kDa Ultrafiltration Membrane Research Articles

Stepwise membrane filtration for Rebaudioside A and Stevioside enrichment in aqueous and ethanolic stevia extracts

Enriching steviol glycosides derived from the stevia plant is an important step in producing stevia natural sweeteners. This study investigated the enrichment of Rebaudioside A and Stevioside compounds of aqueous and ethanol stevia extracts using cascade membrane filtration techniques. Accordingly, extracts from stevia leaves were processed using membrane filtration techniques such as ultrafiltration and nanofiltration. In particular, the 30 kDa ultrafiltration membrane was highly effective in enriching steviol glycosides. The highest concentrations of Rebaudioside A (21.47 g L-1) and Stevioside (19.97 g L-1) compounds were reached at the 30 kDa ultrafiltration retentate fraction in both aqueous and ethanolic extracts. However, it was found that the permeate fluxes and the extracts obtained after the nanofiltration process had very low concentrations of these compounds. The findings highlight the importance of membrane selection in effectively enriching steviol glycosides.

Isolation and purification of bovine N‐glycans from whey protein concentrate (WPC 70)

N‐glycans present in milk glycoproteins (e.g. lactoferrin and immunoglobulin G) can provide infants with nutritional and protective effects similar to breast milk. In this study, we enriched N‐glycoproteins from WPC 70 using ultrafiltration and anion exchange chromatography on DEAE Sepharose Fast Flow column, with N‐glycan content and N‐glycoprotein purity as evaluation indicators. The results showed that changing feed flow rate and molecular weight cut‐off was more effective to improve ultrafiltration separation than changing feed concentration. When using a 100 kDa ultrafiltration membrane with a feed flow rate of 10 rpm and a feed concentration of 10 mg/mL, N‐glycan content could be increased by 200%. However, anion exchange chromatography was more efficient for purification, increasing N‐glycan content by 700%. This study provided a reliable method for N‐glycan enrichment and a scientific basis for further research and potential application of N‐glycans in infant formula and whey products.

Isolation and purification of angiotensin converting enzyme inhibitory peptide from goat milk hydrolysate by ultrafiltration membrane

Hypertension been a significant risk factor for cardiovascular illness. Synthesized angiotensin converting enzyme (ACE) inhibitors are active antihypertensive medicines they frequently generate unwanted side effects. This study describes the isolation and purification of an ACE inhibitory peptide from goat milk hydrolysate (Sahel breed). Hydrolysates containing angiotensin converting enzyme (ACE) inhibitory peptide were produced from goat milk by pepsin and trypsin. The strongest ACE inhibitory action was found in pepsin hydrolysate (57%) at 12h, hence was further purified. Purification was done using a 10 kDa ultrafiltration membrane and three steps of RP-HPLC to obtain ACE inhibitory peptide with activity of 84% and IC50 of 3.25±0.11 μg/ml. Lineweaver–Burks’ plots showed that the inhibitory kinetic mechanism of this peptide was competitive. In conclusion, ACE inhibitory peptide from goat milk hydrolysate treated with pepsin may be positive constituent to serve as drug against hypertension.

Immunoadsorption as a method of antibody donation during the COVID-19 pandemic.

Initial therapeutic efforts to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) included the use of plasma from convalescent donors containing anti-SARS-CoV-2 antibodies. High-neutralizing antibody titres are required for therapeutic efficacy. This study aims to show that immunoadsorption followed by tangential flow filtration can be used to obtain antibody concentrates with high-neutralizing capacities. Eligible donors (n = 10, five males and three females) underwent immunoadsorption using adsorber columns specific for human antibodies. Glycine-washed out eluates of 1.5 L volume were further concentrated by tangential flow filtration using 30 kDa ultrafiltration membranes. The same membranes were applied for diafiltrations to exchange residual glycine for 0.9% normal saline. Antibody concentrates were obtained within 8 h from the start of donation and had 4.58 ± 1.95, 3.28 ± 1.28 and 2.02 ± 0.92 times higher total IgG, IgA and IgM concentrations, 3.29 ± 1.62 and 3.74 ± 0.6 times higher SARS-CoV-2 N and S antibody concentrations and 3.85 ± 1.71 times higher SARS-CoV-2 S-specific IgG concentrations compared to the donors' peripheral blood. The specific SARS-CoV-2 virus neutralization capacities increased in all but one concentrate. All antibody concentrates (50-70 mL final volume) passed microbiological tests, were free of hazardous glycine levels and could be stored at -80°C and 4°C for 1 year with 20 ± 3% antibody loss. Immunoadsorption followed by tangential flow filtration is a feasible procedure to collect IgG, IgA and IgM as well as SARS-CoV-2 N- and S-specific antibody concentrates of low volume, free of albumin and coagulation factors. Whether these concentrates can be used as passive immunisation in infected patients remains to be elucidated.

