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High-throughput sequencing of ribosomal RNA (rRNA) amplicons has served as a cornerstone in microbiome studies. Despite crucial implication of organelle 16S rRNA measurements to host gut microbial activities, genomic DNA (gDNA) was overwhelmingly targeted for amplicon sequencings. Although gDNA could be a reliable resource for gene existing validation, little information is revealed in regard to the activity of microorganisms owing to the limited changes gDNA undertaken in inactive, dormant, and dead bacteria. We applied both rRNA- and gDNA-derived sequencings on mouse cecal contents. Respective experimental designs were verified to be suitable for nucleic acid (NA) purification. Via benchmarking, mainstream 16S rRNA hypervariable region targets and reference databases were proven adequate for respective amplicon sequencing study. In phylogenetic studies, significant microbial composition differences were observed between two methods. Desulfovibrio spp. (an important group of anaerobic gut microorganisms that has caused analytical difficulties), Pediococcus spp., and Proteobacteria were drastically lower as represented by gDNA-derived compositions, while microbes like Firmicutes were higher as represented by gDNA-derived microbiome compositions. Also, using PICRUSt2 as an example, we illustrated that rRNA-derived sequencing might be more suitable for microbiome function predictions since pathways like sugar metabolism were lower as represented by rRNA-derived results. The findings of this study demonstrated that rRNA-derived amplicon sequencing could improve identification capability of specific gut microorganisms and might be more suitable for in silico microbiome function predictions. Therefore, rRNA-derived amplicon sequencings, preferably coupled with gDNA-derived ones, could be used as a capable tool to unveil active microbial components in host gut. KEY POINTS: • Conventional pipelines were adequate for the respective amplicon sequencing study • Groups, such as Desulfovibrio spp., were differently represented by two methods • Comparative amplicon sequencings could be useful in host active microbiota studies.

Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca

Raw skim milk was subjected to different heat treatments: thermization (65°C, 20 s), pasteurization (72°C, 15 s), and no heat treatment (milk was microfiltered using 1.4-µm membranes at 50°C for bacteria removal; 1.4 MF). The milk (thermized, pasteurized, and 1.4 MF) was cooled and stored at 2°C until processing (at least 24 h) with cold (∼6°C) microfiltration using a benchtop crossflow pilot unit (Pall Membralox XLAB 5, Pall Corp., Port Washington, NY) equipped with 0.1-µm nominal pore diameter ceramic Membralox membrane (ET1-070, α-alumina, Pall Corp.). The flux was monitored during the process, and β-casein transmission and removal were calculated. The study aimed to indicate the conditions that should be applied to maximize β-casein passage through the membrane during cold microfiltration (5.6 ± 0.4°C) of skim milk. The proper selection of heat treatment parameters (temperature, time) of the feed before the cold microfiltration process will increase β-casein removal. It is not clear whether the difference in β-casein transmission between 1.4 MF, thermized, and pasteurized milk results from the effect of heat treatment conditions on β-casein dissociation from the casein micelles or on passage of β-casein through the membrane. The values of the major parameters (permeation flux and tangential flow velocity, through the wall shear stress) responsible for a proper membrane separation process were considerably lower than the critical values. It seems that the viscosity of the retentate has a great effect on the performance of the microfiltration membranes for protein separation at refrigerated temperatures.

The Polish market for tvarog (curd cheese)-based products is growing continuously. The as- sortment of these goods include products with different textures, different additives and different flavours. In developing a successful food product one should ensure consumers are sufficiently involved during the development stages. Consumer-led food product development should be a standard procedure. The study aimed to develop a new variants of aerated tvarog spreads.The research was divided into the following steps: evaluation of consumer pref- erences (questionnaire); production of aerated tvarog cheeses (different flavor variants); physico-chemical evaluation of new products (composition, TPA test); evaluation of new products by consumers (questionnaire with a 5-point scale); sensory evaluation of new products by an expert panel (2 types of questionnaire).Sweet tvarog spreads were characterized by a higher degree of aeration than cheeses with a savory flavor. The savory variants had a semi-liquid consistency after aeration. This could result from the addition of salt (6%) to flavor preparation. The products stabilized by gelatine showed a higher degree of aeration than the ones with starch. The savory cheeses showed lower hardness than sweet cheeses (P0.05). Both consum- ers and experts gave higher marks to the sweet variants of manufactured tvarog cheeses.The addition of salt to savory flavor preparation probably affected the texture of these products, which was semi-liquid. Texture plays a very important role in the development of new products and affects consumersrsquo; preferences for the product.

