under study and checked for its viability.

Preparing mare milk sample for proteomic analysis using two-dimensional electrophoresis

There is no universal method to prepare physiological fluids for 2-DE proteomic analysis. Furthermore, interspecies differences in milk composition require the formulation of a species-specific sample preparation procedure. The study was carried out on mare's milk which was prepared for 2-DE in four different methods: the first sample (M1) was defatted, sample M2 was defatted and after casein precipitation, sample M3 was sample M2 after reduction of high molecular proteins and sample M4 was desalted sample M3. The milk samples prepared in different methods were separated by 1-DE and 2-DE. The obtained gels were analysed qualitatively and quantitatively. Furthermore, selected protein spots were identified by MALDI-TOF MS. Analysis of 1-DE and 2-DE gel images indicated that the optimal procedure for preparing mare milk samples for 2-DE and identification of proteins by MS is a method based on defatting and precipitation of caseins. The preparation of mare's milk samples by defatting and then precipitation of caseins is enough to obtain an optimal 2-DE separation for identification of proteins of this body fluid. The method of caseins precipitation should be improved in order to reduce the proportion of these high-abundance protein in milk samples which could increase in the identification of low-abundance proteins in mare's milk.

Casein-assisted enhancement of the compressive strength of biocemented sand

As a soil biomineralization process, casein-assisted enzyme-induced carbonate precipitation (EICP) yielded biocemented specimens with significantly higher compressive strength than specimens cemented by regular or skim-milk-assisted EICP treatments. The compound concentration and curing strategy of casein-assisted EICP were experimentally optimized to maximize the compressive strength of precipitates with low calcium carbonate content. Under the optimized EICP conditions (0.893 M urea, 0.581 M CaCl2, 2.6 g/L urease enzyme, and 38.87 g/L casein), the unconfined compressive strengths reached 2 MPa. The scanning electron micrographs of selected samples provided microscopic evidence that EICP treatments assisted using skim milk and casein impart distinctive strength-enhancement mechanisms. The ammonium ions released from urea hydrolysis created an alkaline environment that makes casein dissociated into the pore water. As the casein-containing pore water became more viscous, the increased contact area with particles facilitated the precipitation of co-bound CaCO3 minerals and casein in the pore water. Casein was identified as a more efficient assisting agent than skim milk for low-level CaCO3 precipitation by EICP treatment.

Production of Milk Clotting Aspartic Protease from Bacterial Species Isolated from Dumping Site of Mehmood Booti, Lahore

Food and dairy industries play a very important role in the economy of every country. Aspartic proteases are important enzyme of dairy industry and is used in cheese making. Previously main sources of protease enzyme were plants, animal or fungi, but due to increased demand globally they are now mostly isolated from bacteria. Objectives: To isolate the milk clotting bacteria from the soil collected from dumping site of Mehmood Booti and produce aspartic protease from them. Methods: Soil sample was collected from Mahmood Booti dumping site near ring road, Lahore. After serial dilutions, sample was inoculated on nutrient agar plates. After 24 hours at 37°C temperature, opaque, round and cream-colored colonies were observed which were sub cultured in LB agar. From there colonies were grown on selective medium made of K2HPO4, (NH4)2 HPO4, casein, MgCl2, yeast extract and agar. After incubation, a colony with clear zone was selected and grown in LB broth for enzyme production. After incubation, broth was centrifuged and supernatant was isolated. While performing protease assay, 3 mL of 5% TCA was added in the mixture. Results: The mixture remained clear which depicted the hydrolysis of casein by protease. While the test tube containing water as blank showed precipitation of casein after the addition of TCA because in this enzyme was not present. Conclusions: This shows that the isolated bacteria had the ability to produce protease which was evident from the protease activity assay and that such bacteria are abundant in dumping site

Tryptophan front-face fluorescence and functional properties of whey: A preliminary study

This study investigated the potential of front-face fluorescence spectroscopy to predict the functional properties of whey. Whey has a commercial interest due to its excellent nutritional value and versatile functional properties. However, its attributes depend dramatically on heat treatment, which may alter its suitability for different food applications. Tryptophan front-face fluorescence of whey and its functional properties, i.e., foaming, gel-forming, and emulsifying properties, were evaluated after milk heat treatment (at 80 °C with seven holding times) to detect correlations between tryptophan fluorescence and functional parameters and generate predictive models. Whey samples were obtained by isoelectric precipitation of caseins from reconstituted skim milk powder enriched with whey protein isolate. As expected, heat treatments induced an increase in whey protein denaturation, as well as a decrease in total whey protein concentration and tryptophan fluorescence intensity. Gel-forming and emulsifying properties of whey significantly correlated with the maximum intensity of tryptophan (P < 0.001). Concerning foaming properties, only the foam stability index revealed a weak correlation with tryptophan maximum intensity (P < 0.05). Consequently, models presented strong significances (P < 0.001). Specifically, using only tryptophan fluorescence allowed successful prediction of emulsifying properties but was not enough for foaming and gel forming properties.

