Feed substrates influence biofilm formation on reverse osmosis membranes and their cleaning efficiency

The dairy industry is increasingly using reverse osmosis (RO) membranes for concentration of various fluid feed materials such as whey and ultrafiltration (UF) permeate. This study compared the effect of UF permeate and whey on membrane biofilm formation. A Bacillus sp., previously isolated in our laboratory from a cleaning-resistant membrane biofilm, was used to develop 48-h-old static biofilms on RO membrane pieces, using the different feed substrates (UF permeate, whey, and an alternating whey/UF feed). Biofilms were analyzed for viable counts by the swab technique, and we used scanning electron and atomic force microscopy for microstructure imaging. The membrane cleaning process included 6 sequential steps. We observed differences in the resistance pattern of the 3 types of biofilms to the typical cleaning process. The mean pretreatment counts of the 48-h UF permeate biofilms were 5.39 log cfu/cm², much higher than the whey biofilm counts of 3.44 log, and alternating whey/UF biofilm counts of 4.54 log. After a 6-step cleaning cycle, we found 2.54 log survivors of the Bacillus isolate on UF biofilms, whereas only 1.82 log survivors were found in whey biofilm, and 2.14 log survivors on whey/UF permeate biofilms. In conclusion, the UF permeate biofilms was more resistant to the biofilm cleaning process compared with the whey or whey/UF permeate biofilms. Scanning electron micrographs showed different microstructures of biofilms based on the type of feed. For UF permeate and whey/UF permeate biofilms, bacilli were present in multilayers of cells in aggregates or irregular clusters with foulant layers. In contrast, those in whey biofilms were in monolayers, with a smoother, flatter appearance. Atomic force microscopy analysis indicated that UF permeate biofilms had the greatest surface roughness among the biofilms, reflecting intensified bacterial colonization. The biofilm micro- and nanostructure variations for the 2 feed substrates and their combination may have resulted in differences in their resistance to the cleaning process.     

Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol

This experiment evaluates the effectiveness of individual steps of a clean-in-place protocol against the biofilm constitutive microflora isolated from the biofilms developed on whey reverse-osmosis membranes, aged 2 to 14 mo, under industrial processing conditions. The isolates used for the in vitro resistance studies included species of Bacillus, Enterococcus, Streptococcus, Staphylococcus, Micrococcus, Aeromonas, Corynebacterium, Pseudomonas, Klebsiella, and Escherichia. The 6 cleaning steps (alkali, surfactant, acid, enzyme, a second surfactant, and sanitizer treatment) revealed resistance of isolates in both planktonic and biofilm-embedded cell states. The most effective step was the acid treatment, which resulted in 4.54 to 7.90 and 2.09 to 5.02 log reductions of the planktonic and biofilm-embedded cells, respectively. Although the sanitizer step causing a reduction of 4.91 to 8.33 log in the case of planktonic cells, it was less effective against the biofilm-embedded cells, resulting in a reduction of 0.59 to 1.64 log. Bacillus spp. showed the highest resistance in both planktonic, as well as embedded cell states.     

Growth potential and biofilm development of nonstarter bacteria on surfaces exposed to a continuous whey stream

Commercial Cheddar cheese production uses an automated, continuous production system that provides favorable conditions for specific undesirable bacterial subpopulations in certain sections of the processing system. The draining and matting conveyor (DMC) is a large, fully enclosed series of conveyor belts that separates curd and whey on the first drain belt and supports the cheddaring process in subsequent sections. In a previous study, we demonstrated that coliforms increase in the draining section of the DMC (pH 6.0–6.3, 36°C) over a typical 18-h production shift and can lead to detectable coliforms in finished cheese. Sampling at the commercial plant indicated 2 sources of very low levels of coliforms: (1) subpasteurized whey and curd entering the DMC and (2) surfaces in the DMC after sanitation. Mitigation of these sources would require different approaches. The aim of this study was to investigate whether naturally low levels of coliforms in whey could increase in the bulk liquid and attach to different surface materials within 18 h. A laboratory-scale system was created to mimic the conditions of the initial draining section of the DMC and consisted of single-pass, naturally contaminated whey (pH 6.3, 35°C) flowing through a bioreactor (1.11 L/h) containing coupons of surface types found in the DMC (stainless steel and polypropylene). Whey inside the bioreactor chamber and surface coupons were enumerated for bacterial subpopulations on selective media for planktonic and attached bacteria, respectively, at 0, 12, 15, and 18 h. Bacterial isolates were identified by 16S rDNA sequencing. Nonstarter bacteria present in the whey at 0 h included coliforms (Enterobacter), Pseudomonas, and Acinetobacter (0.80, 2.55, and 2.32 log cfu/mL, respectively), with each increasing significantly in whey (6.18, 7.00, and 5.89 log cfu/mL) and on coupons (5.20, 6.85, and 5.29 log cfu/cm², respectively) after 18 h in the continuous flowing system. Scanning electron microscopy confirmed bacterial attachment on both surfaces, with early biofilm development evident on polypropylene coupons by 18 h. Results from this laboratory-scale study demonstrated that naturally low levels of coliforms entering the DMC in the whey could replicate within the conditions of the draining section of the DMC to the levels found in the commercial production environment.     