Pasteurized skim milk was subjected to (1) microfiltration (MF) at 50°C and (2) MF at 6°C after storage at 2°C. The products of these treatments were retentate (RMF50) and permeate (PMF50), and retentate (RMF6) and permeate (PMF6), respectively. Additionally, RMF50 was subjected to (3) cold MF after water dilution to produce retentate (RMF6R) and permeate (PMF6R). Calcium migration was monitored by analyzing ionic, soluble, and total calcium content in feed, retentates, and permeates. The influence of calcium partitioning and calcium addition to feed, retentates, and retentates diluted with water was determined. Without CaCl2 addition, only skim milk, RMF50, and RMF6 coagulated after rennet addition. Higher true protein and casein content of RMF50 and RMF6 resulted in shorter time of renneting. The retentates diluted with water showed no signs of coagulation within 40 min. The addition of PMF6R to RMF50 did not affect rennet coagulation time within the observed 40 min in comparison to RMF50 + water. In general, higher CaCl2 addition resulted in shorter rennet coagulation time. Special attention should be paid to calcium partitioning during membrane processing of cheesemilk. The level of calcium addition should be adopted to calcium content in such cheesemilk, which is affected by conditions of the filtration process (i.e., concentration factor and temperature).

The aim of the study was to demonstrate the use of fractal image analysis as a possible tool to monitor the effect of pressurization on the crystallization pattern of anhydrous milk fat. This approach can be useful when developing new products based on milk fat. The samples were subjected to different hydrostatic pressure (100, 200, 300, and 400 MPa) and temperature (10 and 40°C) treatments. The crystallization microphotographs were taken with a scanning electron microscope. The image analysis of scanning electron microscope photographs was done to determine a fractal dimension. Milk-fat pressurization under the applied parameters resulted in slight, but statistically significant, changes in the course of crystallization curves, related to the triacylglycerol fraction crystallizing in the lowest temperature (I exothermic effect). These changes were dependent on the value of pressure but not dependent on the temperatures applied during the process of pressurization (at either 10 or 40°C). In turn, significant differences were observed in crystallization images of milk-fat samples subjected to this process compared with the control sample. The results of additional fractal analysis additionally demonstrated the highest degree of irregularity of the surface of the crystalline form for the nonpressurized sample and the samples pressurized at 200 and 300 MPa at 10°C. The lowest value of fractal dimension—indicative of the least irregularity—was achieved for the fat samples pressurized at 400 MPa, 10°C and at 100 MPa, 40°C. The possibilities of wider application of the fractal analysis for the evaluation of effects of parameters of various technological processes on crystallization properties of milk fat require further extensive investigations.