Identification and characterization of antibacterial peptides produced by Lactobacillus plantarum 1407.

Peptides from lactic acid bacteria provide health benefits and can inhibit the growth of pathogenic organisms. The present work aimed to isolate and characterize peptides with antibacterial activity from Lactobacillus plantarum 1407. Peptides were isolated and purified from L. plantarum 1407. The effect of various physiological parameters on the antibacterial activity of the isolated peptides was analyzed. The mode of action of the peptides on indicator organisms was observed using transmission microscopy analysis and flow cytometry analysis. Antibacterial activity and mode of action of peptides isolated from L. plantarum 1407 against gram-positive and gram-negative bacteria have been studied. L. plantarum culture exhibited maximum antibacterial activity at 40 °C, pH 8, and 0.7% salt concentration. The cell-free supernatant (CFS) was concentrated using a 3 kDa ultrafiltration membrane and the peptide fractions (<3 kDa) were further fractionated using Sephadex G-25 gel filtration chromatography. The antibacterial activity of the eluted fractions (F1 to F4) was evaluated using flow cytometry and transmission electron microscopy. F3 fraction exhibited increased antibacterial activity than F1, F2, and F4 fractions against the indicator organisms. Cell membrane damage and leakage of cytoplasmic content of the bacterial cells treated with the antibacterial F3 fraction peptides were observed. The active peptides from L. plantarum 1407 can be potentially used for the treatment of bacterial infections.

Flavor mechanism of micro-nanoparticles and correlation analysis between flavor substances in thermoultrasonic treated fishbone soup

To study the physicochemical properties of micro-nanoparticles (MNPs) in thermoultrasonic treated fishbone soup, it was subjected to ultra-filtration with a 100kDa ultrafiltration membrane to obtain large MNPs (LMNPs) and small MNPs (SMNPs). LMNPs and SMNPs were treated with force-breakers, and the interactions of the MNPs with five characteristic volatile compounds were investigated. LMNPs covered most proteins (222.66mg/mL) and fatty acids (363.76mg/g), while SMNPs was mostly soluble small molecules with taste substances like total free amino acids (85.26mg/g), organic acids (2.55mg/mL), and 5'-nucleotides (169.17mg/100mL). The stability of LMNPs is significantly higher than raw bone soup, and SMNPs can exist stably in the solution. Correlation analysis between flavor substance content and flavor suggested that the overall flavor profile of halibut bone soup was closely related to the content changes of 72 significant influence variables. The binding of LMNPs to characteristic flavor compounds was largely affected by hydrophobic interactions, hydrogen bonds, and ionic effects. While the binding of SMNPs to characteristic flavor compounds was largely determined by hydrophobic interaction and hydrogen bonding. This study explores the characteristics of MNPs and provides the possibility to clarify the interaction mechanism between MNPs and flavor.

Microplastics in different water samples (seawater, freshwater, and wastewater): Removal efficiency of membrane treatment processes

The distribution and fate of microplastics in different water sources and their treatment plants (seawater, three municipal wastewaters, a pharmaceutical factory wastewater, and three drinking waters) in France were studied. Currently, research in this field is still under exploration since almost no relevant standards or policies have been introduced for the detection, the removal, or the discharge of microplastics. This study used an improved quantitative and qualitative analytical methodology for microplastic detection by μ-FTIR carried out with siMPle analytical software. By investigation, wastewater was determined to contain the most abundant microplastics in quantity (4,203–42,000 MP·L−1), then followed by surface water/groundwater (153–19,836 MP·L−1) and seawater (around 420 MP·L−1). Polyethylene was the dominant material in almost all water types followed by polypropylene, polystyrene, and polyethylene terephthalate. Almost all treatment technologies could remove microplastics whatever the feed water types and concentration of microplastics, though some treatment processes or transport pipes could cause additional contamination from microplastics. The four WWTPs, three DWTPs, and SWTP in France provided, respectively, 87.8–99.8%, 82.3–99.9%, 69.0–96.0% removal/retention of MPs in quantity, and provided 97.3–100%, 91.9–99.9%, 92.2–98.1% removal/retention of MPs in surface area. Moreover, ultrafiltration was confirmed to be an effective technology for microplastic retention and control of dimensions of microplastics in smaller ranges both in field-scale and lab-scale experiments. The 200 kDa ultrafiltration membrane could retain 70–100% and 80–100% of microplastics in quantity and in surface area, respectively.