Preparation and composition of chhana whey powders using membrane processing techniques

Chhana is a traditional Indian product used widely in the confectionery industry. It is produced from cow's milk by a combination of heat and acid coagulation. Chhana whey contains about 6% milk solids yet the vast majority is wasted which leads to pollution problems. This study describes the chemical composition and various options for utilisation of chhana whey using membrane processes. Chhana whey powder containing 956 g kg^?1 total solids, 750 g kg^?1 lactose, 21 g kg^?1 protein. 60 g kg^?1 fat, 65 g kg^?1 ash was produced following concentration of chhana whey by reverse osmosis. Chhana whey protein concentrate powders containing 270, 350, 400 and 580 g kg^?1 protein were produced following ultrafiltration or diafiltration of chhana whey.     

膜技术在处理大豆乳清废水中的应用

大豆乳清废水经预处理后,采用超滤膜技术回收含低聚糖废水中的乳清蛋白,再用纳滤膜脱盐、浓缩低聚糖,滤液过反渗透膜即可达到回用或排放要求。探讨了预处理工序的必要性,考察了不同型号膜的运行情况并进行了选择。实验证明,该工艺简单、节能、易操作,污水零排放,且回收产品质量有较大提高,中试数据可供工业化参考与借鉴。     

Efficiency of removal of whey protein from sweet whey using polymeric microfiltration membranes

Our objective was to measure whey protein removal percentage from separated sweet whey using spiral-wound (SW) polymeric microfiltration (MF) membranes using a 3-stage, 3× process at 50°C and to compare the performance of polymeric membranes with ceramic membranes. Pasteurized, separated Cheddar cheese whey (1,080 kg) was microfiltered using a polymeric 0.3-μm polyvinylidene (PVDF) fluoride SW membrane and a 3×, 3-stage MF process. Cheese making and whey processing were replicated 3 times. There was no detectable level of lactoferrin and no intact α- or β-casein detected in the MF permeate from the 0.3-μm SW PVDF membranes used in this study. We found BSA and IgG in both the retentate and permeate. The β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) partitioned between retentate and permeate, but β-LG passage through the membrane was retarded more than α-LA because the ratio of β-LG to α-LA was higher in the MF retentate than either in the sweet whey feed or the MF permeate. About 69% of the crude protein present in the pasteurized separated sweet whey was removed using a 3×, 3-stage, 0.3-μm SW PVDF MF process at 50°C compared with 0.1-μm ceramic graded permeability MF that removed about 85% of crude protein from sweet whey. The polymeric SW membranes used in this study achieve approximately 20% lower yield of whey protein isolate (WPI) and a 50% higher yield of whey protein phospholipid concentrate (WPPC) under the same MF processing conditions as ceramic MF membranes used in the comparison study. Total gross revenue from the sale of WPI plus WPPC produced with polymeric versus ceramic membranes is influenced by both the absolute market price for each product and the ratio of market price of these 2 products. The combination of the market price of WPPC versus WPI and the influence of difference in yield of WPPC and WPI produced with polymeric versus ceramic membranes yielded a price ratio of WPPC versus WPI of 0.556 as the cross over point that determined which membrane type achieves higher total gross revenue return from production of these 2 products from separated sweet whey. A complete economic engineering study comparison of the WPI and WPPC manufacturing costs for polymeric versus ceramic MF membranes is needed to determine the effect of membrane material selection on long-term processing costs, which will affect net revenue and profit when the same quantity of sweet whey is processed under various market price conditions.     

