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Research| Volume 106, ISSUE 5, P3137-3154, May 2023

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Manufacture of a novel cultured micellar casein concentrate ingredient for emulsifying salt–free process cheese products applications

Open AccessPublished:March 10, 2023DOI:https://doi.org/10.3168/jds.2022-22652

      ABSTRACT

      Micellar casein concentrate (MCC) is a high protein ingredient that is typically produced using 3 stages of microfiltration with a 3× concentration factor and diafiltration. Acid curd is an acid protein concentrate, which can be obtained by precipitating the casein at pH 4.6 (isoelectric point) using starter cultures or direct acids without the use of rennet. Process cheese product (PCP) is a dairy food prepared by blending dairy ingredients with nondairy ingredients and then heating the mixture to get a product with an extended shelf-life. Emulsifying salts are critical for the desired functional characteristics of PCP because of their role in calcium sequestration and pH adjustment. The objectives of this study were to develop a process to produce a novel cultured micellar casein concentrate ingredient (cMCC; culture-based acid curd) and to produce PCP without emulsifying salts using different combinations of protein from cMCC and MCC in the formulations (2.0:1.0, 1.9:1.1, and 1.8:1.2). Skim milk was pasteurized at 76°C for 16 s and then microfiltered in 3 microfiltration stages using graded permeability ceramic membranes to produce liquid MCC (11.15% total protein; TPr and 14.06% total solids; TS). Part of the liquid MCC was spray dried to produce MCC powder (75.77% TPr and 97.84% TS). The rest of the MCC was used to produce cMCC (86.9% TPr and 96.4% TS). Three PCP treatments were formulated with different ratios of cMCC:MCC, including 2.0:1.0, 1.9:1.1, and 1.8:1.2 on the protein basis. The composition of PCP was targeted to 19.0% protein, 45.0% moisture, 30.0% fat, and 2.4% salt. This trial was repeated 3 times using different batches of cMCC and MCC powders. All PCP were evaluated for their final functional properties. No significant differences were detected in the composition of PCP made with different ratios of cMCC and MCC except for the pH. The pH was expected to increase slightly with elevating the MCC amount in the PCP formulations. The end apparent viscosity was significantly higher in 2.0:1.0 formulation (4,305 cP) compared with 1.9:1.1 (2,408 cP) and 1.8:1.2 (2,499 cP). The hardness ranged from 407 to 512 g with no significant differences within the formulations. However, the melting temperature showed significant differences with 2.0:1.0 having the highest melting temperature (54.0°C), whereas 1.9:1.1 and 1.8:1.2 showed 43.0 and 42.0°C melting temperature, respectively. The melting diameter (38.8 to 43.9 mm) and melt area (1,183.9 to 1,538.6 mm2) did not show any differences in different PCP formulations. The PCP made with a 2.0:1.0 ratio of protein from cMCC and MCC showed better functional properties compared with other formulations.

      Key words

      INTRODUCTION

      Microfiltration (MF) is a membrane process that is used to fractionate casein and serum protein (SP) from skim milk using a 0.1-µm semipermeable membrane. The skim milk is driven force through the membrane to separate casein (retentate side) and SP (permeate side) based on their sizes (0.1–0.4 μm vs. 0.003–0.01 μm, respectively). The product on the retentate side is called micellar casein concentrate (MCC), which is mostly native casein. The MCC is a high protein ingredient that is typically manufactured in 3 MF stages using a 3× concentration factor (CF) with diafiltration (DF). Several MF membranes have been studied to produce MCC (
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ), such as spiral-wound membranes (
      • Govindasamy-Lucey S.
      • Jaeggi J.J.
      • Johnson M.E.
      • Wang T.
      • Lucey J.A.
      Use of Cold Microfiltration Retentates Produced with Polymeric Membranes for Standardization of Milks for Manufacture of Pizza Cheese.
      ;
      • Lawrence N.D.
      • Kentish S.E.
      • O'Connor A.J.
      • Barber A.R.
      • Stevens G.W.
      Microfiltration of skim milk using polymeric membranes for casein concentrate manufacture.
      ;
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Efficiency of serum protein removal from skim milk with ceramic and polymeric membranes at 50°C.
      ;
      • Beckman S.L.
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Production efficiency of micellar casein concentrate using polymeric spiral-wound microfiltration membranes.
      ;
      • Beckman S.L.
      • Barbano D.M.
      Effect of microfiltration concentration factor on serum protein removal from skim milk using spiral-wound polymeric membranes.
      ;
      • Marella C.
      • Sunkesula V.
      • Hammam A.R.A.
      • Kommineni A.
      • Metzger L.E.
      Optimization of spiral-wound microfiltration process parameters for the production of micellar casein concentrate.
      ), isoflux ceramic membranes (
      • Adams M.C.
      • Barbano D.M.
      Serum protein removal from skim milk with a 3-stage, 3× ceramic Isoflux membrane process at 50°C.
      ), uniform transmembrane pressure (UTP) ceramic membranes (
      • Vadi P.K.
      • Rizvi S.S.H.
      Experimental evaluation of a uniform transmembrane pressure crossflow microfiltration unit for the concentration of micellar casein from skim milk.
      ;
      • Hurt E.
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Micellar casein concentrate production with a 3X, 3-stage, uniform transmembrane pressure ceramic membrane process at 50°C1.
      ), and graded permeability (GP) ceramic membranes (
      • Zulewska J.
      • Barbano D.M.
      The effect of linear velocity and flux on performance of ceramic graded permeability membranes when processing skim milk at 50°C.
      ;
      • Tremblay-Marchand D.
      • Doyen A.
      • Britten M.
      • Pouliot Y.
      A process efficiency assessment of serum protein removal from milk using ceramic graded permeability microfiltration membrane.
      ;
      • Hammam A.R.A.
      • Beckman S.L.
      • Metzger L.E.
      Production and storage stability of concentrated micellar casein.
      ). The spiral-wound membranes are cheaper and have lower operating costs, but they have a shorter shelf-life, low chemical stability, limited viscosity range, and less efficiency to remove SP as compared with ceramic membranes. The UTP and GP membranes are commonly used to produce MCC due to their high SP removal. The GP membranes have low operating costs (do not require permeate recirculation pump), although they are expensive compared with UTP membranes (
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ).
      The MCC has promising applications in some dairy and nondairy products due to its unique physicochemical and functional characteristics (e.g., foaming, emulsifying, wetting, dispersibility, heat stability, bland flavor, and water-binding ability). The high casein content in MCC makes it heat stable and thereby it can be used in beverages that require sterilization (
      • Beliciu C.M.
      • Sauer A.
      • Moraru C.I.
      The effect of commercial sterilization regimens on micellar casein concentrates.
      ;
      • Sauer A.
      • Moraru C.I.
      Heat stability of micellar casein concentrates as affected by temperature and pH.
      ). The nondairy applications for MCC are pasta, confectionery, meat products, special dietary preparations, textured products, convenience foods, toothpaste, cosmetics, and wound treating preparations (
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ). The dairy applications for MCC include cheddar cheese (
      • Amelia I.
      • Drake M.
      • Nelson B.
      • Barbano D.M.
      A new method for the production of low-fat Cheddar cheese.
      ;
      • Li B.
      • Waldron D.S.
      • Tobin J.T.
      • Subhir S.
      • Kelly A.L.
      • McSweeney P.L.H.
      Evaluation of production of Cheddar cheese from micellar casein concentrate.
      ;
      • Xia X.
      • Tobin J.T.
      • Subhir S.
      • Fenelon M.A.
      • McSweeney P.L.H.
      • Sheehan J.J.
      Effect of thermal treatment on serum protein reduced micellar casein concentrate: An evaluation of rennet coagulability, cheese composition and yield.
      ), Feta cheese (
      • Hammam A.R.A.
      • Kapoor R.
      • Salunke P.
      • Metzger L.E.
      Compositional and functional characteristics of feta-type cheese made from micellar casein concentrate.
      ), Greek-style yogurt (
      • Bong D.D.
      • Moraru C.I.
      Use of micellar casein concentrate for Greek-style yogurt manufacturing: Effects on processing and product properties.
      ), imitation mozzarella cheese (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ;
      • Salunke P.
      • Marella C.
      • Amamcharla J.K.
      • Muthukumarappan K.
      • Metzger L.E.
      Use of micellar casein concentrate and milk protein concentrate treated with transglutaminase in imitation cheese products—Unmelted texture.
      ), recombined MCC (
      • Lu Y.
      • McMahon D.J.
      • Vollmer A.H.
      Investigating cold gelation properties of recombined highly concentrated micellar casein concentrate and cream for use in cheese making.
      ,
      • Lu Y.
      • McMahon D.J.
      • Vollmer A.H.
      Investigating rennet coagulation properties of recombined highly concentrated micellar casein concentrate and cream for use in cheese making.
      ), process cheese (PC) and process cheese products (PCP;
      • Hammam A.R.A.
      • Beckman S.L.
      • Sunkesula V.
      • Metzger L.E.
      Highly concentrated micellar casein: Impact of its storage stability on the functional characteristics of process cheese products.
      ;
      • Salunke P.
      • Metzger L.E.
      Functional characteristics of process cheese product as affected by milk protein concentrate and micellar casein concentrate at different usage levels.
      ,
      • Salunke P.
      • Metzger L.E.
      Impact of transglutaminase treatment given to the skim milk before or after microfiltration on the functionality of micellar casein concentrate used in process cheese product and comparison with rennet casein.
      ), and acid curd (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ). We recently developed a process to produce soft acid curd (basically cottage cheese curd) from MCC using direct acidification (lactic acid;
      • Hammam A.R.A.
      • Kapoor R.
      • Metzger L.E.
      Manufacture of process cheese products without emulsifying salts using acid curd and micellar casein concentrate.
      ) and starter cultures (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ) to set the curd. Although direct acids such as lactic acid take less time to produce the acid curd, those acids are costly and produce acid curd with a bland flavor compared with the varieties of starter cultures that cost less and can be utilized to develop flavors in the curd.
      Acid curd is a protein concentrate, which can be obtained by precipitating the casein at pH 4.6 (isoelectric point) using starter cultures or direct acids without the use of rennet. The colloidal calcium phosphate that exists in the micelles is dissolved at the isoelectric point, which leads to a low mineral (calcium) content in the acid curd. In contrast to the low mineral content of acid curd, MCC has a high level of casein-bound calcium due to its pH of 6.5 to 6.7. Acid curd could be produced from skim milk in a process similar to cottage cheese manufacture (
      • Klei L.
      • Yun J.
      • Sapru A.
      • Lynch J.
      • Barbano D.
      • Sears P.
      • Galton D.
      Effects of milk somatic cell count on cottage cheese yield and quality.
      ). There is a possibility of using MCC instead of skim milk to produce acid curd. Making acid curd from MCC has advantages as compared with skim milk because manufacturing MCC using MF results in milk-derived whey protein as a byproduct, which can be used in many applications, particularly making whey protein isolate. In contrast, acid curd produced from skim milk results in acid whey as a byproduct, which is more difficult to use. The typical composition of MCC (3 stages using a 3× CF with a DF) is > 9% true protein (TP) and > 13% TS (
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Efficiency of serum protein removal from skim milk with ceramic and polymeric membranes at 50°C.
      ). This MCC could be used immediately in making acid curd or diluted to lower protein levels before making acid curd if required. For the rest of this paper, we will refer to acid curd made from micellar casein using cultures as cultured MCC ingredient (cMCC). In our previous studies, we tried to produce acid curd from MCC with different protein content (3, 6, and 9% protein). We found that MCC with 9% protein is the optimum product to produce acid curd (
      • Hammam A.R.A.
      • Kapoor R.
      • Metzger L.E.
      Manufacture of process cheese products without emulsifying salts using acid curd and micellar casein concentrate.
      ).
      Process cheese and PCP are dairy foods prepared by blending dairy ingredients (e.g., natural cheese, protein concentrates, butter, nonfat dry milk, whey powder, permeate) with nondairy ingredients [e.g., sodium chloride, water, emulsifying salts (ES), color, and flavors] and then heating the mixture with continuous agitation to produce a homogeneous product with an extended shelf-life (
      • Meyer A.
      Processed Cheese Manufacture.
      ;
      • Thomas M.A.
      The manufacture of processed cheese-Scientific principles.
      ;
      • Caric M.
      • Gantar M.
      • Kalab M.
      Effects of emulsifying agents on the microstructure and other characteristics of process cheese—A review.
      ;
      • Schmid A.
      Processed cheese manufacture.
      ;
      • Guinee T.P.
      • Carić M.
      • Kaláb M.
      Pasteurized processed cheese and substitute/imitation cheese products.
      ;
      • Mcsweeney P.L.H.
      ;
      • Kapoor R.
      • Metzger L.E.
      Process cheese: Scientific and technological aspects—A review.
      ;
      • Kammerlehner J.
      ;
      • Hammam A.R.A.
      • Beckman S.L.
      • Sunkesula V.
      • Metzger L.E.
      Highly concentrated micellar casein: Impact of its storage stability on the functional characteristics of process cheese products.
      ). Process cheese and PCP are remarkably similar products; however, PC is defined by the Code of Federal Regulations, whereas PCP does not have a Code of Federal Regulations definition (
      • FDA
      21 CFR, Part 133.169 to 133.180.
      ). The major ingredient in PC is natural cheese, whereas PCP may or may not contain cheese. In that case, they typically rely on other ingredients, such as MCC and whey protein concentrate for structure building (
      • Lu Y.
      • Shirashoji N.
      • Lucey J.A.
      Rheological, textural and melting properties of commercial samples of some of the different types of pasteurized processed cheese.
      ;
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ). Process cheese has been made since the late nineteenth and early twentieth century to extend the shelf-life of natural cheeses. Approximately one-third of all-natural cheese produced in the United States is used in making PC (
      • Kommineni A.
      • Amamcharla J.
      • Metzger L.E.
      Effect of xylitol on the functional properties of low-fat process cheese.
      ), which is considered one of the leading varieties of cheese in the world and has several applications (

