kDa Retentate Research Articles

Production and Characterization of ACE Inhibitory and Anti-Diabetic Peptides from Buffalo and Camel Milk Fermented with Lactobacillus and Yeast: A Comparative Analysis with In Vitro, In Silico, and Molecular Interaction Study.

The investigation aimed at assessing a comparative study on the production and characterization of ACE inhibitory, anti-diabetic, and anti-inflammatory activities, along with the production of ACE inhibitory and anti-diabetic peptides through the fermentation of buffalo and camel milk by Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A). The angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic properties were evaluated at particular time intervals (12, 24, 36, and 48 h) at 37 °C, and we discovered maximum activity at 37 °C after 48 h of incubation. The maximum ACE inhibitory, lipase inhibitory activities, alpha-glucosidase inhibitory, and alpha-amylase inhibitory activities were found in the fermented camel milk (77.96 ± 2.61, 73.85 ± 1.19, 85.37 ± 2.15, and 70.86 ± 1.02), as compared to the fermented buffalo milk (FBM) (75.25 ± 1.72, 61.79 ± 2.14, 80.09 ± 0.51, and 67.29 ± 1.75). Proteolytic activity was measured with different inoculation rates (1.5%, 2.0%, and 2.5%) and incubation times (12, 24, 36, and 48 h) to optimize the growth conditions. Maximum proteolysis was found at a 2.5% inoculation rate and at a 48 h incubation period in both fermented buffalo (9.14 ± 0.06) and camel milk (9.10 ± 0.17). SDS-PAGE and 2D gel electrophoresis were conducted for protein purification. The camel and buffalo milk that had not been fermented revealed protein bands ranging from 10 to 100 kDa and 10 to 75 kDa, respectively, whereas all the fermented samples showed bands ranging from 10 to 75 kDa. There were no visible protein bands in the permeates on SDS-PAGE. When fermented buffalo and camel milk were electrophoresed in 2D gel, 15 and 20 protein spots were detected, respectively. The protein spots in the 2D gel electrophoresis ranged in size from 20 to 75 kDa. To distinguish between different peptide fractions, water-soluble extract (WSE) fractions of ultrafiltration (3 and 10 kDa retentate and permeate) of fermented camel and buffalo milk were employed in RP-HPLC (reversed-phase high-performance liquid chromatography). The impact of fermented buffalo and camel milk on inflammation induced by LPS (lipopolysaccharide) was also investigated in the RAW 264.7 cell line. Novel peptide sequences with ACE inhibitory and anti-diabetic properties were also analyzed on the anti-hypertensive database (AHTDB) and bioactive peptide (BIOPEP) database. We found the sequences SCQAQPTTMTR, EMPFPK, TTMPLW, HPHPHLSFMAIPPK, FFNDKIAK, ALPMHIR, IPAVFK, LDQWLCEK, and AVPYPQR from the fermented buffalo milk and the sequences TDVMPQWW, EKTFLLYSCPHR, SSHPYLEQLY, IDSGLYLGSNYITAIR, and FDEFLSQSCAPGSDPR from the fermented camel milk.

Fractionation and characterization of poly(β-L-malic acid) produced by Aureobasidium melanogenum ipe-1

Poly (β-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries, whose functions primarily depend upon its molecular size and chemical structure. Up to now, the fractionation and characterization of PMLA produced by Aureobasidium spp. were still unclear. In this study, the product from A. melanogenum ipe-1 was effectively fractionated using 300 and 50 kDa membranes. During the filtration, the mechanisms of membrane fouling were illegible since the PMLA can both reject and permeate the membrane, while the main fouling mechanism varied between standard blocking and complete blocking during the diafiltration. After fractionation, 14.0, 8.4 and 77.6 % of the PMLAs with Mws of 75,134, 21,344 and 10,056 Da were distributed in the 300 kDa retentate after diafiltrating, 50 kDa retentate after diafiltrating, and the 50 kDa permeate, respectively. The Mw/Mns of the PMLAs were 4.12, 1.92, and 1.12 in the three fractions. Based on characteristic spectra of NMR, HPLC and FTIR, the product was not usual L-malic acid monomers, but glucose-terminated PMLA. The glucose was located at the terminal hydroxyl of PMLA. These results would serve as a valuable guide for process design and practical operation in subsequent industrial application.