溶液pH对不同超滤膜超滤大豆黄浆水的影响

实验研究溶液pH值对不同超滤膜超滤大豆黄浆水的影响。结果表明,当溶液pH值偏离大豆蛋白等电点时,渗透通量增大,膜衰减系数减小,蛋白质截留率变化不大,总糖透过率略有上升,PES-10膜的适宜pH为11,PS-10膜的适宜pH为6.2。     

Survey of salty and sweet whey composition from various cheese plants in Wisconsin

Salty whey is currently underutilized in the dairy industry because of its high salt content and increased processing and disposal costs. Salty whey accounts for 2 to 5% of the total whey generated during Cheddar and other dry-salted cheese manufacture. Because relatively little information is available on salty whey, this study was conducted to determine the range of compositional components in salty whey from commercial cheese plants. Gross compositional differences in percent protein, salt, solids, and fat between sweet whey and salty whey from various dry-salted cheeses from 8 commercial plants were determined. Differences between individual whey protein compositions were determined using sodium dodecyl sulfate-PAGE. Average total solids, fat, and salt content were significantly greater in the salty whey compared with the corresponding sweet whey. True protein was reduced in salty whey although great variability existed among samples. Individual whey proteins identified included lactoferrin (Lf), BSA, immunoglobulin G, β-lactoglobulin, and α-lactalbumin. Salty whey showed an increase in Lf content and a decrease in α-lactalbumin and β-lactoglobulin content when compared with sweet whey. Salty whey may be a source of Lf, potentially increasing its value to whey processors. However, the compositional assessments showed that commercial salty whey is a highly variable waste stream.     

Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms

This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.     

Determination of the efficiency of removal of whey protein from sweet whey with ceramic microfiltration membranes

Our research objective was to measure percent removal of whey protein from separated sweet whey using 0.1-µm uniform transmembrane pressure ceramic microfiltration (MF) membranes in a sequential batch 3-stage, 3× process at 50°C. Cheddar cheese whey was centrifugally separated to remove fat at 72°C and pasteurized (72°C for 15 s), cooled to 4°C, and held overnight. Separated whey (375 kg) was heated to 50°C with a plate heat exchanger and microfiltered using a pilot-scale ceramic 0.1-µm uniform transmembrane pressure MF system in bleed-and-feed mode at 50°C in a sequential batch 3-stage (2 diafiltration stages) process to produce a 3× MF retentate and MF permeate. Feed, retentate, and permeate samples were analyzed for total nitrogen, noncasein nitrogen, and nonprotein nitrogen using the Kjeldahl method. Sodium dodecyl sulfate-PAGE analysis was also performed on the whey feeds, retentates, and permeates from each stage. A flux of 54 kg/m² per hour was achieved with 0.1-µm ceramic uniform transmembrane pressure microfiltration membranes at 50°C. About 85% of the total nitrogen in the whey feed passed though the membrane into the permeate. No passage of lactoferrin from the sweet whey feed of the MF into the MF permeate was detected. There was some passage of IgG, bovine serum albumen, glycomacropeptide, and casein proteolysis products into the permeate. β-Lactoglobulin was in higher concentration in the retentate than the permeate, indicating that it was partially blocked from passage through the ceramic MF membrane.     

反渗透浓缩籽瓜汁的实验研究

采用反渗透技术对籽瓜汁进行浓缩,分别研究了膜通量、压力、料液温度、籽瓜汁浓度等参数之间的关系,结果表明:反渗透膜对籽瓜汁有效成分的截留率接近100%,对无机盐截留率大于98%,籽瓜汁的最终浓缩浓度为20Brix;其它条件不变的情况下,膜通量与压力、料液温度成正比关系,与料液浓度成反比关系。      

乳清多肽的制备及其发酵饮料的开发

研究了乳清多肽的制备、性质及其发酵饮料的开发,结果表明,碱性蛋白酶比中性蛋白酶水解乳清蛋白的能力强,且更经济,水解最佳条件为加酶量为7000(U/g蛋白)、底物添加量为5%、水解温度为60℃、水解初始pH值为8.5,最大乳清蛋白水解度可达到22.45%。最优酒精发酵条件为接种量5%、初始pH7.5、温度22℃、时间45h。乳清多肽发酵饮料的配方为酸量0.1%,蔗糖量为8%,-β环状糊精量为0.5%。     

酪蛋白与乳清蛋白比例对酸奶凝胶性质的影响

研究了乳中酪蛋白和乳清蛋白比例对凝固型酸奶流变学特性和微观结构的影响,结果表明,固定蛋白质质量分数、降低酪蛋白和乳清蛋白的比例,可以明显提高酸奶凝胶的质量.乳中蛋白质质量分数一致时,酸奶凝胶的硬度、黏度、持水力随着酪蛋白和乳清蛋白比例的减小而增大,凝胶网络结构变得更规则、致密,孔隙更小.在低蛋白质质量分数下,降低乳中酪...     