      Sorensen, H. H. 2001. The World Market for Cheese, Document No. 359.

      ;
      • Kapoor R.
      • Metzger L.E.
      Process cheese: Scientific and technological aspects—A review.
      ).
      A critical reaction that occurs during PC and PCP manufacture is calcium sequestration using ES (sodium citrate, disodium phosphate, and so on). Emulsifying salts are critical for the functional characteristics of PC and PCP due to their role in improving the emulsification characteristics of casein by sequestrating a portion of the calcium from the calcium-casein-phosphate network in natural cheese or casein containing ingredients (Figure 1). As shown in Figure 1, ES such as disodium phosphate sequester the calcium from the calcium-casein-phosphate network by donating their sodium ions. As a result, the major molecular forces that cross-link the various monomers of casein are partially disrupted. This disruption leads to hydration and dispersion of caseins. The partially dispersed monomers of casein, such as any protein, have hydrophilic and hydrophobic regions. These amphiphilic dispersed caseins, in the presence of heat and mixing, get hydrated via hydrophobic interactions and at the same time emulsify fat phase via hydrophobic interactions to produce an emulsified PC (
      • Guinee T.P.
      • Carić M.
      • Kaláb M.
      Pasteurized processed cheese and substitute/imitation cheese products.
      ).
      Figure thumbnail gr1
      Figure 1Emulsifying salts interaction during making process cheese or process cheese products.
      If cMCC is mixed with MCC (Figure 2), it may be possible to create a partially de-aggregated casein network without the use of ES. The ratio of cMCC to MCC will have an effect on the level of de-aggregation and the pH of the final PC product. We recently patented a method of producing PCP with no ES using cMCC and MCC (

      Metzger, L. E., and A. R. A. Hammam. 2020. Process for Manufacture of Process Cheese Without Emulsifying Salt. US Pat. No. US20200260753A1.

      ). We hypothesized that a ratio of 2 parts of protein from cMCC to 1 part of protein from MCC will create a partially de-aggregated casein network similar to a typical PC that uses ES (Figure 2).
      Figure thumbnail gr2
      Figure 2Cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC) interaction in making clean label process cheese products.
      No studies have used MCC in making cMCC powder as well as using this ingredient in making PCP with no ES. Therefore, the objectives of this study were to develop a process to produce a novel cMCC powder ingredient and to produce PCP without ES using different combinations of protein from cMCC and MCC in the formulations (2.0:1.0, 1.9:1.1, and 1.8:1.2).

      MATERIALS AND METHODS

      Experimental Design

      Manufacture of MCC was completed in approximately 10 h in 1 d at Davis Dairy Plant at South Dakota State University (Brooking, SD). The experiment was repeated 3 times with different lots of skim milk. Part of the final MCC was dried using a spray dryer to produce MCC powder, whereas the rest of the MCC was fermented using starter culture to produce cMCC, which was eventually dried in the fluid bed. The MCC and cMCC powders were mixed in different ratios to produce PCP with no ES.
      No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.

      Preparation of Skim Milk

      The MCC was manufactured as described in our previous study with some modifications (
      • Hammam A.R.A.
      • Beckman S.L.
      • Metzger L.E.
      Production and storage stability of concentrated micellar casein.
      ). Approximately 750 kg of whole bovine milk was separated (Model MSE 140–48–177 AirTight centrifuge; GEA Co.) at 4°C at the South Dakota State University Davis Dairy Plant and then pasteurized in a plate heat exchanger (model PR02-SH, AGC Engineering) at 76°C for 16 s. The pasteurized skim milk was then kept at ≤4°C until MF was conducted.

      Microfiltration Operation

      To fractionate skim milk into casein and SP to produce MCC, a pilot-scale ceramic GP MF system (TIA, Rond-point des, Portes de Provence, Rue Robert Schumann 84500) was used. The GP MF system was equipped with 7 ceramic tubes (19 channels with a diameter of 3.3 mm) mounted in the system vertically. The ceramic GP MF membranes had a 0.1-μm pore size, 1.68 m2 surface areas, and a 1.02-m membrane length. The GP MF system was also equipped with a feed pump and a retentate recirculation pump (TIA). The MF of skim milk (approximately 670 kg) was performed in 3 stages to produce MCC.

      Manufacture of MCC

      First Stage

      The GP MF system was started with soft water at 50°C using 3× CF (1 kg of retentate:2 kg of permeate) in a feed and bleed mode (1-way pass) with 400 kPa retentate pressure inlet (Rpi), 200 kPa retentate pressure outlet (Rpo), and 200 kPa permeate pressure outlet (Ppo). The skim milk (∼10.3% Brix) was heated to 50°C with a heat plate exchanger (SABCO Plate-pro Sanitary Chiller; NP925–41) before processing. When the processing conditions were stable while running with water, the system was transitioned to milk. The skim milk was MF with the GP MF system at a constant flux (71.42 L/m2 per h) using a 3× CF in a feed and bleed mode at 50°C (Figure 3). The water at the beginning of the process was flushed out with skim milk by collecting about 36.0 kg of permeate and 17.0 kg of retentate. The permeate flow rate was set at 120 L/h (flux of 71.42 L/m2 per h) and the retentate flow rate was 60 L/H to produce a 3× retentate. After this startup, retentate and permeate were collected and weighed continuously. During MF of skim milk, Rpi, Rpo, and Ppo were targeted to maintain 400, 200, and 200 kPa, respectively. The CF was calculated every 15 min by collecting permeate and retentate samples. The composition of retentate and permeate during MF was monitored using an infrared spectrophotometer (FT-IR spectroscopy, Model Dairyspec FT, Bentley Instrument Inc). A composite permeate sample was collected during the process. The collected retentate was kept in tanks during the MF process. The processing time of the first stage was approximately 4 h.
      Figure thumbnail gr3
      Figure 3Manufacture of micellar casein concentrate (MCC) using microfiltration (MF) graded permeability ceramic membranes using 3 MF stages with 3× concentration factor (CF) and diafiltration (DF).