Membrane-based approach for the removal of pigment impurities secreted by Pichia pastoris

During recombinant protein expression in Pichia pastoris under the alcohol oxidase promoter, the culture supernatant developed green and yellow pigments, complicating downstream processing. Currently, a chromatography-based purification approach is one of the most efficient strategies to eliminate these pigments, but this method is complicated and often the most expensive step during processing. In this study, we designed a sequential cross-flow filtration by the combination of a microfiltration membrane with a pore size of 0.2 micrometers, followed by an ultrafiltration membrane with a molecular weight cut-off of 10 and 2 kilodaltons (kDa), respectively, and finished with diafiltration of the 10 and 2 kDa retentate fractions. The results show that the microfiltration membrane eliminated host cell impurities and significantly reduced the green pigment, which was measured using the 1976 CIE LAB system, while the 10 and 2 kDa membranes allowed the yellow pigment to pass through. The diafiltration steps also significantly reduced the yellow pigment. The host cell proteins were removed by recirculating in the retentate fraction of the 10 kDa membrane, while the 4.1 kDa target peptide, the candidate cell-penetrating antimicrobial peptide, was recovered by recirculation in the retentate fraction of the 2 kDa membrane.

Anti-Inflammatory, ACE Inhibitory, Antioxidative Activities and Release of Novel Antihypertensive and Antioxidative Peptides from Whey Protein Hydrolysate with Molecular Interactions

Objective The aim of the study was to evaluate the whey protein hydrolysate with bio-functional attributes viz. antioxidative, anti-inflammatory and ACE inhibition efficacy and release of bioactive peptides with antioxidative and ACE-inhibitory activity by employing Pepsin. Method The antioxidant, Anti-inflammatory, ACE inhibitory and proteolytic activities of the whey protein hydrolysates were studied followed by SDS-PAGE analysis and IEF. Anti-inflammatory activity of whey protein hydrolysate was also studied on RAW 264.7 cell line. The separation of the bioactive peptides from whey protein hydrolysate was achieved by RP-HPLC. The purified bioactive peptides were identified and characterized using RPLC/MS. Results WPC (Whey protein concentrate) hydrolysate with pepsin showed proteolytic activity ranging between 14.46 and 18.87 mg/ml. Using the ABTS assay, the highest antioxidative activity was observed in 10 kDa retentate (84.50%) and 3 kDa retentate (85.96%), followed by the highest proteolytic activity (13.83 mg/ml) and ACE inhibitory activity (58.37%) in a 5% WPC solution at 65 °C after 8 h of pepsin hydrolysis. When the protein hydrolysate concentration was low, the production of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages (RAW 264.7) was reduced. SDS-PAGE results exhibited very little protein bands when comparing with WPC hydrolysates to insoluble WPC. There were no protein spots on 2 D gel electrophoresis and “in-solution trypsin digestion” technique have been utilized to digest protein samples directly from WPC hydrolysates. Novel antioxidative peptides and ACE inhibitory peptides were also observed by comparing two databases, i.e., BIOPEP and AHTPDB respectively. The peptide sequences used in this study were found to have excellent potential to be used as inhibitors of hACE as all of them were able to show substantial interactions against the enzyme’s active site. Conclusions The antihypertensive and antioxidative peptides from whey protein hydrolysates may be beneficial for the future development of physiologically active functional foods. Further, in vivo investigations are required to establish the health claim for each individual bioactive peptide from whey protein hydrolysate. Supplemental data for this article is available online at

Antioxidative, antimicrobial and anti-inflammatory activities and release of ultra-filtered antioxidative and antimicrobial peptides during fermentation of sheep milk: In-vitro, in-silico and molecular interaction studies