S-13-3: AN APPROACH TO IDENTIFY THE UNDETERMINED ADIPOCYTE-DERIVED FACTOR(S) THAT STIMULATE ALDOSTERONE SYNTHASE GENE (CYP11B2) EXPRESSION

Objectives: Hypertension is a frequently observed complication among obese patients. Recently, undetermined adipocyte-derived factor(s) have been recognized as one of the causes of the obesity-related hypertension independent of angiotensin (A) II. In this study, we aim to identify the undetermined adipocyte-derived factor(s) that stimulate aldosterone synthase gene (CYP11B2) expression aldosterone secretion that may induce hypertension. Methods: We first differentiated mouse fibroblast 3T3-L1 cells into adipocytes and collected their supernatants. The supernatants were incubated with human adrenocortical carcinoma H295R cells, and their CYP11B2 mRNA expression was measured by quantitative PCR with reverse transcription (qRT-PCR). Thereafter, the supernatants were fractionated by ammonium sulfate precipitation to purify adipocyte-derived factor(s) that stimulate CYP11B2 expression. The precipitates were collected in 30%, 60% and 90% saturated concentration of ammonium sulfate. Each of precipitates was concentrated using 100 kDa ultrafiltration membrane and incubated with H295R cells. CYP11B2 mRNA expression in H295R cells was measured by qRT-PCR. Then, we purified the precipitation of the supernatants that obtained in 60% ammonium sulfate by anion-exchange chromatography. Each of the fractions was incubated with human adrenocortical carcinoma H295R cells, and their CYP11B2 mRNA expression was measured by qRT-PCR. Results: The supernatants obtained from 3T3-L1 adipocytes were demonstrated to stimulate CYP11B2 expression. Fractionation of the supernatants by ammonium sulfate precipitation demonstrated that the ability to stimulate CYP11B2 expression was detected in fractions precipitated in 60% saturated concentration. Fractionation of the supernatants by anion-exchange chromatography demonstrated that the ability to stimulate CYP11B2 expression was detected in two specific fractions. Specific fractions were analyzed by mass spectrometry, resulting in the identification of 39 specific proteins. We are now investigating that the relationship between CYP11B2 gene expression and 39 proteins.

Dry polishing of enzymatically produced fatty acid methyl esters through polymeric membranes

Industrial production of enzymatic biodiesel is based on the use of low-quality feedstocks with usually high water and free fatty acid contents. For this reason, a polishing step is essential to remove impurities and meet the required quality for downstream processing, storage, or commercialization of the product. In this work, we propose the application of membrane separation to polish crude industrial enzymatic fatty acid methyl esters (FAME). Polymeric membranes of different features were selected and tested for crude industrial FAME filtration. Results showed that the 10 kDa ultrafiltration (UF) membrane provided the best performance resulting in the exemption of soap content in the permeate. Moreover, the UF membrane was successfully able to drastically reduce the color, total contamination, and phosphorus content of FAME samples. The permeate flux was found stable during the ultrafiltration process in crossflow mode and no severe fouling was observed, thus making the membrane separation process a promising approach for FAME polishing.