      Second Stage

      The retentate (∼17.0% Brix) from the first stage was diluted with soft water (approximately 212.0 kg of retentate was mixed with 424.0 kg of water) to obtain a DF of 3× to get back the original volume of skim milk. After mixing, the diluted retentate (∼5.5% Brix) was heated to 50°C and processed with the GP MF system using a 3× CF, as described in the first stage. The water at the beginning of the process was flushed out of the system with the diluted retentate by collecting about 36.0 kg of permeate and 18.0 kg of retentate. The processing conditions in the second stage were similar to the first stage. The Rpi, Rpo, and Ppo were set at 400, 200, and 200 kPa, respectively. The permeate flow rate was 120 L/h (flux of 71.42 L/m2 per h) and the retentate flow rate was 60 L/h. Permeate and retentate were weighed continuously, as described in the first stage. A composite permeate sample was collected during the process. The retentate was collected in sanitized cans. The processing time of the second stage was approximately 3.5 h.

      Third Stage

      Approximately 175.0 kg of the retentate (∼13.0% Brix) was MF in a recirculation mode. The retentate of the second stage was placed in the tank of MF unit and then proceed to the third stage using a 3× CF at 50°C. The processing conditions in the third stage were similar to the first and second stages. The following conditions were applied: Rpi, Rpo, and Ppo were 400, 200, and 200 kPa, respectively, whereas the permeate flow rate was 120 L/h (flux of 71.42 L/m2 per h) and the retentate flow rate was 60 L/h. The retentate was recirculated while permeate was collected until the TS reached approximately 14 to 15% (CEM Smart System5 SL7199) or ∼16.0 to 17.0% Brix. Increasing the solids content of MCC during MF led to decreasing the Ppo. The decrease of Ppo is related to the concentration polarization and membrane fouling that accumulated on the membrane during recirculation. The final MCC resulting from the third stage (approximately 125.0 kg) was collected. A composite sample of the permeate was sampled for compositional analysis. The processing time for the third stage was around 1 h. The MCC was then pasteurized at 63°C/30 min. Approximately 50.0 kg of the liquid MCC was dried using a spray dryer to produce MCC powder, whereas the rest of the liquid MCC (75.0 kg) was used in making cMCC. This trial was replicated 3 times using 3 separate lots of raw milk.

      Cleaning the Membrane

      After processing, the GP MF system was flushed with soft water to remove all retentate residues from the system. The initial flux was measured with approximately 60 kg of soft water at 27°C. During the flux measurement, the retentate valves were closed and the permeate valves were completely opened with the feed pump running. Subsequently, 30 kg of soft water was added to the system and heated to 74°C, then 900 mL of Ultrasil 110 Alkaline cleaner (Ecolab Inc.) and 200 mL of XY 12 (Ecolab Inc.) were added to get pH 11 (Accumet, Fisher Scientific). This solution was recirculated for 30 min at a 350 L/h permeate flow rate (flux of 208 L/m2 per h). After cleaning the MF system with the alkaline solution, the membrane was cooled to 50°C (less than 10°C per min). The alkaline solution was flushed out of the MF system with soft water until the pH of outlet water ranged from 8.3 to 8.5. The flux was measured again, as described previously. The system was cleaned with an acid solution (Ultrasil 78 acid cleaner) by adding 30 kg of soft water and heated to 52°C; subsequently, 400 mL of Ultrasil 78 (Ecolab Inc.) was added to get a pH of 2. The recirculation of the acid solution was applied for 20 min at a flux of 208 L/m2 per h. Subsequently, the machine was turned off, and the acid was retained inside the system. Before using the system again, the acid solution was flushed out with soft water until the pH reached 8.3 to 8.5. The flux was measured again after flushing the acid solution. Within each MF stage, membrane was flushed using water, and flux was measured. The membrane was cleaned within stages using the abovementioned procedures when the flux did not show the original flux.

      Drying of MCC

      A pilot-scale spray dryer at Davis Dairy Plant at South Dakota State University was used to dry the MCC. The nozzle used to dry the MCC had a core size of 21 and an orifice size of 66. The inlet pressure was set at 2,250.0 psi using a high-pressure pump speed, and it was adjusted manually through the fan (30.0%). The supply fan and exhaust fan were set at 80.0 and 90.0%, respectively. The inlet temperature was 175°C, whereas the outlet temperature was 82°C. The dryer was connected to a fluid bed (Dahmes Stainless INC: DSI, Model no 10011–11) that was equipped with a sieve. The fluid bed was attached to 3 fans (hot = 71°C and 40.0% speed; warm = 50°C and 50.0% speed; cool = 21°C and 40.0% speed). Approximately 50.0 kg of the liquid MCC was heated to 50°C in a water bath before feeding into the dryer. The powder was collected in an airtight container and stored at room temperature until further analysis. The weight of the final powder was about 7.0 kg.

      Manufacture of cMCC

      The rest of the liquid MCC (75.0 kg) was fortified with lactose by adding 4.7% milk permeate powder (IdaPro milk permeate, Idaho Milk Products) to add approximately extra 3% lactose to ensure there was enough lactose for fermentation using starter cultures. The milk permeate was hydrated in the MCC and then the mix was pasteurized at 65°C for 30 min and cooled to 43°C to be ready for fermentation. The fortified MCC was then inoculated with 0.5% starter cultures (i455, Batch No 3489654, Chr. Hansen) at 43°C to get the pH of 4.6 in 12 to 14 h. A small amount (100 mL) of lactic acid (88% Lactic Acid FCC, product code: 175820, lot number:1501277028) was added before cutting if the pH did not drop to the desired pH (4.6). Once the pH of 4.6 was reached, the curd was completely cut and left to heal for 30 min with gentle mixing, and then it was heated gradually to 50°C in 1 h. The whey was subsequently drained from the curd and sampled. The water at 50°C was used to wash the curd for 5 min at a ratio of 1:1 and then the water was drained. When the curd was washed with water, the pH of the curd increased slightly due to the buffering capacity. As a result, approximately 50 mL of lactic acid was added to the water (∼100 lb) before washing the curd to keep the pH at 4.6. The washing step was repeated 3 times. The water was drained, and the curd was pressed for 6 h at 80 psi. The pressed curd was kept at ≤4°C until the next day.

      Drying of cMCC

      After pressing, the curd was milled into small pieces using a grinder (Humboldt Model 5DPJ3, split phase motor from Dayton) equipped with a 4.5-mm diameter stainless sieve.
      A pilot-scale spray dryer at Davis Dairy Plant at South Dakota State University was used to dry the cMCC. The dryer was not turned on only the exhaust fan and 3 fans connected to the fluid bed were running manually. The exhaust fan was set at 20%. The 3 fans attached to the fluid bed were set as follows: hot air fan = 71.0°C and 40.0% speed, warm air fan = 71.0°C and 50.0% speed, and cool air fan = 21.0°C and 15.0% speed.
      The curd was then placed on the sieve of the fluid bed, which is part of the spray dryer. The curd was evenly distributed on the surface of the fluid bed. The temperature of fans was elevated or decreased by controlling the speed of fans manually. The curd was moved and mixed every 30 min upside down. The curd was left in the fluid bed for around 4 h to ensure the curd was completely dried. The dried curd was collected in an airtight container and stored at room temperature until grinding. The weight of the dried curd was approximately 14.0 kg. The dried curd was then turned into powder using a high-speed multifunctional grinder (item no. HC-700Y). The powder was then separated using a metal sieve (item no. 8RTG5, Sieve #70, Grainger) with a 212-µm pore size to have the typical particle size of powders. Powder particles with over 212 µm were collected and milled again in the Retsch Ultracentrifugal mill (Brinkmann Retsch ZM1) equipped with a 0.2-mm stainless steel ring sieve to have a fine powder. The cMCC powder was collected in bags and stored at room temperature until further analysis.