Kazein Bazlı Yapıştırıcıların Üretimi ve Fizikokimyasal Özellikleri

In this study, different adhesives manufactured by using various casein powders (micellar casein, αS-, β- and ĸ-casein fractions, sodium caseinate and calcium caseinate) were investigated for their properties and potential application in the food industry. Casein-based adhesives using different sources of caseins were produced and the differences in their adhesive strength were determined. For the isolation of casein fractions, the methods of selective solubility, precipitation and a decanter centrifuge (for separation of precipitated casein and supernatant) were used. Achieved purities of αS-, β- and κ-casein fractions were higher than 25, 91 and 54%, respectively. Results showed that the type of casein raw material used in the production of adhesives had an influence on the adhesive properties and the highest adhesive strength was achieved with the enriched αS-casein fraction and micellar casein.

El suero de leche, subproducto de la industria de queso: Composición, recuperación de proteínas y aplicaciones

Whey is the liquid by-product obtained after the precipitation of casein in the manufacture of cheese. The composition and type of whey vary considerably depending on the type of milk, the type of cheese made, and the treatment used. The main nutrient is lactose (4.5% w/v), then protein (0.8% w/v) and fat (0.5%). For the food industry, whey is a low-cost protein by-product that offers several properties in a wide variety of foods. Whey improves texture, flavor, and color, emulsifies, and stabilizes, improves flowability, and exhibits many other food properties. Based on the nutritional value of whey, many commercial applications have been found, such as the production of ethanol, organic acids, carbonated beverages, fermented beverages, biomass, protein concentrates, isolation and hydrolysis, edible films, infusions, xanthan production, enzymes, and lactose separation. Future research is envisioned around the application of cheese whey in the creation of animal feed, as well as the creation of mass consumption foods that replace junk food.

Определение казеина в молоке

In the present study the precipitation of casein from the various milk samples such as cow milk, goat milk were studied. The technique of precipitation of casein is used to predict the protein content in the milk samples. It was found that the main components of casein have genetic variants that differ in several amino acid residues. These proteins have a molecular weight of about 20 thousand, an isoelectric point of about 4.7, contain an increased amount of proline (the polypeptide chain has a b-structure) and are resistant to denaturants.

Dynamic modeling of milk acidification: an empirical approach

Acidification is an important process in the production of several dairy products and has an impact in taste, viscosity, and shelf life of the products. This paper deals with the development of an empirical mathematical model to describe milk acidification in terms of pH dynamics. The model is built upon experiments of pasteurized skimmed milk acidification for acid-induced casein precipitation and extended to predict the partitioning of calcium and phosphates in the whey and the dynamics of pH during fermentation of yoghurt manufacture. Furthermore, the model can be used directly or in optimization problems to provide recommendations about product design.

Effect of rainfall and soil fertility on total phenol and tannin contents in Cenostigma microphyllum (Mart. ex G. Don) E. Gagnon &amp; G.P. Lewis (Fabaceae)

The study examined the total phenol and tannin contents in stem bark and leaf tissues of the medicinal species Cenostigma microphyllum (Mart ex. G. Don) E. Gagnon & G. P. Lewis distributed under different rainfall amounts and soil fertility levels in the Caatinga, the largest nucleus of Seasonally Dry Tropical Forest and Woodlands in the Neotropics. Using colorimetric method (Folin–Ciocalteu to total phenol content and casein precipitation to total tannin content), it was shown that both phenol and tannin content was significantly higher in the leaves than in the stem bark. Soil fertility affected negatively only the total phenol content. No relationship was observed between rainfall and compounds, and between soil fertility and total tannin content. Thus, our findings indicate that although water-deficit stress is a limiting factor in arid and semi-arid regions, soil fertility is also an abiotic factor that affect the production of phenolic compounds. Moreover, the concentration of such metabolites changes according to the class of compound and plant tissue analyzed.

Przygotowanie prób siary i mleka klaczy do rozdziału 2-DE z pominięciem precypitacji acetonem

Before electrophoretic separation is performed, the samples must be dissolved in a lysis buffer (necessary to keep proteins dissolved and unbound during proteomic analyses during for a separation of proteins on polyacrylamide gels). The first step in preparing samples for proteomic analyses is their precipitation using e.g. acetone. The aim of precipitation is to obtain proteins from the sample and to remove the compounds interfering with 2-D electrophoresis. Due to difficulties in dissolving some colostrum and mare's milk samples in buffer lysis electrophoretic separation of this biological material was performed without acetone precipitation of proteins. To assess the effectiveness of the applied method, after two-dimensional separation of proteins (2-DE), the obtained gels were stained and archived. The preparation of mare's colostrum and milk samples for proteomic analyses, consisting of defatting, then precipitation of caseins and separation 2-DE, which was not preceded by precipitation of the samples with acetone, resulted in the loss of many protein spots which made it impossible to identify them later using the mass spectrometer.

Whey proteins inhibit food intake and tend to improve oxidative balance in obese zucker rats.