Raw milk (approximately 1,800 kg) was separated at 4°C, pasteurized (at 72°C for 16s), and split into 2 batches. One batch (620 kg) was microfiltered (MF) using pilot-scale ceramic uniform transmembrane pressure Membralox membranes (model EP1940GL0.1 μA, 0.1-μm alumina; Pall Corp., East Hills, NY) to produce retentate and permeate. The permeate from the MF uniform transmembrane pressure was casein-free skim milk (CFSM). The CFSM was MF using polymeric spiral-wound (SW) membranes (model FG7838-OS0x-S, 0.3 μm; Parker-Hannifin Corp., Process Advanced Filtration Division, Tell City, IN) at a concentration factor of 3× and temperature of 50°C. Following the processing of CFSM, the second batch of skim milk (1,105 kg) was processed using the same polymeric membranes to determine how casein content in the feed material for MF with polymeric membranes affects the performance of the system. There was little resistance to passage of milk serum proteins (SP) through a 0.3-μm polyvinylidene fluoride (PVDF) SW membrane at 50°C and no detectable increase in hydraulic resistance of the membrane when processing CFSM. Therefore, milk SP contributed little, if any, to fouling of the PVDF membrane. In contrast, when processing skim milk containing a normal concentration of casein, the flux was much lower than when processing CFSM (17.2 vs. 80.2 kg/m(2) per hour, respectively) and the removal of SP from skim milk with a single-pass 3× bleed-and-feed MF system was also much lower than from CFSM (35.2 vs. 59.5% removal, respectively). Thus, when processing skim milk with a PVDF SW membrane, casein was the major protein foulant that increased hydraulic resistance and reduced passage of SP through the membrane.

The goals of this study were to determine if adding annatto color to milk or applying a bleaching process to whey or microfiltration (MF) permeate influenced ultrafiltration (UF) flux, diafiltration (DF) flux, or membrane fouling during production of 80% whey protein concentrate (WPC80) or 80% serum protein concentrate (SPC80). Separated Cheddar cheese whey (18 vats using 900kg of whole milk each) and MF permeate of skim milk (18 processing runs using 800kg of skim milk each) were produced to make WPC80 and SPC80, respectively. The 6 treatments, replicated 3 times each, that constituted the 18 processing runs within either whey or MF permeate UF were as follows: (1) no annatto; (2) no annatto+benzoyl peroxide (BPO); (3) no annatto+hydrogen peroxide (H 2 O 2 ); (4) annatto; (5) annatto+BPO; and (6) annatto+H 2 O 2 . Approximately 700kg of whey or 530kg of MF permeate from each treatment were heated to 50°C and processed in 2 stages (UF and DF) with the UF system in batch recirculation mode using a polyethersulfone spiral-wound UF membrane with a molecular weight cutoff of 10,000Da. Addition of annatto color had no effect on UF or DF flux. The processes of bleaching whey or MF permeate with or without added color improved flux during processing. Bleaching with H 2 O 2 usually produced higher flux than bleaching with BPO. Bleaching with BPO increased WPC80 flux to a greater extent than it did SPC80 flux. Though no differences in mean flux were observed for a common bleaching treatment between the WPC80 and SPC80 production processes during the UF stage, mean flux during WPC80 DF was higher than mean flux during SPC80 DF for each bleaching treatment. Water flux values before and after processing were used to calculate a fouling coefficient that demonstrated differences in fouling which were consistent with flux differences among treatments. In both processes, bleaching with H 2 O 2 led to the largest reduction in fouling. No effect of annatto on fouling was observed. The reasons for flux enhancement associated with bleaching treatments are unclear.

The production of serum protein (SP) and micellar casein from skim milk can be accomplished using microfiltration (MF). Potential commercial applications exist for both SP and micellar casein. Our research objective was to determine the total SP removal and SP removal for each stage, and the composition of retentates and permeates, for a 3×, continuous bleed-and-feed, 3-stage, uniform transmembrane pressure (UTP) system with 0.1-μm ceramic membranes, when processing pasteurized skim milk at 50°C with 2 stages of water diafiltration. For each of 4 replicates, about 1,100 kg of skim milk was pasteurized (72°C, 16 s) and processed at 3× through the UTP MF system. Retentate from stage 1 was cooled to