      Manufacture of PCP

      Techwizard (Excel-based-formulation software program provided by Owl Software) was used to develop PCP formulations (
      • Metzger L.E.
      Nutrition Labeling Using a Computer Program.
      ) with different ratios of cMCC and MCC (2.0:1.0 formulation = FR-2.0:1.0, 1.9:1.1 formulation = FR-1.9:1.1, and 1.8:1.2 formulation = FR-1.8:1.2) to produce PCP with 45% moisture, 30% fat, 19% protein, and 2.4% salt. The percentage of ingredients used in PCP formulations is shown in Table 1. In each formulation, the amount of protein from cMCC and MCC was adjusted based on the formulation. As a result, the amount of cMCC was decreased, whereas the amount of MCC was increased from 2.0:1.0 to 1.8:1.2 formulations. The ingredients included aged natural cheddar cheese (Kraft Heinz Cheddar), salted butter (Land O'Lakes Inc.), cMCC, MCC, milk permeate (product lot: 19113D40, Idaho Milk Products), and salt (Morton Salt Inc.). Approximately 15% of aged cheddar was added into PCP formulations to get mild cheddar flavor in the final PCP. Milk permeate powder was used to standardize the solids content in all formulations. Process cheese products formulations were prepared by mixing cMCC powder, MCC powder, and water for 10 min in a kitchenaid at room temperature. Then other ingredients were added and mixed for 30 min to produce a homogeneous paste. A 20-g sample of the paste was weighed in a canister, warmed in a 40°C water bath for 30 min, and then cooked in the rapid visco analyzer (RVA; Perten RVA 4500) for 3 min at 95°C. We added 0.5 g of water into each canister to compensate for the water that evaporated during mixing and cooking in the RVA. The stirring speed was 1,000 rpm for the first 2 min and 160 rpm for the last min. Each batch was divided into 10 canisters. The cooked PCP was then poured into copper molds (diameter = 20 mm; height = 30 mm) to measure the hardness by using texture profile analysis. The cooked PCP was poured into plastic molds (diameter = 28.3 mm; height = 25 mm), which was used to measure the melt temperature by using dynamic stress rheometry (DSR) and melt diameter by using the Schreiber melt test. This experiment was repeated 3 times using different batches of cMCC and MCC powders.
      Table 1Mean (n = 3) composition of process cheese products (PCP) formulations made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      Ingredient (%)Treatment
      Treatments: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      FR-2.0:1.0FR-1.9:1.1FR-1.8:1.2
      Cheddar cheese (aged)15.0015.0015.00
      Salt1.621.621.62
      Water34.0234.0334.04
      Milk permeate powder0.400.310.22
      Butter (salted)30.8630.8630.86
      MCC powder6.607.257.90
      cMCC powder11.5010.9310.36
      Total100100100
      1 Treatments: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.

      Chemical Analyses

      Skim milk, liquid MCC, MF permeate (composite from all 3 stages), MCC powder, fortified MCC (used in making curd), cMCC prior drying, acid whey, and cMCC powder were analyzed for TS (
      • AOAC International
      Official Methods of Analysis.
      ; method 990.20; 33.2.44), total protein (TPr = total nitrogen × 6.38;
      • AOAC International
      Official Methods of Analysis.
      ; method 991.20; 33.2.11), noncasein nitrogen (NCN;
      • AOAC International
      Official Methods of Analysis.
      ; method 998.05; 33.2.64;
      • Zhang H.
      • Metzger L.E.
      Noncasein nitrogen analysis of ultrafiltration and microfiltration retentate.
      ), NPN (
      • AOAC International
      Official Methods of Analysis.
      ; method 991.21; 33.2.12), lactose content and lactic acid (
      • Amamcharla J.K.
      • Metzger L.E.
      Development of a rapid method for the measurement of lactose in milk using a blood glucose biosensor.
      ), ash (
      • AOAC International
      Official Methods of Analysis.
      ; method 945.46; 33.2.10), and mineral profile (
      • AOAC International
      Official Methods of Analysis.
      ; method 985.01). The skim milk, MCC powder, and cMCC powder were also analyzed for fat using Mojonnier method (
      • AOAC International
      Official Methods of Analysis.
      ; method 989.05; 33.2.26). Also, TS, ash, and pH (Hannah Edge Blu) of the final PCP were determined.

      Functional Analyses

      End Apparent Viscosity

      The end apparent viscosity of the PCP was set to be measured by the end of the cooking time in the RVA at 95°C after 3 min by calculating the mean of the last 5 apparent viscosity readings from the RVA curve. However, due to the high viscosity of the treatments in this study, the RVA automatically stopped after 1 min of cooking, so this cooking time was standardized in all treatments and the last point referred to the end apparent viscosity. The end apparent viscosity was measured in all canisters of the batch.

      Texture Profile Analysis.

      The hardness of the PCP was measured by using the texture profile analysis. The PCP was prepared by pulling the cheese out from the copper cylinders and then cutting the cheese into 200-mm height. The PCP was analyzed for hardness using a TA.XT-Plus Texture Analyzer (TA.XT-Plus, 6 Patton Drive) equipped with a 38-mm diameter cylindrical flat probe (TA-4) and using uniaxial double bite 10% compression with 1 mm/s crosshead speed. The maximum force of the first compression was referred to as the hardness of the cheese. This test was repeated 6 times for each batch.

      Dynamic Stress Rheometry

      The PCP was prepared by cutting the cheese into slices (2 mm thick and a 28.3-mm diameter) using a wire cutter (
      • Hammam A.R.A.
      • Beckman S.L.
      • Sunkesula V.
      • Metzger L.E.
      Highly concentrated micellar casein: Impact of its storage stability on the functional characteristics of process cheese products.
      ). A stress sweep test of the PCP was performed at a frequency of 1.5 Hz, and stress ranged from 1 to 1,000 Pa at 20°C using a rheometer with parallel plate geometry (MSR 92, Anton Paar). The stress sweep experiment determined that the maximum stress limit for the linear viscoelastic region was 500 Pa. The dynamic rheological properties of the PCP were then analyzed with a dynamic temperature ramp test that ranged from 20 to 90°C with a ramp rate of 1°C/min using a frequency of 1.5 Hz and constant stress of 500 Pa. The temperature at which tan δ = 1 [loss modulus (G′′)/storage modulus (G′)] was referred to as the cheese melting temperature. A duplicate was performed on each batch.

      Schreiber Melt Test

      The PCP samples were cut into cylinders (diameter = 28.3 mm and height = 7 mm) and placed on 0.75 mm thick aluminum plates (100 mm × 90 mm). The plates were transferred to a forced draft oven at 90°C for 7 min (
      • Hammam A.R.A.
      • Kapoor R.
      • Metzger L.E.
      Manufacture of process cheese products without emulsifying salts using acid curd and micellar casein concentrate.
      ). After cooling the plates, the diameter of the melted PCP samples was measured in 4 different places using a vernier caliper and reported in millimeters. This test was repeated 4 times for each batch.

      Statistical Analysis

      Statistical analysis was performed to study the effect of the ratio of protein from MCC and cMCC on the functional properties of PCP. Each replicate was considered as a block for the design, which was manufactured on a different day. Analysis of variance was done to obtain the mean squares and P-values using the GLM procedure available in R software (R x64 3.3.3 using R studio). Differences were tested using the least significant difference comparison test when a significant difference was detected at P < 0.05.

      RESULTS AND DISCUSSION

      Microfiltration to Produce MCC

      Composition of Skim Milk

      The mean composition of skim milk used in this study is shown in Table 2. The skim milk showed an average of 3.37, 0.29, 0.87, 0.58, 9.01, 4.41, 0.20, and 0.21% for TPr, NPN, NCN, SP, TS, lactose, lactic acid, and fat, respectively. The TP in skim milk was 3.08% with 2.50% casein. The casein as a percentage of TP was approximately 81.08%, whereas the ash content in skim milk was 0.72% including 0.12% Ca and 0.09% P. No noticeable differences were detected in the composition of skim milk within the 3 replicates. The mean composition of skim milk did not differ from the skim milk reported in other MF studies (
      • Beckman S.L.
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Production efficiency of micellar casein concentrate using polymeric spiral-wound microfiltration membranes.
      ;
      • Hurt E.
      • Barbano D.M.
      Processing factors that influence casein and serum protein separation by microfiltration1.
      ;
      • Adams M.C.
      • Barbano D.M.
      Serum protein removal from skim milk with a 3-stage, 3× ceramic Isoflux membrane process at 50°C.
      ;
      • Hammam A.R.A.
      • Beckman S.L.
      • Metzger L.E.
      Production and storage stability of concentrated micellar casein.
      ). The high pasteurization temperature can result in denaturation of β-LG on κ-CN through disulfide bonding (
      • Singh H.
      ), which can increase the percent of casein to TP (CN%TP) and thereby decrease the SP removal during MF (
      • Hurt E.
      • Barbano D.M.
      Processing factors that influence casein and serum protein separation by microfiltration1.
      ). The CN%TP in this study was typically similar to other studies (
      • Hurt E.
      • Barbano D.M.
      Processing factors that influence casein and serum protein separation by microfiltration1.
      ;
      • Adams M.C.
      • Barbano D.M.
      Serum protein removal from skim milk with a 3-stage, 3× ceramic Isoflux membrane process at 50°C.
      ;
      • Beckman S.L.
      • Barbano D.M.
      Effect of microfiltration concentration factor on serum protein removal from skim milk using spiral-wound polymeric membranes.
      ) because the pasteurization temperature did not exceed the recommended value. The extensive pasteurization can be noticed in the NCN values, which is increasing with higher temperature because SP is denaturated on casein. It has been found that the Ca and P in milk were around 0.11 and 0.09%, respectively (
      • Wong N.P.
      • LaCroix D.E.
      • Mattingly W.A.
      • Vestal J.H.
      • Alford J.A.
      The effect of manufacturing variables on the mineral content of cottage cheese.
      ), and those values are similar to the Ca and P values reported in Table 2.
      Table 2Mean (n = 3) composition (% by weight) of ingredients used in manufacture of micellar casein concentrate (MCC) and cultured micellar casein concentrate (cMCC)
      IngredientComposition
      Composition: Total protein (TPr) = total nitrogen × 6.38; NPN = nonprotein nitrogen × 6.38; NCN = noncasein nitrogen × 6.38; serum proteins (SP) = NCN − NPN; true protein (TP) = TPr − NPN; CN = TPr − NCN; CN%TP = CN as a percentage of TP = (CN/TP) × 100.
      (%)
      AshTPrNPNNCNSPTSLactoseLactic acidFatCaPTPCNCN%TPpH
      Skim milk0.723.370.290.870.589.014.410.200.210.120.093.082.5081.08
      Permeate0.320.440.160.430.264.013.110.170.010.020.280.014.28
      MCC1.1811.150.161.261.1014.061.320.180.340.2010.999.8989.95
      Fortified MCC1.3910.950.251.451.2017.374.540.240.350.2310.709.5088.78
      Acid whey1.373.820.371.521.1410.612.492.300.330.163.442.3065.55
      Soft cMCC0.9636.450.741.330.5840.370.801.340.110.1835.7135.1398.364.60
      MCC powder8.0375.772.079.667.6097.848.691.732.982.421.2973.7066.1189.69
      cMCC powder2.0586.882.222.290.0896.421.412.552.550.170.2984.6684.5899.91
      1 Composition: Total protein (TPr) = total nitrogen × 6.38; NPN = nonprotein nitrogen × 6.38; NCN = noncasein nitrogen × 6.38; serum proteins (SP) = NCN − NPN; true protein (TP) = TPr − NPN; CN = TPr − NCN; CN%TP = CN as a percentage of TP = (CN/TP) × 100.