The antioxidative, antimicrobial, and anti-inflammatory properties of fermented sheep milk with L. fermentum (KGL4), as well as the generation of antioxidative and antimicrobial peptides, are evaluated in the study. Antioxidative and antibacterial activities in sheep milk fermented with KGL4 increased with incubation hours, along with varied antioxidative properties (ABTS assay: 35.12%, hydroxyl free radical scavenging assay: 29.12%, superoxide free radical scavenging activity: 36.38%). Antimicrobial activity of fermented sheep milk against E. faecalis (19 mm), S. typhimurium (15.67 mm), B. cereus (14 mm), and E. coli (13 mm) was also observed. Furthermore, after 48 h, the KGL4 showed maximum proteolysis (10.40 mg/ml) at 2.5% rate of addition sheep milk. The antioxidative and antimicrobial activities of fermented sheep milk fractions (3 kDa and 10 kDa permeates and retentates) were also investigated. The highest ABTS activity (26.90%) was found in 10 kDa permeate, whereas the 3 kDa retentate had higher hydroxyl free radical scavenging activity (69.20%) and 3 kDa permeate showed maximum superoxide free radical scavenging activity (32.85%). The 10 kDa retentate had shown maximum antimicrobial activity against S. typhimurium (13.67 mm) and E. faecalis (17.00 mm). Proteins spotted on 2D gel electrophoresis of KGL4 were varied from 10 to 70 kDa. RP-LC/MS was used to identify 5 novel peptide sequences from 2-D gel spots. Searches in the BIOPEP database confirmed the antioxidative and antimicrobial effects of the novel fermented sheep milk peptides. Fermented sheep milk with KGL4 (SMKGL4) significantly reduced excessive TNF-α, IL-6, & IL-1β production in LPS-activated RAW 264.7 cells.

Rheological and microstructural characterisation of heat-induced whey protein isolate gels affected by the addition of caseinomacropeptide.

Caseinomacropeptide (CMP) is derived from the chymosin cleavage of κ-casein during cheese production. This study developed gels from CMPs, which were isolated by different ultrafiltration systems, and whey protein isolate (WPI), and studied their rheological and ultrastructural characteristics. The 30% WPI gel showed high elastic modulus (G') values and stronger structure than the other samples with CMP. Another gel, with 50% protein, 30% WPI and 20% CMP sample isolated from the 30 kDa retentate, had a weaker structure and lower G' value. The third gel, with 30% WPI and 20% CMP sample from the 5 kDa retentate derived from the 30 kDa retentate, presented intermediate structural strength. Despite the increase in protein concentration from the addition of CMP, there was a decrease in the strength of the gel network. Different CMP isolation processes also contributed to differences in the microscopic analysis of gel structures with the same protein content.

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.

Evaluation of the in vitro effects of the increasing inclusion levels of yeast β-glucan, a casein hydrolysate and its 5 kDa retentate on selected bacterial populations and strains commonly found in the gastrointestinal tract of pigs.

Previously, the 5 kDa retentate (5kDaR) of a casein hydrolysate (CH) and yeast β-glucan (YBG) were identified as promising anti-inflammatory dietary supplements for supporting intestinal health in pigs post-weaning. However, their direct effects on intestinal bacterial populations are less well-known. The main objectives of this study were to determine if the increasing concentrations of the CH, 5kDaR and YBG individually, can: (1) alter the bacterial and short-chain fatty acid profiles in a weaned pig faecal batch fermentation assay, and (2) directly influence the growth of selected beneficial (Lactobacillus plantarum, L. reuteri, Bifidobacterium thermophilum) and pathogenic (Enterotoxigenic Escherichia coli, Salmonella Typhimurium) bacterial strains in individual pure culture growth assays. The potential of CH as a comparable 5kDaR substitute was also evaluated. The 5kDaR increased lactobacilli counts and butyrate concentration in the batch fermentation assay (P < 0.05) and increased L. plantarum (linear, P < 0.05), L. reuteri (quadratic, P < 0.05) and B. thermophilum (linear, P < 0.05) counts and reduced S. typhimurium (quadratic, P = 0.058) counts in the pure culture growth assays. CH increased butyrate concentration (P < 0.05) in the batch fermentation assay. YBG reduced Prevotella spp. counts (P < 0.05) and butyrate concentration (P < 0.05) in the batch fermentation assay. Both CH and YBG had no major effects in the pure culture growth assays. In conclusion, the 5kDaR had the most beneficial effects associated with increased counts of Lactobacillus and Bifidobacterium genera and butyrate production and reduced S. typhimurium counts in vitro indicating its potential to promote gastrointestinal health.