The objectives of this study were to identify and compare the composition, flavor, and volatile components of serum protein concentrate (SPC) and whey protein concentrate (WPC) containing about 34% protein made from the same milk to each other and to commercial 34% WPC from 6 different factories. The SPC and WPC were manufactured in triplicate with each pair of serum and traditional whey protein manufactured from the same lot of milk. At each replication, SPC and WPC were spray dried (SD) and freeze dried (FD) to determine the effect of the heat used in spray drying on sensory properties. A trained sensory panel documented the sensory profiles of rehydrated SD or FD powders. Volatile components were extracted by solid-phase microextraction (SPME) and solvent extraction followed by solvent-assisted flavor evaporation (SAFE) with gas chromatography-mass spectrometry and gas chromatography-olfactometry. Whey protein concentrates had higher fat content, calcium, and glycomacropeptide content than SPC. Color differences (Hunter L, a, b) were not evident between SPC and WPC powders, but when rehydrated, SPC solutions were clear, whereas WPC solutions were cloudy. No consistent differences were documented in sensory profiles of SD and FD SPC and WPC. The SD WPC had low but distinct buttery (diacetyl) and cardboard flavors, whereas the SD SPC did not. Sensory profiles of both rehydrated SD products were bland and lower in overall aroma and cardboard flavor compared with the commercial WPC. Twenty-nine aroma impact compounds were identified in the SPC and WPC. Lipid and protein oxidation products were present in both products. The SPC and WPC manufactured in this study had lower total volatiles and lower concentrations of many lipid oxidation compounds when compared with commercial WPC. Our results suggest that when SPC and WPC are manufactured under controlled conditions in a similar manner from the same milk using the same ultrafiltration equipment, there are few sensory differences but distinct compositional and physical property differences that may influence functionality. Furthermore, flavor (sensory and instrumental) properties of both pilot-scale manufactured protein powders were different from commercial powders suggesting the role of other influencing factors (e.g., milk supply, processing equipment, sanitation).

Raw milk (about 500 kg) was cold (4°C) separated and then the skim milk was pasteurized at 72°C and a holding time of 16s. The milk was cooled to 4°C and stored at ≤ 4°C until processing. The skim milk was microfiltered using a pilot-scale ceramic graded permeability (GP) microfilter system equipped with 0.1-µm nominal pore diameter ceramic Membralox membranes. First, about 155 kg of pasteurized skim milk was flushed through the system to push the water out of the system. Then, additional pasteurized skim milk (about 320 kg) was microfiltered (stage 1) in a continuous feed-and-bleed 3× process using the same membranes. The retentate from stage 1 was diluted with pasteurized reverse osmosis water in a 1:2 ratio and microfiltered (stage 2) with a GP system. This was repeated 3 times, with total of 3 stages in the process (stage 1 = microfiltration; stages 2 and 3 = diafiltration). The results from first 3 stages of the experiment were compared with previous data when processing skim milk at 50°C using ceramic uniform transmembrane pressure (UTP) membranes. Microfiltration of skim milk using ceramic UTP and GP membranes resulted in similar final retentate in terms of serum proteins (SP) removed. The SP removal rate (expressed by kilogram of SP removed per meter-squared of membrane area) was higher for GP membranes for each stage compared with UTP membranes. A higher passage of SP and SP removal rate for GP than UTP membranes was achieved by using a higher cross-flow velocity when processing skim milk. Increasing flux in subsequent stages did not affect membrane permeability and fouling. We operated under conditions that produced partial membrane fouling, due to using a flux that was less than limiting flux but higher than critical flux. Because the critical flux is a function of the cross-flow velocity, the difference in critical flux between UTP and GP membranes resulted only from operating under different cross-flow velocities (6.6 vs 7.12 for UTP and GP membranes, respectively). Conditions that allow microfiltration operation at higher flux will reduce the membrane surface area required to process the same amount of milk in the same length of time. Less membrane surface area reduces investment costs and uses less energy, water, and chemicals to clean the microfiltration system.