      Composition of Permeate

      The composite permeate composition collected from the 3 stages of MF is shown in Table 2. The composite permeate had 0.44, 0.16, 0.43, 0.26, 4.01, 3.11, and 0.17% for TPr, NPN, NCN, SP, TS, lactose, and lactic acid, respectively. We found 0.01% casein in the permeate, whereas TP was 0.28%, therefore the CN%TP was 4.28%. The ash content in the permeate was 0.32% with 0.01% Ca and 0.02% P. The composition of permeate was similar within replicates. We have produced MCC previously using the same procedures to have >25% TS and >20% protein (
      • Hammam A.R.A.
      • Beckman S.L.
      • Metzger L.E.
      Production and storage stability of concentrated micellar casein.
      ). The composite permeate of the first and second stages in our previous study can be used for comparison reason to this study because we did not go further to produce highly concentrated MCC. No differences were detected in the composition of permeate of our previous study relative to the current study. It showed 4.01% TS, 0.43% TPr, 0.14% NPN, 0.29% SP, and 0.32% ash, which are similar to the current study. The composition of permeate in this study was also similar to the permeate reported in other studies (
      • Hurt E.
      • Barbano D.M.
      Processing factors that influence casein and serum protein separation by microfiltration1.
      ;
      • Hurt E.
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Micellar casein concentrate production with a 3X, 3-stage, uniform transmembrane pressure ceramic membrane process at 50°C1.
      ;
      • Zulewska J.
      • Barbano D.M.
      The effect of linear velocity and flux on performance of ceramic graded permeability membranes when processing skim milk at 50°C.
      ;
      • Tremblay-Marchand D.
      • Doyen A.
      • Britten M.
      • Pouliot Y.
      A process efficiency assessment of serum protein removal from milk using ceramic graded permeability microfiltration membrane.
      ). The slight differences in the composition of permeate within studies can be due to the differences in membrane type, DF steps, as well as the composition of skim milk.

      Composition of MCC

      The composition of MCC made from MF of skim milk is illustrated in Table 2. The average composition of MCC made from the 3 replicates was 11.15% TPr, 0.16% NPN, 1.26% NCN, 1.10% SP, 14.06% TS, 1.32% lactose, and 0.18% lactic acid. The ash content was 1.18% with 0.34% Ca and 0.20% P. The CN%TP was approximately 90% with an average of 9.89% casein and 10.99% TP. We previously produced MCC with similar composition using the GP MF ceramic membranes following the same procedures (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ). We found that the TS, TPr, and ash were 13.79, 9.54, and 1.03%, respectively. Similar results were also found by
      • Tremblay-Marchand D.
      • Doyen A.
      • Britten M.
      • Pouliot Y.
      A process efficiency assessment of serum protein removal from milk using ceramic graded permeability microfiltration membrane.
      . The latter study reported that the retentate of 3× MF GP membranes had 89.59% CN%TP with 0.92% SP and 15.31% TS. However, the CN%TP of MCC produced in other studies was high relative to our study due to the extra DF stage that those studies used between the second and third stages (
      • Hurt E.
      • Barbano D.M.
      Processing factors that influence casein and serum protein separation by microfiltration1.
      ;
      • Hurt E.
      • Zulewska J.
      • Newbold M.
      • Barbano D.M.
      Micellar casein concentrate production with a 3X, 3-stage, uniform transmembrane pressure ceramic membrane process at 50°C1.
      ;
      • Zulewska J.
      • Barbano D.M.
      The effect of linear velocity and flux on performance of ceramic graded permeability membranes when processing skim milk at 50°C.
      ). This led to increasing the CN%TP in MCC due to the high SP amount that was removed during MF.

      Culture-Based Acid Curd

      Composition of Fortified MCC

      The composition of MCC used in making cMCC is exemplified in Table 2. The same MCC product produced from MF was used in making cMCC after fortification with milk permeate powder. The TPr, NPN, NCN, SP, TS, lactose, and lactic acid in fortified MCC were 10.95, 0.25, 1.45, 1.20, 17.37, 4.54, and 0.24%, respectively. The ash content was 1.39% including 0.35% Ca and 0.23% P. The CN%TP was 88.78% with 9.50 and 10.70% casein and TP, respectively. No differences were detected between the MCC solutions before and after fortification with milk permeate powder except for TS and lactose. It was expected that lactose will be elevated by >3% due to the addition of milk permeate powder as a source of lactose to ensure there is enough lactose for fermentation using starter cultures. Additionally, the TS increased by around 3.3% for the same reason.

      Composition of Acid Whey

      The composition of acid whey produced as a byproduct of making cMCC from MCC is presented in Table 2. The whey showed approximately 3.82, 0.37, 1.52, 1.14, 10.61, 2.49, and 2.30% for TPr, NPN, NCN, SP, TS, lactose, and lactic acid, respectively. The whey showed around 1.37, 0.33, and 0.16% ash, Ca, and P. The TPr presented in whey includes 3.44% TP and 2.30% casein to show 65.55% CN%TP. We previously produced cMCC from MCC (13.02% TS, 1.96% lactose, 1.01% NCN, and 9.18% TPr) using starter cultures at a laboratory scale (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ). The composition of acid whey produced as a byproduct of making cMCC at a laboratory scale did not differ much from that produced on the pilot scale. The percentage of TPr to TS in acid whey produced on a laboratory scale was 28.1%, which is close to the value in this study (36.0%). Approximately 1.4 to 1.8% lactose was required to reach a pH of 4.6 in the acid curd/whey. As a result, around 2.5% lactose from a total of 4.23% was left in the whey. Using 2.0% lactose MCC left around 0.6% of lactose in the laboratory scale acid whey (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ). The loss of components in whey especially protein depends on the composition of initial material as well as handling the curd in the cheese vat and this loss gets higher with increasing the scale. The composition of acid whey produced as a byproduct of making cMCC was different from the acid whey produced from milk in previous studies (
      • Peri C.
      • Dunkley W.L.
      Reverse osmosis of cottage cheese whey. 1. Influence of composition of the feed.
      ;
      • Durham R.J.
      • Hourigan J.A.
      ;
      • Saffari M.
      • Langrish T.
      Effect of lactic acid in-process crystallization of lactose/protein powders during spray drying.
      ;
      • Chandrapala J.
      • Duke M.C.
      • Gray S.R.
      • Zisu B.
      • Weeks M.
      • Palmer M.
      • Vasiljevic T.
      Properties of acid whey as a function of pH and temperature.
      ;
      • Lievore P.
      • Simões D.R.S.
      • Silva K.M.
      • Drunkler N.L.
      • Barana A.C.
      • Nogueira A.
      • Demiate I.M.
      Chemical characterisation and application of acid whey in fermented milk.
      ;
      • Chen G.Q.
      • Eschbach F.I.I.
      • Weeks M.
      • Gras S.L.
      • Kentish S.E.
      Removal of lactic acid from acid whey using electrodialysis.
      ;
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ) due to the differences in the composition of starting material (MCC), which was expected. As a result, TS, ash, TPr, lactose, and lactic acid contents in acid whey can be changed based on the composition of MCC. Those studies found that the TS of acid whey can range from 5.0 to 7.0%, whereas TPr and ash can be 0.5 to 1.0 and 0.5 to 1.0%, respectively. The TS, TPr, and ash in acid whey produced from MCC were high because they were high in MCC compared with acid whey produced from using milk in curd manufacture.