Process for selective extraction of pigments and functional proteins from Chlorella vulgaris

Extraction of multiple high-value products is recommended for sustainability of the microalgal production platform. This study proposes a process for selective extraction of carotenoids and chlorophylls with ethanol, followed by alkaline pH extraction of proteins from wet, freeze-thawed Chlorella vulgaris biomass. A biomass-to-solvent ratio of 1:5 and 3 extraction stages were required to achieve maximum extraction yield of chlorophylls and carotenoids. The main compounds in the ethanol extract were identified as lutein, chlorophyll a and chlorophyll b. The lutein and total chlorophyll yield in the extracts were 5.4 mg/g and 15.4 mg/g dry biomass respectively. Effective protein release from freeze-thawed biomass was contact-time dependent and >76% of total protein could be extracted in 15 min via bead-milling, and in 6 min by high-pressure homogenization at 15000 psi. Ethanol extraction of pigments affected protein solubility, and an alkaline pH was required to release the same total protein. Concentration and fractionation of proteins was carried out using a two-stage membrane filtration process and 78–80% of proteins remained in the 300 kDa retentate. Ethanol treatment and higher pH conditions did not negatively impact membrane filtration, nutritive value, or the emulsification properties of protein concentrates.

Fractionation of polyphenols from thermomechanical pulp mill process water by flotation and membrane integrated process

ABSTRACTFractionation of phenolic compounds in thermomechanical pulp mills was performed with a coupling of a prior treatment realized by flotation and a ceramic membrane process. Two lines of membranes filtration were tested. After a common 150 kDa clarification, 1 kDa filtration was performed with or without previous 5 kDa filtration. Flotation was shown to be inevitable to retain lipophilic compounds which cause severe membrane fouling. 150 kDa permeate flux was 20% higher when process water was firstly floated and was around 260 L h−1 m−2. 1 kDa membrane was fouled with 31% of irreversible fouling without previous 5 kDa filtration and phenolic compounds purity reached only 26% in this 1 kDa permeate. Phenolic compounds as lignin-like substances which might be attached to hemicelluloses were recovered in 5 kDa retentate. Retentate of 1 kDa might contain a major fraction of lignin derivatives with molecular weights around 1 kDa free or linked with phenolic acids. Permeate of 1 kDa contained 14% of phenolic compounds such as lignans and free phenolic acids purified at 50%.

In Vitro and In Silico Analysis of Novel ACE-Inhibitory Bioactive Peptides Derived from Fermented Goat Milk

In the study, two Lactobacillus cultures i.e. L. casei (NK9) and L. fermentum (LF) were studied for their proteolytic activity, di and tripeptidase activity, ACE-inhibitory activity and peptides production under optimized growth condition from fermented goat milk (Capra aegagrus hircus). NK9 and LF were found to be a strong proteolytic culture with 2.0% rate of inoculation after 48 h. LF (10 kDa retentate) produced maximum peptides among all the retentates of the fermented goat milk. Goat milk fermented with NK9 (10 kDa permeates) exhibited peptide sequence i.e. AFPEHK which had ACE inhibitory activity, matched with goat milk protein databases of AHTPDB. However, L. casei (NK9) and L. fermentum (LF) could be explored for the production of ACE inhibitory peptides from fermented goat milk.