Effect of whey concentration on protein recovery in fresh ovine ricotta cheese

Ricotta cheese, particularly the ovine type, is a typical Italian dairy product obtained by heat-coagulation of the proteins in whey. The aim of this work was to investigate the influence of whey protein concentration, obtained by ultrafiltration, on yield of fresh ovine ricotta cheese. Ricotta cheeses were obtained by thermocoagulation of mixtures with protein content of 1.56, 3.10, 4.16, and 7.09 g/100 g from the mixing of skim whey and ultrafiltered skim whey. A fat-to-protein ratio of 1.1 (wt/wt) was obtained for all mixtures by adding fresh cream. The initial mixtures, as well as the final ricotta cheeses, were analyzed for their composition and by SDS-PAGE. Protein bands were quantified by QuantityOne software (Bio-Rad, Hercules, CA) and identified by liquid chromatography-tandem mass spectrometry. Significant differences in the composition of the ricotta cheese were observed depending on protein concentration. Particularly, ricotta cheese resulting from the mixture containing 7.09 g/100 g of protein presented higher moisture (72.88 ± 1.50 g/100 g) and protein (10.18 ± 0.45 g/100 g) contents than that prepared from the mixture with 1.56 g/100 g of protein (69.52 ± 1.75 and 6.70 ± 0.85 g/100 g, respectively), and fat content was lower in this sample (12.20 ± 1.60 g/100 g) compared with the other treatments, with mean values between 15.72 and 20.50 g/100 g. Each protein fraction presented a different behavior during thermocoagulation. In particular, the recovery of β-lactoglobulin and α-lactalbumin in the cheese increased as their content increased in the mixtures. It was concluded that concentrating ovine rennet whey improved the extent of heat-induced protein aggregation during the thermal coagulation process. This resulted in a better recovery of each protein fraction in the product, and in a consequent increase of ricotta cheese yield.     

Selective separation of the major whey proteins using ion exchange membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate β-lactoglobulin, BSA, and α-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for β-lactoglobulin with less than 1% of the eluate consisting of α-lactalbumin or BSA. The binding capacity of a pure β-lactoglobulin solution was in excess of 1.5 mg/cm² of membrane. This binding capacity was reduced to approximately 1.2 mg/cm² when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that α-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by β-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10 mM sodium phosphate buffer (pH 6.4). Binding capacities of β-lactoglobulin, α-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.     

Comparison of the behavior of two nanofiltration membranes for sweet whey demineralization

Nanofiltration is a process used to separate mineral salts from lactose, having previously removed the proteins by ultrafiltration. Both proteins and lactose can be used as raw materials to prepare a variety of products. In this paper, we studied the feasibility of demineralizing sweet whey obtained from the cheese industry of the Comunidad Valenciana (Spain) using membrane technologies. The NF200 membrane showed the highest volumetric flux and solute rejection values, whereas the DS-5 DL membrane showed the lowest values. The volumetric fluxes obtained with the NF200 and DS-5 DL membranes in these experiments with the ultra-filtered whey demonstrated significant differences between membranes. Concerning solute rejection, the highest values were obtained using the NF200 membrane. The chosen parameter to evaluate the demineralization capability was solute flux. In this way, the values obtained for chloride ion were 9.90 and 32.42 g/ (m²·h) for the NF200 and DS-5 DL membranes, respectively, with the highest demineralization rates being achieved with the DS-5 DL membrane.     