Novel antioxidant peptides from protein hydrolysates of scallop (Argopecten irradians) mantle using enzymatic and microbial methods: Preparation, purification, identification and characterization

The aim of this study was to isolate, purify, identify, and characterize novel antioxidant peptides from protein hydrolysates of scallop (Argopecten irradians) mantle. Antioxidant peptides were obtained following protein hydrolysis, using a neutral protease, and microbial fermentation which was carried out by Bacillus licheniformis CICC 20033 under optimized conditions. Antioxidant peptides from the scallop mantle hydrolysates were then purified through fractionation using ultrafiltration membranes, and the fraction (<3 kDa) showed the highest oxygen radical absorbance capacity (ORAC) with the value of 0.362 μM trolox equivalent (TE)/mg. Subsequently, this fraction furtherly purified by Sephadex G-15 chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) showed that the ORAC value was 2.96 ± 0.10 μM TE/mg. The ORAC value of fermented hydrolysates increased from 0.359 μM TE/mg to 0.425 μM TE/mg through 3 kDa ultrafiltration membrane. Five peptides from hydrolysates by enzyme treatment and twelve peptides from fermented scallop mantle hydrolysates were identified using LC-MS/MS. All selected sequences except Lys-Asp-Ala-Ala-Lys-Thr-Lys showed clear differences in scavenging activity for various radicals, which were dependent on the content and position of charged polar amino acids, aromatic amino acids and sulfur-containing amino acid in sequences.

Ultrafiltration significantly increased the scaling potential of municipal secondary effluent on reverse osmosis membranes

Ultrafiltration (UF) was often used as pretreatment in front of reverse osmosis (RO) unit because of its high rejection efficiency of microbes and particles. However, in some cases UF pretreatment might show adverse effects on the RO membrane flux. In this study, the effects of UF pretreatment on secondary effluent water quality and its RO membrane fouling characteristics were explored. There was almost no change of water quality after UF with different molecular weight cut-off (MWCO) membranes (100, 30 and 10 kDa), including total dissolved solid (TDS), alkalinity, conductivity, ion concentrations, etc., while pH increased a little and dissolved organic carbon (DOC) declined by about 1 mg/L. On the contrary, the RO membrane flux of UF permeates presented clear decline in comparison to the secondary effluent. The membrane fouling velocity and steady-state flux of secondary effluent was 0.052 and 0.656, while fouling velocity increased (0.077, 0.071, 0.067) and steady-state flux decreased significantly (0.397, 0.416, 0.448) after 100, 30, 10 kDa UF membrane pretreatment. Scanning electron microscope (SEM) images showed many crystals on the fouled membrane surfaces, which turned out to be CaCO3 by Energy dispersive spectrometer (EDS) analysis and precipitation calculation. After the addition of UF retentates to UF permeates, scaling was prevented and crystals on the RO membrane almost disappeared, which implied the anti-scaling effect of the UF retentates with low concentration. According to anti-scaling performance experiments, the anti-scaling performance of 100 k, 30 k, 10 k retentates was 2.7%, 4.0% and 7.3%, respectively. Excitation emission matrix (EEM) and fourier transform infra-red (FTIR) results showed that these retentates retained by different MWCO membranes were similar and composed of protein-like substances and soluble microbial products. The effect of key minority components in RO system deserved further exploration.

Mathematical analysis of cake layer formation in an ultrafiltration membrane of a phycobiliprotein-containing solution extracted from Nostoc commune

Phycobiliproteins have been used as a natural dye, an analytical probe for fluorescence studies, and a chemical for medicinal use. An ultrafiltration membrane is useful for the purification of phycobiliproteins. This study addressed the purification of a phycobiliprotein-containing solution, extracted from Nostoc commune, which was individually performed by two kinds of ultrafiltration membrane (MWCO 150 kDa and 10 kDa) in cross-flow. Three proteins were mainly included in the extractant and analyzed by gel permeation chromatography. Molecular weight of included proteins were 60 kDa, 20 kDa, and 200 Da. The formation of a cake layer was analyzed mathematically by solving ordinary differential equation to fit the experimental data and obtain the 5 parameters needed to mechanistically predict cake-formation. The obtained packed coefficient (K4) and permeation coefficient of protein (K5) demonstrated that the proteins of the phycobiliprotein-containing solution were filtered by the formation of a cake layer in the 150-kDa membrane, while the proteins filtered by the 10-kDa membrane resulted in the clogging of the membrane pores. Transport coefficient of each protein through 10 kDa- and 150 kDa-ultrafiltration membrane showed 0.40 and 0.94 of phycobiliprotein, respectively, demonstrating that 10 kDa-ultrafiltration membrane had the ability for separation of 60 kDa and 10 kDa proteins. The 10-kDa ultrafiltration membrane showed the sensitive purification performance of phycobiliprotein, having the potential for scale-up modules.