Pilot scale production of a phospholipid-enriched dairy ingredient by means of an optimised integrated process employing enzymatic hydrolysis, ultrafiltration and super-critical fluid extraction

Abstract Pilot scale production of a dairy ingredient enriched in phospholipids (PLs) was generated from a buttermilk powder (BMP) substrate utilising a combined process of targeted enzymatic hydrolysis of the innate milk proteins followed by ultrafiltration with a 50 kDa membrane. An 8.5 fold increase in PL was achieved in the 50 kDa retentate (50 R) compared to the BMP, 11.05 ± 0.02% and 1.30 ± 0.00% total PL, respectively. Simultaneously, total lipid content in the retentate increased 8.7 fold with reference to the BMP, 60.07 ± 0.54% and 6.84 ± 0.17% total lipid respectively. Protein reduced to 10.58 ± 0.09% (50 R) from 31.40 ± 0.57% in BMP. Supercritical CO 2 fluid extraction (SFE) was employed to generate a purified lipid fraction. SFE with ethanol as a co-solvent yielded a purified lipid extract with enriched PLs level of 56.24 ± 0.07% on a dry matter basis. Industrial relevance PLs have many associated health and nutritional benefits including those related to cognitive development and repair. Generation of an ingredient enriched in dairy PLs would be advantageous from an industrial view to allow fortification of nutritionals, both infant and geriatric, in promoting brain health. The present work demonstrates a novel pilot scale process for the generation of a PL enriched ingredient from a BMP substrate. Utilising a combined process of targeted protein hydrolysis followed by selective removal by ultrafiltration of the smaller molecular weight peptide material, an ingredient with 8.5 fold increase in PL material was achieved. SFE technology was utilised to generate a purified lipid extract with greater PL levels for future applications in biological assays to determine these pathways. The need for investigate and further develop the knowledge relating to the modes of action of these bioactive compounds would be beneficial from a nutritional perspective.

Selective enrichment of dairy phospholipids in a buttermilk substrate through investigation of enzymatic hydrolysis of milk proteins in conjunction with ultrafiltration

Extensive enzymatic hydrolysis of milk proteins in reconstituted buttermilk powder was combined with ultrafiltration to generate a phospholipid (PL) enriched fraction with maximum permeation of hydrolysed peptides. Buttermilk, naturally high in PLs, is the ideal substrate for enrichment of these bio- and techno-functionally active compounds. A 7.8 fold increase in PL was achieved in the 50 kDa retentate; 6.16±0.02% total PL compared with 0.79±0.01% in the starting substrate, an increase considerably greater than previously reported. Total lipid content (% dry matter) increased 6.3 fold in the retentate, 43.43±0.61%, from the starting substrate, 6.84±0.17%. This combined strategic approach enabled maximum enrichment of PLs with no transmission of lipid material into the permeate, 0.09±0.02% total lipid, and non-detectable levels of PLs recovered in the permeate, 0.00±0.01% total PL.

Molecular characterization of whey protein hydrolysate fractions with ferrous chelating and enhanced iron solubility capabilities.

The ferrous (Fe2+) chelating capabilities of WPI hydrolysate fractions produced via cascade membrane filtration were investigated, specifically 1 kDa permeate (P) and 30 kDa retentate (R) fractions. The 1 kDa-P possessed a Fe2+ chelating capability at 1 g L(-1) equivalent to 84.4 μM EDTA (for 30 kDa-R the value was 8.7 μM EDTA). Fourier transformed infrared (FTIR) spectroscopy was utilized to investigate the structural characteristics of hydrolysates and molecular interactions with Fe2+. Solid-phase extraction was employed to enrich for chelating activity; the most potent chelating fraction was enriched in histidine and lysine. The solubility of ferrous sulfate solutions (10 mM) over a range of pH values was significantly (P<0.05) improved in dispersions of hydrolysate fraction solutions (10 g protein L(-1)). Total iron solubility was improved by 72% in the presence of the 1 kDa-P fraction following simulated gastrointestinal digestion (SGID) compared to control FeSO4·7H2O solutions.