      Composition of Soft Acid Curd

      The composition of soft cMCC produced from MCC is shown in Table 2. The TPr, NPN, NCN, SP, TS, lactose, and lactic acid content in produced cMCC was 36.45, 0.74, 1.33, 0.58, 40.37, 0.80, and 1.34%, respectively. The ash content was 0.96% including 0.11% Ca and 0.18% P. The casein in cMCC was 35.13%, whereas TP was 35.71% to present 98.36% CN%TP. The average pH of cMCC was targeted to have 4.60. The composition of cMCC depends on the composition of initial materials, final pH, and process conditions (e.g., cooking temperature, washing curds, pressing) (
      • Wong N.P.
      • LaCroix D.E.
      • Mattingly W.A.
      • Vestal J.H.
      • Alford J.A.
      The effect of manufacturing variables on the mineral content of cottage cheese.
      ). The step of washing the curd has a significant role in increasing the ratio of casein to TP and decreasing the ash, Ca, P, lactose, and lactic acid content in the final cMCC. Because the lactose is converted to lactic acid using starter cultures as a result of fermentation, lactose decreases and lactic acid increases as the pH reaches 4.6. It is also expected that the ash content decreases when the MCC is turned into cMCC (
      • McDowall F.H.
      • Dolby R.M.
      107. Studies on the chemistry of cheddar cheese making. I. The Mineral content of cheese and whey.
      ;
      • Czulak J.
      • Conochie J.
      • Sutherland B.J.
      • Van Leeuwen H.J.M.
      Lactose, lactic acid and mineral equilibria in Cheddar cheese manufacture.
      ;
      • Wong N.P.
      • LaCroix D.E.
      • Mattingly W.A.
      • Vestal J.H.
      • Alford J.A.
      The effect of manufacturing variables on the mineral content of cottage cheese.
      ;
      • Hill A.R.
      • Bullock D.H.
      • Irvine D.M.
      Composition of cheese whey: Effect of pH and temperature of dipping.
      ;
      • Lucey J.A.
      • Fox P.F.
      Importance of calcium and phosphate in cheese manufacture: A review.
      ). The Ca is converted from insoluble (colloidal form) to soluble form and released in the whey as the pH decreases (
      • Davies D.T.
      • White J.C.D.
      The use of ultrafiltration and dialysis in isolating the aqueous phase of milk and in determining the partition of milk constituents between the aqueous and disperse phases.
      ;
      • Wong N.P.
      • LaCroix D.E.
      • Mattingly W.A.
      • Vestal J.H.
      • Alford J.A.
      The effect of manufacturing variables on the mineral content of cottage cheese.
      ;
      • Dalgleish D.G.
      • Law A.J.R.
      pH-Induced dissociation of bovine casein micelles. II. Mineral solubilization and its relation to casein release.
      ;
      • Guinee T.P.
      • Pudja P.D.
      • Farkye N.Y.
      ). As a result, the Ca and P drop as the pH decreases with having a low ratio of Ca to P (
      • Kindstedt P.S.
      • Kosikowski F.V.
      Calcium, phosphorus, and sodium concentrations in cheddar cheese.
      ;
      • Lucey J.A.
      • Fox P.F.
      Importance of calcium and phosphate in cheese manufacture: A review.
      ).
      • Wong N.P.
      • LaCroix D.E.
      • Mattingly W.A.
      • Vestal J.H.
      • Alford J.A.
      The effect of manufacturing variables on the mineral content of cottage cheese.
      have reported that the Ca in cottage curd is reduced by 68.2% (washed 3 times) relative to the Ca content in skim milk, which is similar to our results (68.6%). Another review reported that the Ca and P in cottage cheese were 0.08 and 0.16%, respectively (
      • Lucey J.A.
      • Fox P.F.
      Importance of calcium and phosphate in cheese manufacture: A review.
      ).

      Composition of MCC powder, cMCC powder, and PCP

      Composition of MCC Powder

      The liquid MCC was spray dried to produce MCC powder. Table 2 presents the composition of MCC powder. The average composition of MCC powder showed 75.77% TPr, 2.07% NPN, 9.66% NCN, 7.60% SP, 97.84% TS, 8.69% lactose, 1.73% lactic acid, and 2.98% fat. The ash content was 8.03% with 2.42% Ca and 1.29% P. The CN%TP was approximately 89.70% with an average of 66.10% casein and 73.70% TP. The composition of MCC powder is relatively similar to the liquid MCC on a dry basis. The composition of MCC powder in this study is similar to the composition of commercial MCC 80 reported previously (
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ). The MCC 80 has approximately 80.0% TPr, 95.0% TS, 4.5% lactose, 3.0% fat, and 8.0% ash. Similar results were reported in several studies (
      • Nasser S.
      • Moreau A.
      • Jeantet R.
      • Hédoux A.
      • Delaplace G.
      Influence of storage conditions on the functional properties of micellar casein powder.
      ,
      • Nasser S.
      • De Sa Peixoto P.
      • Moreau A.
      • Croguennec T.
      • Bray F.
      • Rolando C.
      • Tessier F.J.
      • Hédoux A.
      • Delaplace G.
      Storage of micellar casein powders with and without lactose: Consequences on color, solubility, and chemical modifications.
      ;
      • Carter B.
      • Patel H.
      • Barbano D.M.
      • Drake M.
      The effect of spray drying on the difference in flavor and functional properties of liquid and dried whey proteins, milk proteins, and micellar casein concentrates.
      ). Those studies found that TPr, TS, lactose, and ash ranged from 80.0 to 83.0, 94.0 to 95.0, 1.0 to 2.5, and 7.0 to 8.0%, respectively. The lactose content in the commercial MCC and other studies is low due to the multiple DF stages that are used to deplete more lactose and low molecular weight components in the permeate during MF. The slight differences can be related to the composition of initial material, MF process conditions (e.g., temperatures, pressures, CF, DF), and types of membrane (e.g., ceramic membranes, spiral-wound membranes) (
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ).

      Composition of cMCC Powder

      The composition of cMCC powder is exemplified in Table 2. The cMCC powder showed 86.88, 2.22, 2.29, 0.08, 96.42, 1.41, 2.55, and 2.55% for TPr, NPN, NCN, SP, TS, lactose, lactic acid, and fat. The ash content in cMCC was 2.05% with 0.17% Ca and 0.29% P. The majority of TP in cMCC was casein. The cMCC had 84.58% casein and 84.66% TP to present approximately 99.90% CN%TP. The composition of cMCC powder was relative to the composition of soft cMCC on a dry basis and similar to this range reported in previous studies (
      • Huffman L.M.
      • James Harper W.
      Maximizing the value of milk through separation technologies.
      ;
      • Hammam A.R.A.
      • Martínez-Monteagudo S.I.
      • Metzger L.E.
      Progress in micellar casein concentrate: Production and applications.
      ). The acid curd made from milk could have less ash content as well as Ca and P due to the low content of those components in milk as compared with MCC, which has higher ash, Ca, and P contents. The slight differences in the composition of cMCC are due to the differences in the composition of the initial material and process used to make the curd.

      Composition of PCP

      The composition of PCP made from cMCC and MCC is shown in Table 3. The mean squares and the P-values for the composition of PCP are indicated in Table 4. The moisture content was 44.19, 44.21, and 44.15% in PCP made from FR-2.0:1.0, FR-1.9:1.1, and FR-1.8:1.2, respectively. The ash content of PCP made from the 2.0:1.0 ratio of protein from cMCC to MCC was 3.28%, whereas it was 3.42 and 3.40% in the PCP made from 1.9:1.1 and 1.8:1.2, respectively. No differences (P > 0.05) were detected in the moisture and ash contents of PCP because all formulations were standardized before cooking in the RVA to have the same composition. The PCP exhibited 0.31, 0.33, and 0.41% Ca in formulations of FR-2.0:1.0, FR-1.9:1.1, and FR-1.8:1.2, respectively. Although a slight increase in the Ca content was noticed with decreasing the cMCC and increasing the MCC, no significant difference (P > 0.05) was detected in the Ca content. It was reported that the Ca of PC made using ES and natural cheeses ranged from 0.3 to 0.4% (
      • Kapoor R.
      • Metzger L.E.
      • Biswas A.C.
      • Muthukummarappan K.
      Effect of natural cheese characteristics on process cheese properties.
      ), which is similar to our values. However, increasing the amount of MCC led to differences (P < 0.05) in the pH of final PCP, which was 5.25 in 2.0:1.0 formulation, 5.32 in 1.9:1.1 formulation, and 5.37 in 1.8:1.2 formulation. This was expected as the MCC had higher pH when compared with cMCC. The ratio of cMCC to MCC has an effect on the level of de-aggregation and the pH of the final PCP. It was indicated that the Ca and P, residual lactose, and salt-to-moisture ratio in natural cheeses affect the pH of PC (
      • Kapoor R.
      • Metzger L.E.
      • Biswas A.C.
      • Muthukummarappan K.
      Effect of natural cheese characteristics on process cheese properties.
      ). It has been reported that the typical pH of PC made from natural cheeses and ES can fall in the range of 5.4 to 5.8 (
      • Marchesseau S.
      • Gastaldi E.
      • Lagaude A.
      • Cuq J.-L.
      Influence of pH on protein interactions and microstructure of process cheese.
      ;
      • Kapoor R.
      • Metzger L.E.
      Process cheese: Scientific and technological aspects—A review.
      ;
      • Lu Y.
      • Shirashoji N.
      • Lucey J.A.
      Effects of pH on the textural properties and meltability of pasteurized process cheese made with different types of emulsifying salts.
      ;
      • Hammam A.R.A.
      • Beckman S.L.
      • Sunkesula V.
      • Metzger L.E.
      Highly concentrated micellar casein: Impact of its storage stability on the functional characteristics of process cheese products.
      ). The pH of PCP in this study was close to the typical values. However, it has been found that pH of PC can range from 5.1 to 5.2 and still shows good functional properties (
      • Kommineni A.
      • Amamcharla J.
      • Metzger L.E.
      Effect of xylitol on the functional properties of low-fat process cheese.
      ). We previously have found that the pH of PCP or imitation mozzarella cheese produced with no ES using cMCC and MCC ranged from 5.3 to 5.5 (
      • Hammam A.
      Manufacture of Ingredients for Use in Clean Label Process Cheese Products.
      ;
      • Hammam A.R.A.
      • Kapoor R.
      • Metzger L.E.
      Manufacture of process cheese products without emulsifying salts using acid curd and micellar casein concentrate.
      ), which is similar to this study. The differences in pH could affect the structure and quality of final PCP and thereby its functional properties due to its effects on the protein interactions (
      • Palmer H.J.
      • Sly W.H.
      Oil separation in processed cheese.
      ;
      • Meyer A.
      Processed Cheese Manufacture.
      ;
      • Marchesseau S.
      • Gastaldi E.
      • Lagaude A.
      • Cuq J.-L.
      Influence of pH on protein interactions and microstructure of process cheese.
      ).
      • Palmer H.J.
      • Sly W.H.
      Oil separation in processed cheese.
      stated that the emulsion of PC is low when the pH is lower than 5.4 or higher than 5.8. It was found that as the pH of PC drops to 5.2, the protein-protein interaction increases (
      • Marchesseau S.
      • Gastaldi E.
      • Lagaude A.
      • Cuq J.-L.
      Influence of pH on protein interactions and microstructure of process cheese.
      ) because the pH is close to the isoelectric point of caseins (4.6). This induces the aggregation of the protein, which in turn, results in a poor emulsion of fat in PCP. On the other hand, the PC had an open structure when the pH elevated to 6.1, which eventually led to weaker emulsification (
      • Marchesseau S.
      • Gastaldi E.
      • Lagaude A.
      • Cuq J.-L.
      Influence of pH on protein interactions and microstructure of process cheese.
      ).
      • Marchesseau S.
      • Gastaldi E.
      • Lagaude A.
      • Cuq J.-L.
      Influence of pH on protein interactions and microstructure of process cheese.
      also found that as the pH increased up to 5.7 it resulted in PC with better uniform fat emulsion with a closely knit protein network.
      Table 3Mean (n = 3) composition of process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Moisture (%)pHAsh (%)Ca (%)
      FR-2.0:1.044.195.25
      Means in the same column not sharing a common superscript are different (P < 0.05).
      3.280.31
      FR-1.9:1.144.215.32
      Means in the same column not sharing a common superscript are different (P < 0.05).
      3.420.33
      FR-1.8:1.244.155.37
      Means in the same column not sharing a common superscript are different (P < 0.05).
      3.400.41
      SEM0.040.020.040.02
      a–c Means in the same column not sharing a common superscript are different (P < 0.05).
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Table 4Mean squares and P-values (in parentheses) for the composition of the process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      FactordfMoisture (%)pHAsh (%)Ca (%)
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      20.003 (0.90)0.011 (<0.05)0.016 (0.27)0.009 (0.17)
      Replication20.008 (0.75)0.001 (<0.05)0.036 (0.10)0.002 (0.61)
      Error40.0260.00004440.0080.003
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.