Studies on the Separation and Purification of the Caulis sinomenii Extract Solution Using Microfiltration and Ultrafiltration

The separation and purification process of alkaloids faces great challenges of pollution, high energy consumption and low continuity. In this study, the effects of ceramic microfiltration (MF) membrane (membrane pore size of 0.50 μm, 0.20 μm, 0.05 μm) and organic ultrafiltration (UF) membrane (membrane molecular weight cut-off of 10 KDa and 1 KDa) on the separation and purification of Caulis sinomenii extract solution in pilot scale were studied. The cleaning effects of different cleaning methods (pure water, 1% HCl-NaOH, 1% sodium hypochlorite) were investigated. The experimental results indicated that 0.05 μm ceramic membrane and 1 kDa UF membrane have higher sinomenine hydrochloride (SH) permeabilities and total solids (TS) removal rates. The ceramic membrane was circulating cleaned by 1% sodium hypochlorite solution for 1 h; the membrane flux can be restored to more than 90% of the original, the membrane flux of 1 kDa UF membrane can be restored to 99.2% of the original by pure water washing. From the above study, the optimal technic parameters was determined in which 0.05 μm ceramic MF membrane and 1 kDa UF membrane were used to separate and purify the Caulis sinomenii extract solution to remove the invalid ingredients, and the two kinds of membranes were cleaned with 1% sodium hypochlorite solution and pure water, respectively, to keep satisfactory membrane fluxes. The study provided an environment-friendly alternative for the separation and purification of alkaloids in natural products, which has a good prospect for the industrial application.

Recombinant expression and molecular characterization of buffalo sperm lysozyme-like protein 1

Several sperm lysozyme-like genes evolved from lysozyme by successive duplications and mutations; however their functional role in the reproduction of farm animals is not well understood. To understand the function and molecular properties of buffalo sperm lysozyme-like protein 1 (buSLLP1), it was expressed in E. coli; however, it partitioned to inclusion bodies. Lowering of temperature and inducer concentration did not help in the recovery of the expressed protein in the biologically active form. Therefore, buSLLP1 was cloned and expressed in Pichiapink system based on auxotrophic Pichia pastoris in a labscale fermenter. The expressed protein was obtained in flow-through by using a 30 kDa ultrafiltration membrane followed by MonoQ anion exchange chromatography, resulting in a homogenous preparation of 40 mg recombinant buSLLP1 per liter of initial spent culture-supernatant. Circular dichroism spectroscopy showed that recombinant buSLLP1 possessed a native-like secondary structure. The recombinant buSLLP1 also showed thermal denaturation profile typical of folded globular proteins; however, the thermal stability was lower than the hen egg white lysozyme. Binding of buSLLP1 to chitin and zona pellucida of buffalo oocytes showed that the recombinant buSLLP1 possessed a competent binding pocket, therefore, the produced protein could be used to study its functional role in the reproduction of farm animals.

Forward Black Liquor Acid Precipitation: Lignin Fractionation by Ultrafiltration.

Lignin recovery from black liquor is an important task for producing valuable chemical products. Acidification processes are currently applied by pulp and paper industries for black liquor treatment, in which two main streams are produced: the precipitated lignin fraction and a lignin-lean black liquor. Membrane filtration is a suitable alternative for lignin recovery from black liquor, but studies on lignin-lean black liquor filtration are scarce. Here, we evaluated the ultrafiltration process for lignin recovery from the both fractions of black liquor acidification. The lignin-lean black liquor presented 22 wt% of total solids with 4.6 wt% of lignin. Lignin retention from the lignin-lean black liquor by the 5 kDa ultrafiltration membrane was equal to 85%, with reduction in total solid concentration from 219.8 to 68.1 g L-1. Due to the relatively high solid concentration in the lignin-lean black liquor, cake formation was the main fouling mechanism during ultrafiltrations. The precipitated lignin solution presented 4.8 wt% of total solids with equivalent lignin concentration (4.7 wt%). The used membrane was able to retain almost 100% of solids and lignin from the solution prepared from the precipitated lignin. All fouling mechanisms were responsible for flux decay in ultrafiltration of the precipitated lignin solution. Steady state fluxes for lignin-lean black liquor and precipitated lignin solution were 0.9 and 15.9 L h-1 m-2, respectively. According to TGA analyses up to 800 °C, precipitated lignin and lignin-lean black liquor presented total mass losses of 63.5% and 44.3%, respectively. Also, the permeate samples presented lower mass losses than their respective feed samples. The ultrafiltration process reduced the average weight molar mass (Mw) of the precipitated lignin solution and lignin-lean black liquor from 1817 to 486 g mol-1and from 2876 to 1095 g mol-1, respectively. Thus, the 5 kDa ultrafiltration membrane was efficient for lignin recovery from the lignin-lean black liquor, while membranes with lower cut-off should be proposed for lignin purification from the precipitated fraction.