Physical properties of yogurt fortified with various commercial whey protein concentrates

The effects of whey protein concentrates on physical and rheological properties of yogurt were studied. Five commercial whey protein concentrates (340 g kg^?1 protein nominal) were used to fortify milk to 45 g protein kg^?1. Fermentation was performed with two different starters (ropy and non‐ropy). Resulting yogurts were compared with a control yogurt enriched with skim milk powder. The water‐holding capacity of the yogurt fortified with skim milk powder was 500 g kg^?1 and ranged from 600 to 638 g kg^?1 when fortified with whey protein concentrates. Significant rheological differences have been noticed between the yogurts fortified with different whey protein concentrates, independent of the starter used. Three whey protein concentrates generated yogurts with a behavior similar to the control. The two others produced yogurt with lower firmness (15 g compared with 17 g), lower Brookfield viscosity (6 Pa s compared with 9 Pa s), lower yield stress (2 Pa compared with 4 Pa), lower complex viscosity (13 Pa s compared with 26 Pa s), and lower apparent viscosity (0.4 Pa s compared with 1 Pa s) than the control, respectively. The yogurts with the lowest firmness and viscosity were produced with concentrates which contained the highest amount of non‐protein nitrogen fraction (160 g kg^?1 versus 126 g kg^?1 of the total nitrogen), and the highest amount of denaturation of the whey protein (262 versus 200 g kg^?1 of the total nitrogen). Copyright © 2004 Society of Chemical Industry     

热处理温度及蛋白浓度对乳清分离蛋白-黄油乳液体系的影响

稳定的乳清分离蛋白（WPI）-黄油乳液体系在乳制品加工及乳制品营养传递系统中有良好的应用前景。对不同质量分数（2%、4%、6%、8%）的WPI分别进行不同的热处理（未加热、80 ℃和90 ℃）,加入黄油并进行超声波处理,制备成乳液,对乳液体系粒径、絮凝指数（FI）、乳化活性（EA）、乳化稳定性（ES）、物理稳定性、储藏期粒径变化和脂肪上浮情况进行分析。结果表明：随着热处理温度的升高,乳液的平均粒径增大,未加热乳液平均粒径均小于1 μm,经加热处理后,不同蛋白质量分数乳液的粒径均有不同程度的增大;经过热处理,乳液的EA和ES均有所改善;随着蛋白质量分数的增大,乳液的物理稳定性提高,其中WPI质量分数为6%和8%时,90 ℃热处理样品的稳定性指数（TSI）均小于0.6,稳定性最好,同一蛋白质量分数下,热处理温度越高,蛋白对乳液的稳定作用越强;乳液储藏期脂肪上浮情况与热处理温度和蛋白质量分数显著相关,较高的蛋白质量分数及热处理温度能够改善乳液体系中脂肪上浮情况。研究表明,通过控制蛋白质量分数和WPI热处理温度可以有效提高WPI-黄油乳液体系的乳化特性及稳定性。     

A study of various chemical pretreatments to fractionate lipids from whey protein phospholipid concentrate

Dairy-derived lipids such as phospholipids (PL) have been gaining interest due to their functional and nutritional properties. Our research goal was to develop a separation process (nonsolvent based) to produce an enriched dairy lipid fraction from whey protein phospholipid concentrate (WPPC). Various chemical pretreatments (i.e., adjustment of pH, calcium, or temperature) were applied to rehydrated commercial WPPC solutions. These treatments were done on a bench-top scale to aid in the precipitation of proteins or PL. The chemically treated solutions were centrifuged and fractionated into the following 3 layers: (1) top fat layer, (2) supernatant in the middle zone, and (3) sediment at the bottom of the centrifuge tubes. The thickness and size of the layers varied with the treatment parameters. Compositional analysis of each layer showed that the proteins, fat, and PL always appeared to fractionate in similar proportions. The proteins in each layer were characterized using sodium dodecyl sulfate–PAGE under reducing and nonreducing conditions. Different proteins including whey proteins, caseins, and milk fat globule membrane proteins and lipoproteins were identified, and no specific type of protein had an affinity for either the top or bottom layer. All types of proteins were present in each of the layers after centrifugation, and there were no major differences in fractionation of the proteins between layers with respect to the chemical treatment applied. The microstructure of protein and fat in WPPC was investigated using confocal laser scanning microscopy. Dual staining of the rehydrated WPPC solution with Fast Green FCF (proteins) and Nile Red (lipids) showed the presence of very large protein aggregates that varied in size from 20 to 150 μm, with fat trapped within these aggregates. The confocal laser scanning microscopy images of liquid WPPC revealed fine strands of a weak protein network surrounding the fat globules. This indicated that there were specific interactions between the proteins, as well as between the fat and proteins in WPPC. Sodium dodecyl sulfate treatment was performed to understand the nature of the interactions between protein and fat. We found that about 35% of the fat present in WPPC was in the form of free fat, which was only physically entrapped within the protein aggregates. The remaining fat had some form of association with the proteins in WPPC. Other fractionation techniques would be needed to obtain an enriched dairy lipid fraction.