Whey proteins (WP), obtained from milk after casein precipitation, represent a heterogeneous group of proteins. WP are reported to inhibit food intake in diet-induced experimental obesity; WP have been proposed as adjuvant therapy in oxidative stress-correlated pathologies. This work evaluates the effects of WP in comparison with casein, as a source of alimentary proteins, on food intake, weight growth and some indexes of oxidative equilibrium in Zucker Rats, genetically prone to obesity. We monitored food intake and weight of Zucker Rats during the experiment, and some markers of oxidative equilibrium. WP induced significant decrease of food intake in comparison to casein (WP 80.41 ± 1.069ml/day; CAS: 88.95 ± 1.084ml/day; p < 0.0005). Body weight growth was slightly reduced, and the difference was just significant (WP 128.2 ± 6.56g/day; CAS 145.2 ± 3.29g/day; p = 0.049), while plasma HNE level was significantly lower in WP than in CAS (WP 41.2 ± 6.3 vs CAS 69.61 ± 4.69pmol/ml, p = 0.007). Mild amelioration of oxidative equilibrium was indicated by a slight increase of total glutathione both in the liver and in the blood and a significant decrease of plasma 4-hydroxynonenal in the group receiving WP. The effect of WP on food intake and weight growth in Zucker Rats is particularly noteworthy since the nature of their predisposition to obesity is genetic; the possible parallel amelioration of the oxidative balance may constitute a further advantage of WP since oxidative stress is believed to be interwoven to obesity, metabolic syndrome and their complications.

Phytochemical analysis and in vitro antioxidant and antimicrobial activities of hydroalcoholic extracts of the leaves of Salacia kraussii

Abstract The present work was aimed at identifying the bioactive compounds and determine the in vitro antioxidant and antimicrobial activities of the hydroethanolic extract of S. kraussii leaves. Phytochemical analysis was performed using gas chromatography-mass spectrometry (GC-MS) and classical colorimetric methods. Folin-Ciocalteu method, aluminum chloride precipitation, and casein precipitation were employed for quantification of total phenols, flavonoids, and tannins, respectively. The antioxidant activity was estimated by DPPH scavenging, phosphomolybdate (TAC), ABTS radical scavenging, and reducing power assays (FRAP-1 and FRAP-2). Antimicrobial activity was determined by the disk diffusion method against Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumoniae, and Candida albicans. Phytochemical tests revealed the presence of flavonoids, quinones, alkaloids, steroids, tannins, and saponins. GC-MS analysis identified 17 biologically important compounds belonging to different classes, including fatty acids, carbohydrates, and terpenoids. The extract showed relatively low content of total phenols, tannins, and flavonoids of 5.00 mgEAG g−1, 0.08 mgEAT g−1, and 0.67 ± 0.29 mgEQ g−1, respectively. Its antioxidant potential was considerably stronger than that of the other Salacia sp. with EC50 values of 64.28 ± 0.01, 36.44 ± 0.67, 35.78 ± 0.09, 33.91 ± 0.12, and 2.22 ± 0.25 μg mL−1 as measured by FRAP-1, TAC, DPPH, FRAP-2, and ABTS radical scavenging assays, respectively. Regarding the antimicrobial activity, the extract inhibited the growth of all the test organisms with minimum inhibitory concentrations of 125 μg mL−1 and 250 μg mL−1 for the bacteria and fungus, respectively. This insightful study is a significant step towards the exploitation of S. kraussii leaves as a cheap source of broad-spectrum antimicrobial agents.

Purification of Antibodies From Human Milk and Infant Digestates for Viral Inhibition Assays.

Oral administration of enteric pathogen-specific immunoglobulins may be an ideal approach for preventing infectious diarrhea in infants and children. For oral administration to be effective, antibodies must survive functionally intact within the highly proteolytic digestive tract. As an initial step toward assessing the viability of this approach, we examined the survival of palivizumab, a recombinant monoclonal antibody (IgG1κ), across infant digestion and its ability to neutralize respiratory syncytial virus (RSV). Human milk and infant digestive samples contain substances known to interfere with the RSV neutralization assay (our selected functional test for antibody survival through digestion), therefore, antibody extraction from the matrix was required prior to performing the assay. The efficacy of various approaches for palivizumab purification from human milk, infant's gastric and intestinal digestates, including casein precipitation, salting out, molecular weight cut-off, and affinity chromatography (protein A and G) were compared. Affinity chromatography using protein G with high-salt elution followed by 30-kDa molecular weight cut-off centrifugal filtration was the most effective technique for purifying palivizumab from human milk and infant digestates with a high yield and reduced background interference for the viral neutralization assay. This work is broadly applicable to the optimal isolation of antibodies from human milk and infant digesta for viral neutralization assays, enables the examination of how digestion affects the viral neutralization capacity of antibodies within milk and digestive samples, and paves the way for assessment of the viability of oral administration of recombinant antibodies as a therapeutic approach to prevent enteric pathogen-induced infectious diarrhea in infants.