Comparison of bovine milk fat globule membrane protein retention by different ultrafiltration membranes using a label-free proteomic approach

Ultrafiltration (UF) is an effective method for separating and enriching milk proteins. This study aims to analyze the protein profile of milk fat globule membrane (MFGM) materials enriched by UF membranes from butter serum with different molecular mass cutoffs through a label-free proteomic approach. In total, 616 MFGM proteins were identified in all groups, of which 395, 399, 399 and 484 proteins were identified with a 30 kDa, 50 kDa and 100 kDa UF membrane and without UF, respectively. It was more efficient to enrich fatty acid binding protein and mucin 15 using a 30 kDa membrane, whereas mucin 1 was more abundant when using a 50 kDa membrane. Compared with the 30 kDa and 50 kDa membranes, the 100 kDa membrane led to the optimal efficiency with low permeation of the most major MFGM proteins and high permeation of non-MFGM proteins. Butyrophilin, adipophilin, xanthine oxidase/dehydrogenase, and periodic acid Shiff6/7 were concentrated approximately twofold in the 100 kDa retentate. Meanwhile, the relative abundances of β-lactoglobulin and α-lactalbumin were reduced by approximately 17.3% and 16.2%, respectively. This work will facilitate the development of MFGM protein isolation using membrane separation processes and provide a theoretical reference for the production of MFGM proteins.

Effects of membrane pore size and transmembrane pressure on ultrafiltration of red‐fleshed dragon fruit (Hylocereus polyrhizus) juice

AbstractBACKGROUNDRed‐fleshed dragon fruit contains high amounts of heat‐sensitive betacyanin pigments that require non‐thermal technologies in their processing. Ultrafiltration (UF) can be a promising alternative technique for fruit juice clarification and cold sterilization.RESULTSThis study reported clarification of red‐fleshed dragon fruit juice by UF employing polyethersulfone UF membranes with pore sizes of 5, 10, and 20 kDa operated at three different transmembrane pressures of 1, 2 and 3 bar. Findings indicated that dragon fruit juice was best ultrafiltrated with the 10 kDa UF membrane at 3 bar, which resulted in the highest permeate flux (around 7.9 kg m−2 h−1), lowest retention of betacyanins (30.6%) and phenolic compounds (11.3%), as well as having the closest 2,2‐diphenyl‐2‐picrylhydrazyl hydrate scavenging ability as compared to that of the control. Resistance analyses presented that cake resistance (69–94%) played the major role in decreasing flux and was the dominant fouling cause in the UF process, while reversible (5–25%) and irreversible resistances (1–16%) had lesser impacts. Membrane characterizations using scanning electron microscopy and Fourier‐transform infrared spectroscopy suggested that the chemical structure and surface morphology of the UF membrane were hardly altered after being used and washed with detergents. Liquid chromatographic–mass spectroscopic analyses demonstrated that UF was successfully employed for juice clarification without affecting heat‐sensitive compounds of fruit juices such as betacyanins.CONCLUSIONSimultaneous selection of suitable membrane pore size and operation pressure is crucial for UF of fruit juice. UF is a promising technique in processing bioactive compound‐containing liquid foods. © 2021 Society of Chemical Industry

The ultrafiltration molecular weight cut-off has a limited effect on the concentration and protein profile during preparation of human milk protein concentrates