Anti-inflammatory effects of a casein hydrolysate and its peptide-enriched fractions on TNFα-challenged Caco-2 cells and LPS-challenged porcine colonic explants.

Bioactive milk peptides are reported to illicit a range of physiological benefits and have been proposed as potential functional food ingredients. The objective of this study was to characterize the anti-inflammatory properties of sodium caseinate (NaCAS), its enzyme hydrolysate (EH) and peptide-enriched fractions (5 kDa retentate [R], 1 kDaR and 1 kDa permeate [P]), both in vitro using a Caco-2 cell line, and also ex vivo using a porcine colonic tissue explant system. Caco-2 cells were stimulated with tumour necrosis factor alpha (TNFα) and co-treated with casein hydrolysates for 24 h. Following this, interleukin (IL)-8 concentrations in the supernatant were measured using enzyme-linked immunosorbent assay. Porcine colonic tissue was stimulated with lipopolysaccharide and co-treated with casein hydrolysates for 3 h. The expression of a panel of inflammatory cytokines was measured using qPCR. While dexamethasone reduced the IL-8 concentration by 41.6%, the 1 kDaR and 1 kDaP fractions reduced IL-8 by 68.7% and 66.1%, respectively, relative to TNFα-stimulated Caco-2 cells (P < 0.05). In the ex vivo system, only the 1 kDaR fraction elicited a decrease inIL1-α,IL1-β,IL-8,TGF-β andIL-10 expression (P < 0.05). This study provides evidence that the bioactive peptides present in the 1 kDaR fraction of the NaCAS hydrolysate possess anti-inflammatory properties in vitro and ex vivo. Further in vivo analysis of the anti-inflammatory properties of the 1 kDaR is proposed.

Production of whey protein isolate hydrolysate fractions with enriched ACE-inhibitory activity

A study on the enrichment of angiotensin-converting enzyme (ACE) inhibitory activity in whey protein isolate (WPI) hydrolysate fractions is presented. A previously identified low molecular mass fraction (1 kDa permeate) of an enzymatically hydrolysed heat-treated WPI with elevated ACE-inhibition (IC50 = 0.23 g L−1) was subjected to cascade membrane ultrafiltration (UF) and diafiltration steps at lab-scale. Assaying for ACE-inhibition revealed that the 1 kDa retentate demonstrated the highest ACE-inhibitory activity (IC50 = 0.17 g L−1). Isoelectric focussing (IEF) of the hydrolysate fraction further increased ACE-inhibition in fractions collected within the pH range 6.1–6.6. Overall, both UF and IEF enriched the ACE inhibitory activity in the original fraction by ∼52%, demonstrating the potential for enrichment of bio-functional activities in enzymatic hydrolysates of whey proteins.

Enhancing the lipolysis-stimulating activity of soy protein using limited hydrolysis with Flavourzyme and ultrafiltration.

The aim of this study was to explore the lipolysis-stimulating activity of soy protein isolate (SPI) hydrolysate using 3T3-L1 adipocytes. Intracellular triglyceride residue (TR) was employed as a marker for lipolysis in cells. The lower TR represents the better lipolysis-stimulating activity. SPI was hydrolysed with Flavourzyme for 2 h to obtain the hydrolysate FH2h, which showed lipolysis-stimulating activity in adipocytes at 400-1600 ppm levels. The sequential fractionation of FH2h with 30-0.3 kDa molecular weight cut-off (MWCO) membranes in order to obtain a 1 kDa retentate resulted in further enhancement of its lipolysis-stimulating activity in the cells. The TR decreased significantly from 2.73 to 2.30 μmole/mg protein at the 400 ppm level (p<0.05). Based on the western immunoblot and immunostaining analysis, the 1 kDa retentate promotes lipolysis by increasing phosphorylation and translocation of the hormone-sensitive lipase in 3T3-L1 adipocytes.