      Functional Characteristics of PCP

      End Apparent Viscosity of PCP

      The end apparent viscosity of PCP made from cMCC and MCC is illustrated in Table 5. The mean squares and the P-values for the end apparent viscosity and hardness of PCP are presented in Table 6. The end apparent viscosity of PCP made from FR-2.0:1.0, FR-1.9:1.1, and FR-1.8:1.2 was approximately 4,305.0, 2,408.8, and 2,499.3 cP, respectively. The end apparent viscosity was different (P < 0.05) within treatments showing the highest viscosity in the FR-2.0:1.0 PCP. The highest viscosity in FR-2.0:1.0 could be due to the differences in pH. The viscosity of PC increased as the pH decreased. As the pH dropped to the isoelectric point (4.6), the protein-protein interactions increased which led to high viscosity (
      • Kapoor R.
      Effect of Intact Casein, Total Calcium and Ph on the Functional Properties and Microstructure of Process Cheese.
      ).
      Table 5Mean values (n = 3) of end apparent viscosity (cP) and hardness (g) of the process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      End apparent viscosity (cP)Hardness (g)
      FR-2.0:1.04,305.06
      Means in the same column not sharing a common superscript are different (P < 0.05).
      424.09
      FR-1.9:1.12,408.78
      Means in the same column not sharing a common superscript are different (P < 0.05).
      511.89
      FR-1.8:1.22,499.33
      Means in the same column not sharing a common superscript are different (P < 0.05).
      407.10
      SEM339.9828.98
      a,b Means in the same column not sharing a common superscript are different (P < 0.05).
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Table 6Mean squares and P-values (in parentheses) for the end apparent viscosity (cP) and hardness (g) of the process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      FactordfEnd apparent viscosity (cP)Hardness (g)
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      234,323,39 (<0.05)9,489 (0.43)
      Replication2149,899 (0.63)2,763.7 (0.75)
      Error4289,5128,992
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.

      Hardness of PCP

      The hardness of PCP made from cMCC and MCC is shown in Table 5. The mean squares and the P-values for the hardness of PCP are exemplified in Table 6. The hardness was approximately 424.0 g for the 2.0:1.0 ratio, 512.0 g for the 1.9:1.1 ratio, and 407.0 g for the 1.8:1.2 ratio. The hardness of PCP did not show any significant differences (P > 0.05) between different formulations made from different ratios of protein from cMCC and MCC. Although the hardness should be correlated with viscosity (
      • Kapoor R.
      • Metzger L.E.
      Process cheese: Scientific and technological aspects—A review.
      ) and pH (
      • Kapoor R.
      Effect of Intact Casein, Total Calcium and Ph on the Functional Properties and Microstructure of Process Cheese.
      ), we did not find differences in the hardness of the PCP made from different ratios of protein from cMCC and MCC. The hardness of our PCP looks better compared with those PCP made using ES. In our previous study, we found that the hardness of PCP made using MCC and ES ranged from 100.0 to 212.0 g (
      • Hammam A.R.A.
      • Beckman S.L.
      • Sunkesula V.
      • Metzger L.E.
      Highly concentrated micellar casein: Impact of its storage stability on the functional characteristics of process cheese products.
      ). Another study reported that the hardness of PCP made from MCC using ES was 110.0 to 135.0 g (
      • Salunke P.
      Impact of transglutaminase on the functionality of milk protein concentrate and micellar casein concentrate.
      ).

      Melting Characteristics of PCP

      The melting characteristics of PCP made from cMCC and MCC are shown in Table 7. The mean squares and the P-values for the melting characteristics of PCP are illustrated in Table 8. We detected a significant difference (P < 0.05) in the melting temperature of PCP made from 2.0:1.0, 1.9:1.1, and 1.8:1.2 formulations. The highest melting temperature was 54.0°C in the 2.0:1.0 ratio, whereas it was 43.0 and 42.0°C in the 1.9:1.1 and 1.8:1.2 ratios, respectively. It was expected that 2.0:1.0 formulation will have the highest melting temperature because the PCP showed higher end apparent viscosity than other formulations. We found a correlation between viscosity and melt temperature. It was mentioned that the melt temperature elevated as the end apparent viscosity increased (
      • Kapoor R.
      • Metzger L.E.
      Process cheese: Scientific and technological aspects—A review.
      ), which our work pointed out in Table 5. Additionally, the melting of PC increased as the pH decreased. As the pH decreased to the isoelectric point (4.6), the protein-protein interactions increased, which required more temperature to be melted (
      • Kapoor R.
      Effect of Intact Casein, Total Calcium and Ph on the Functional Properties and Microstructure of Process Cheese.
      ).
      Table 7Mean values (n = 3) of melting properties of the process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Melt temperature (°C)Melt diameter (mm)Melt area (mm2)
      FR-2.0:1.054.02
      Means in the same column not sharing a common superscript are different (P < 0.05).
      38.801,183.95
      FR-1.9:1.143.02
      Means in the same column not sharing a common superscript are different (P < 0.05).
      43.901,538.62
      FR-1.8:1.242.01
      Means in the same column not sharing a common superscript are different (P < 0.05).
      42.361,415.34
      SEM2.181.53107.17
      a,b Means in the same column not sharing a common superscript are different (P < 0.05).
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Table 8Mean squares and P-values (in parentheses) for the melting properties of the process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC)
      FactordfMelt temperature (°C)Melt diameter (mm)Melt area (mm2)
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      2133.0 (<0.05)20.54 (0.48)97,263 (0.49)
      Replication229.3 (0.056)17.33 (0.53)85,511 (0.53)
      Error44.5623.07115,371
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      It has been reported that the melt diameter or melt area should increase as the melt temperature decreases (
      • Kapoor R.
      • Metzger L.E.
      Process cheese: Scientific and technological aspects—A review.
      ). Although we detected a significant difference in the melting temperature of PCP, no differences (P > 0.05) were detected in the melt diameter or area. We found an increase in the melt diameter of PCP, but this increase was not significant. The melt diameter and melt area increased with elevating the protein ratio from MCC compared with cMCC. The melt diameter was 38.8, 43.9, and 42.4 mm for PCP made with 2.0:1.0, 1.9:1.1, and 1.8:1.2 of protein from cMCC to MCC, respectively. The melt area was approximately 1,184.0, 1,539.0, and 1,415.0 mm2 for PCP made with 2.0:1.0, 1.9:1.1, and 1.8:1.2 of protein from cMCC to MCC, respectively. The melting temperature is an indicator of the melt diameter. As a result, a similar trend was found in the melt diameter compared with the melt temperature.
      The differences in the onset of melting can be explained by the differences in pH of those cheeses. As the pH drops to the isoelectric point, the net negative charges on caseins reduce which increase the protein-protein interactions, and this leads to aggregation of protein and thereby low meltability. This led to a higher melting temperature with less melted diameter and area.