Optimizing protein intake for very low birth weight (<1,500 g) infants is fundamental to prevent faltering postnatal growth with the potential association of impaired neurodevelopment. The protein content of human milk is not sufficient to support the growth of very low birth weight infants. To meet their elevated protein requirements, human milk is currently fortified using typically bovine milk-based protein isolates (>85% on a dry basis). However, these products have several limitations for use in this vulnerable population. To overcome the shortcomings of bovine milk-based protein supplement, a human milk protein concentrate (HMPC) was developed. In preliminary attempts using 10 kDa ultrafiltration (UF) membranes, it was not possible to reach the protein content of commercial protein isolates, presumably due to the retention of human milk oligosaccharides (HMO). Consequently, it was hypothesized that the use of a UF membrane with a higher molecular weight cut-off (50 kDa rather than 10 kDa) could improve the transmission of carbohydrates, including HMO, in the permeate, thus increasing the protein purity of the subsequent HMPC. The results showed that permeate fluxes during the concentration step were similar to either UF molecular weight cut-off, but the 50-kDa membrane had a higher permeate flux during the diafiltration sequence. However, it was not sufficient to increase the protein purity of the human milk retentate, as both membranes generated HMPC with similar protein contents of 48.8% (10 kDa) and 50% (50 kDa) on a dry basis. This result was related to the high retention of HMO, mainly during the concentration step, although the diafiltration step was efficient to decrease their content in the HMPC. As the major bioactive proteins (lactoferrin, lysozyme, bile salt stimulated lipase, and α1-antitrypsin) in human milk were detected in both HMPC, the 50-kDa membrane seems the most appropriate to the preparation of HMPC in terms of permeation flux values. However, improving the separation of HMO from proteins is essential to increase the protein purity of HMPC.

Effect of Pectinolytic Enzyme Pretreatment on the Clarification of Cranberry Juice by Ultrafiltration.

Cranberries, mainly processed as juice, have garnered interest over the past decade due to their high content of phytochemical compounds related to promising health benefits. To meet consumer expectations, a juice clarification step is usually incorporated to remove suspended solids. The use of pectinolytic enzyme and membrane processes are commonly applied to the production of clarified juices, but no studies have been done on cranberry juice. In this study, the effects of 60 (D60) and 120 min (D120) of depectinization by pectinolytic enzymes coupled to clarification by ultrafiltration (UF) (membrane molecular weight cut-off (MWCO) of 50, 100 and 500 kDa) was evaluated on the filtration performance, membrane fouling and cranberry juice composition. Compared to fresh juice, depectinization for 60 and 120 min reduced the UF duration by 16.7 and 20 min, respectively. The best filtration performance, in terms of permeate fluxes, was obtained with the 500 kDa MWCO UF membrane despite the highest total flux decline (41.5 to 57.6%). The fouling layer at the membrane surface was composed of polyphenols and anthocyanins. Compared to fresh juice, anthocyanin decreased (44% and 58% for D60 and D120, respectively) in depectinized juices whereas proanthocyanidin (PAC) content increased by 16%. In view of the industrial application, a 60 min depectinization coupled to clarification by a 500 kDa UF membrane could be viewed as a good compromise between the enhancement of filtration performance and the loss of polyphenols and their fouling at the membrane surface.

Ultrafiltration behavior of recombinant adeno associated viral vectors used in gene therapy

Recombinant Adeno-Associated Viruses (AAVs) are increasingly being used as the choice of vectors for gene therapy applications. While ultrafiltration is the unit operation in place to concentrate the AAV and place vector in the final formulation buffer, not enough data, and fundamental understanding of ultrafiltration of AAV vectors exists to date. This work examines the effect of membrane molecular weight cut-off and operating conditions on the flux and transmission of AAV2 and AAV9 serotypes through composite regenerated cellulose (CRC) ultrafiltration membranes. The transmission of AAV through more permeable ultrafiltration membranes was observed to vary as a function of the operating conditions and membrane properties. The effect of operating conditions, membrane permeability, and scale were examined for a completely retentive 30 kDa ultrafiltration membrane. It was found that a 100 kDa ultrafiltration membrane examined in this work could perform as a completely retentive membrane or, conversely, as a membrane significantly permeable to both AAV serotypes. It was found that operating the 30 kDa membrane closer to the pressure independent regime maximized flux and reduced process variability. This study also demonstrated that a flat sheet cassette may be successfully used as a scale-down model for a holderless spiral wound device, enabling users to choose those devices for small scale process development. This is the first study to provide a quantitative understanding of the ultrafiltration behavior of AAVs for gene therapy to guide process development.