Separation and Purification of Hemicellulose by Ultrafiltration

During hydrolysis treatments, where the main objective is the solubilization of hemicelluloses, several undesirable reactions may occur. As a result, monomeric sugars and nonsaccharide compounds are obtained in the liquor. In the present study, autohydrolysis liquor resulting from the corn waste hydrolysis treatment (180 °C, 30 min) was filtered successively increasing the membrane cutoff (1, 5, and 10 kDa) to observe the potential of this technology for hemicellulose purification and fractionation by molecular weight. The experimental results showed that the 10 kDa retentate liquor fraction has the highest hemicellulose concentration (6.10 g/L) and it was constituted by defined molecular weight components (Mw = 2880), with low content in smaller compounds and inorganic material. Hemicelluloses have been isolated from each fraction and were analyzed by different analytical techniques (FT-IR, TGA, 1H NMR, and CHNS–O elemental analysis) to determine that it was possible to improve the physicochemical charac...

Impact of white wine macromolecules on fractionation performance of ultrafiltration membranes

&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Aim&lt;/strong&gt;: The aim of this study was to investigate the performance of ultrafiltration (UF) membranes to achieve a selective separation of macromolecules from Sauvignon blanc wine into stable and unstable fractions and to characterize the main compounds separated.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Methods and results&lt;/strong&gt;: The macromolecules from two Sauvignon blanc wines (Curicó Valley and Casablanca Valley, Chile) were separated by a cascade of UF membranes into three nominal fractions (10-30, 30-100 and 100-300 kDa). These fractions were characterized by native and SDS electrophoresis and membrane performance was evaluated by protein rejection and transmission. Separation by UF allowed the concentration of thermally unstable proteins in the 10-30 kDa retentate fraction, increasing heat induced haze by 8.9 fold, while heat stable glycoproteins were concentrated into the 100-300 kDa retentate fraction, reducing heat induced haze by 5.3 fold compared to unfiltered wine. The retention of high macromolecular species by the UF membrane with a 100 kDa molecular weight cut-off contributed to increased protein aggregation in the filtered wines.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: The concentration and purification of anti-hazing compounds by membrane filtration seem to be a new technology to improve the protein stability of white wines.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Significance and impact of the study&lt;/strong&gt;: Specific wine proteins are responsible for wine instability, resulting in haze formation in white wines. On the other hand, glycoproteins prevent protein aggregation and precipitation, thereby improving wine stability. Fractionation of wine macromolecules by UF membranes may help us to improve our knowledge about the contribution of specific proteins and glycoproteins to the haze stability of white wines.&lt;/p&gt;

Three-dimensional fluorescence characteristics of dissolved organic matter produced by Prorocentrum donghaiense Lu

Filtration and cross-flow ultrafiltration techniques were used to separate culture media of Prorocentrum donghaiense at the exponential growth, stationary and decline stages into <0.45 µm filtrate, 100 kDa-0.45 µm, 10–100 kDa and 1–10 kDa retentate and <1 kDa ultrafiltrate fractions. The fluorescence properties of different molecular weights of dissolved organic matter (DOM) were measured by excitation-emission matrix spectra. Protein-like and humic-like fluorophores were observed in the DOM produced by P. donghaiense. The central positions of protein-like fluorophores showed a red shift with prolonged growth duration, shifting from tyrosine-like properties at the exponential growth stage to tryptophan-like properties at the stationary and decline stages. The excitation wavelengths of protein-like fluorophores exhibited some change in the exponential growth and stationary stages with increased molecular size, but showed little change in the decline stage. However, the emission wavelengths in the decline stage exhibited a blue shift. Very distinct C type and A type peaks in humic-like fluorophores were observed. With a prolonged culture time, the intensities of both of the peaks became strong and the excitation wavelengths of peak A showed a red shift, while the A:C ratios fell. More than 94% of fluorescent DOM was in the lower than 1 kDa molecular weight fraction.