      The Rheological Properties of PCP

      The G′ (elastic) and G′′ (viscous) moduli of PCP measured during heating from 20 to 90°C at 10°C increments are shown in Table 9, Table 10, respectively. The ANOVA table with mean squares and P-values at 20, 70, and 90°C for both elastic (G′: Pa) and viscous (G″: Pa) moduli of the PCP made from cMCC and MCC are shown in Table 11. The effect of using different ratios of protein cMCC and MCC (2.0:1.0, 1.9:1.1, and 1.8:1.2) in manufacture of PCP with no ES on G′ and G″ during heating from 20 to 90°C is presented in Figure 4, Figure 5, respectively.
      Table 9Mean elastic modulus (G′: Pa) of process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC) during heating from 20 to 90°C using dynamic rheological analysis (DSR)
      Temperature (°C)Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      FR-2.0:1.0FR-1.9:1.1FR-1.8:1.2
      2069,179.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      70,611.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      72,193.2
      Means in the same row not sharing a common superscript are different (P < 0.05).
      3030,019.5
      Means in the same row not sharing a common superscript are different (P < 0.05).
      27,004.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      27,120.2
      Means in the same row not sharing a common superscript are different (P < 0.05).
      4011,594.9
      Means in the same row not sharing a common superscript are different (P < 0.05).
      5,982.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      3,556.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      506,055.7
      Means in the same row not sharing a common superscript are different (P < 0.05).
      2,182.2
      Means in the same row not sharing a common superscript are different (P < 0.05).
      623.8
      Means in the same row not sharing a common superscript are different (P < 0.05).
      60926.6
      Means in the same row not sharing a common superscript are different (P < 0.05).
      353.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      164.9
      Means in the same row not sharing a common superscript are different (P < 0.05).
      70201.5
      Means in the same row not sharing a common superscript are different (P < 0.05).
      68.6
      Means in the same row not sharing a common superscript are different (P < 0.05).
      60.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      80103.5
      Means in the same row not sharing a common superscript are different (P < 0.05).
      24.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      36.1
      Means in the same row not sharing a common superscript are different (P < 0.05).
      9040.7
      Means in the same row not sharing a common superscript are different (P < 0.05).
      7.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      10.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      a–c Means in the same row not sharing a common superscript are different (P < 0.05).
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Table 10Mean viscous modulus (G′′: Pa) of process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC) during heating from 20 to 90°C using dynamic rheological analysis (DSR)
      Temperature (°C)Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      FR-2.0:1.0FR-1.9:1.1FR-1.8:1.2
      2023,228.8
      Means in the same row not sharing a common superscript are different (P < 0.05).
      24,522.5
      Means in the same row not sharing a common superscript are different (P < 0.05).
      25,684.8
      Means in the same row not sharing a common superscript are different (P < 0.05).
      3011,385.6
      Means in the same row not sharing a common superscript are different (P < 0.05).
      11,052.7
      Means in the same row not sharing a common superscript are different (P < 0.05).
      11,450.5
      Means in the same row not sharing a common superscript are different (P < 0.05).
      406,881.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      4,258.1
      Means in the same row not sharing a common superscript are different (P < 0.05).
      2,926.8
      Means in the same row not sharing a common superscript are different (P < 0.05).
      504,661.1
      Means in the same row not sharing a common superscript are different (P < 0.05).
      2,491.5
      Means in the same row not sharing a common superscript are different (P < 0.05).
      1,029.9
      Means in the same row not sharing a common superscript are different (P < 0.05).
      601,345.6
      Means in the same row not sharing a common superscript are different (P < 0.05).
      739.2
      Means in the same row not sharing a common superscript are different (P < 0.05).
      421.6
      Means in the same row not sharing a common superscript are different (P < 0.05).
      70438.7
      Means in the same row not sharing a common superscript are different (P < 0.05).
      229.8
      Means in the same row not sharing a common superscript are different (P < 0.05).
      214.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      80299.1
      Means in the same row not sharing a common superscript are different (P < 0.05).
      99.4
      Means in the same row not sharing a common superscript are different (P < 0.05).
      150.0
      Means in the same row not sharing a common superscript are different (P < 0.05).
      90143.3
      Means in the same row not sharing a common superscript are different (P < 0.05).
      53.2
      Means in the same row not sharing a common superscript are different (P < 0.05).
      70.9
      Means in the same row not sharing a common superscript are different (P < 0.05).
      a–c Means in the same row not sharing a common superscript are different (P < 0.05).
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Table 11Mean squares and P-values (in parentheses) for elastic modulus (G′: Pa) and viscous modulus (G′′: Pa) of process cheese products (PCP) made with different ratios of cultured micellar casein concentrate (cMCC) and micellar casein concentrate (MCC) during heating from 20 to 90°C using dynamic rheological analysis (DSR)
      FactordfG′G′′
      20°C70°C90°C20°C70°C90°C
      Treatment
      Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      26,818,056 (0.69)18,829.8 (0.02)1,032.8 (0.15)4,528,264 (0.20)47,191 (0.048)6,836.4 (0.15)
      Replication295,627,858 (0.06)1,057.6 (0.54)642.0 (0.25)6,119,707 (0.14)7,533 (0.40)5282 (0.20)
      Error416,655,3301,470.1327.01,889,4236,5992,203
      1 Treatment: FR-2.0:1.0 = PCP formulation made with 2.0:1.0 ratio of protein from cMCC to MCC; FR-1.9:1.1 = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC.
      Figure thumbnail gr4
      Figure 4Elastic modulus (G′: Pa) of process cheese products (PCP) made from FR-2.0:1.0 (•) = PCP formulation made with 2.0:1.0 ratio of protein from cultured micellar casein concentrate (cMCC) to micellar casein concentrate (MCC); FR-1.9:1.1 (▼) = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 (▪) = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC during heating from 20 to 90°C using dynamic rheological analysis.
      Figure thumbnail gr5
      Figure 5Viscous modulus (G′′: Pa) of process cheese products (PCP) made from FR-2.0:1.0 (•) = PCP formulation made with 2.0:1.0 ratio of protein from cultured micellar casein concentrate (cMCC) to micellar casein concentrate (MCC); FR-1.9:1.1 (▼) = PCP formulation made with 1.9:1.1 ratio of protein from cMCC to MCC; FR-1.8:1.2 (▪) = PCP formulation made with 1.8:1.2 ratio of protein from cMCC to MCC during heating from 20 to 90°C using dynamic rheological analysis.
      The G′ values of PCP made using different ratios of protein from cMCC and MCC (2.0:1.0, 1.9:1.1, and 1.8:1.2) were not different (P > 0.05) at a temperature range of 20 to 30°C (Figure 4). However, we found differences (P < 0.05) in G′ values of PCP made using different ratios of protein from cMCC and MCC from 40 to 80°C (Table 9). These differences in G′ values of PCP made using different ratios of protein from cMCC and MCC were not found (Table 11) at 90°C (Table 9). Process cheese product made from 2.0:1.0 (protein from cMCC to MCC) gave the highest G′ values at temperatures of 40 to 80°C, followed by 1.9:1.1 and 1.8:1.2 formulations.
      The G′′ of PCP made using different ratios of protein from cMCC and MCC (2.0:1.0, 1.9:1.1, and 1.8:1.2) followed the same trend of G′ at a temperature range of 20 to 90°C (Figure 5). The G′′ values of PCP made using different ratios of protein from cMCC and MCC (2.0:1.0, 1.9:1.1, and 1.8:1.2) were not different (P > 0.05) at a temperature range of 20 to 30°C (Table 10). However, we detected differences (P < 0.05) in G′′ values made using different ratios of protein from cMCC and MCC from 40 to 80°C (Table 10). These differences in G′′ values of PCP made using different ratios of protein from cMCC and MCC were not found at 90°C (Table 11). Process cheese product made from 2.0:1.0 (protein from cMCC to MCC) gave the highest G′ values at temperatures of 40 to 80°C, followed by 1.9:1.1 and 1.8:1.2 formulations.
      The G′ of PCP made using different ratios of protein from cMCC and MCC (2.0:1.0, 1.9:1.1, and 1.8:1.2) before melting was higher than G′′. This indicates that the PCP has elastic behavior (gel) more than the viscous behavior (liquid). The G′ and G′′ are decreased during measuring the melting using the DSR. Both moduli are decreased until the cross point, which is the cheese melting point. The same trend was found in other studies (
      • Hennelly P.J.
      • Dunne P.G.
      • O'Sullivan M.
      • O'Riordan D.
      Increasing the moisture content of imitation cheese: effects on texture, rheology and microstructure.
      ;
      • Subramanian R.
      • Muthukumarappan K.
      • Gunasekaran S.
      Linear viscoelastic properties of regular- and reduced-fat pasteurized process cheese during heating and cooling.
      ;
      • Zhong Q.
      • Daubert C.R.
      • Velev O.D.
      Physicochemical variables affecting the rheology and microstructure of rennet casein gels.
      ;
      • Guinee T.P.
      • O'Kennedy B.T.
      The effect of calcium content of Cheddar-style cheese on the biochemical and rheological properties of processed cheese.
      ;
      • Kommineni A.
      • Amamcharla J.
      • Metzger L.E.
      Effect of xylitol on the functional properties of low-fat process cheese.
      ;
      • Hosseini-Parvar S.H.
      • Matia-Merino L.
      • Golding M.
      Effect of basil seed gum (BSG) on textural, rheological and microstructural properties of model processed cheese.
      ). The improvement in the functional characteristics of PCP with no ES made using different ratios of protein from cMCC and MCC (2.0:1.0, 1.9:1.1, and 1.8:1.2) may be a result of a better emulsion of cMCC and MCC during making the cheese.

      CONCLUSIONS

      The GP MF membranes can be effectively used to produce MCC with >9% TP and >13% TS using 3 stages MF with a 3× CF and DF. Additionally, cMCC was produced from the liquid MCC. A unique method was developed to produce cMCC powder by drying and grinding the curd. Different ratios of protein from cMCC powder to MCC can be used in making PCP with no ES. The cMCC and MCC protein of different ratios create a partially de-aggregated casein network that results in a PC with functionality similar to PC produced with ES. We also found that PCP made from the 2.0:1.0 formulation showed better functional properties as compared with other formulations. The novel ingredients (cMCC and MCC powders) produced in this study can be mixed in a specific ratio and market to be ready for the manufacture of clean label imitation mozzarella cheese and PCP with no ES.

      ACKNOWLEDGMENTS

      The authors acknowledge and thank the Midwest Dairy Foods Research Center (St. Paul, MN) for their financial support. The authors have not stated any conflicts of interest.

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