Most current research has focused on using ceramic microfiltration (MF) membranes for micellar casein concentrate production, but little research has focused on the use of polymeric spiral-wound (SW) MF membranes. A method for the production of a serum protein (SP)–reduced micellar casein concentrate using SW MF was compared with a ceramic MF membrane. Pasteurized (79°C, 18s) skim milk (1,100kg) was microfiltered at 50°C [about 3 × concentration] using a 0.3-μm polyvinylidene fluoride spiral-wound membrane, bleed-and-feed, 3-stage process, using 2 diafiltration stages, where the retentate was diluted 1:2 with reverse osmosis water. Skim milk, permeate, and retentate were analyzed for SP content, and the reduction of SP from skim milk was determined. Theoretically, 68% of the SP content of skim milk can be removed using a single-stage 3× MF. If 2 subsequent water diafiltration stages are used, an additional 22% and 7% of the SP can be removed, respectively, giving a total SP removal of 97%. Removal of SP greater than 95% has been achieved using a 0.1-μm pore size ceramic uniform transmembrane pressure (UTP) MF membrane after a 3-stage MF with diafiltration process. One stage of MF plus 2 stages of diafiltration of 50°C skim milk using a polyvinylidene fluoride polymeric SW 0.3-μm membrane yielded a total SP reduction of only 70.3% (stages 1, 2, and 3: 38.6, 20.8, and 10.9%, respectively). The SP removal rate for the polymeric SW MF membrane was lower in all 3 stages of processing (stages 1, 2, and 3: 0.05, 0.04, and 0.03 kg/m 2 per hour, respectively) than that of the comparable ceramic UTP MF membrane (stages 1, 2, and 3: 0.30, 0.11, and 0.06 kg/m 2 per hour, respectively), indicating that SW MF is less efficient at removing SP from 50°C skim milk than the ceramic UTP system. To estimate the number of steps required for the SW system to reach 95% SP removal, the third-stage SP removal rate (27.4% of the starting material SP content) was used to extrapolate that an additional 5 water diafiltration stages would be necessary, for a total of 8 stages, to remove 95% of the SP from skim milk. The 8-plus stages necessary to remove >95% SP for the SW MF membrane would create more permeate and a lengthier process than required with ceramic membranes.

Raw milk (2,710 kg) was separated at 4 degrees C, the skim milk was pasteurized (72 degrees C, 16 s), split into 3 batches, and microfiltered using pilot-scale ceramic uniform transmembrane pressure (UTP; Membralox model EP1940GL0.1microA, 0.1 microm alumina, Pall Corp., East Hills, NY), ceramic graded permeability (GP; Membralox model EP1940GL0.1microAGP1020, 0.1 microm alumina, Pall Corp.), and polymeric spiral-wound (SW; model FG7838-OS0x-S, 0.3 microm polyvinylidene fluoride, Parker-Hannifin, Process Advanced Filtration Division, Tell City, IN) membranes. There were differences in flux among ceramic UTP, ceramic GP, and polymeric SW microfiltration membranes (54.08, 71.79, and 16.21 kg/m2 per hour, respectively) when processing skim milk at 50 degrees C in a continuous bleed-and-feed 3x process. These differences in flux among the membranes would influence the amount of membrane surface area required to process a given volume of milk in a given time. Further work is needed to determine if these differences in flux are maintained over longer processing times. The true protein contents of the microfiltration permeates from UTP and GP membranes were higher than from SW membranes (0.57, 0.56, and 0.38%, respectively). Sodium-dodecyl-sulfate-PAGE gels for permeates revealed a higher casein proportion in GP and SW permeate than in UTP permeate, with the highest passage of casein through the GP membrane under the operational conditions used in this study. The slight cloudiness of the permeates produced using the GP and SW systems may have been due to the presence of a small amount of casein, which may present an obstacle in their use in applications when clarity is an important functional characteristic. More beta-lactoglobulin passed through the ceramic membranes than through the polymeric membrane. The efficiency of removal of serum proteins in a continuous bleed-and-feed 3x process at 50 degrees C was 64.40% for UTP, 61.04% for GP, and 38.62% for SW microfiltration membranes. The SW polymeric membranes had a much higher rejection of serum proteins than did the ceramic membranes, consistent with the sodium-dodecyl-sulfate PAGE data. Multiple stages and diafiltration would be required to produce a 60 to 65% serum protein reduced micellar casein concentrate with SW membranes, whereas only one stage would be needed for the ceramic membranes used in this study.