Pilot scale production of micellar casein concentrate using stainless steel membrane

International Dairy Journal 80 (2018) 26e34

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Pilot scale production of micellar casein concentrate using stainless steel membrane Shuwen Zhang a, b, 1, Jianhang Chen b, 1, Xiaoyang Pang b, Jing Lu b, Mingxing Yue c, Lu Liu b, Jiaping Lv b, * a b c

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China Institute of Food Science and Technology, Chinese Academy of Agricultural Science, Beijing 100193, China Beijing Polytechnic, Beijing 100176, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 October 2017 Received in revised form 6 January 2018 Accepted 6 January 2018 Available online 31 January 2018

A stainless steel (SS) membrane was used for separation of micellar casein and serum proteins. A 4
concentration process was used for comparing separation performance, after which a 0.02 mm SS membrane was chosen for further studies. Furthermore, serum protein reduction efficiency between the 0.02 mm SS membrane was compared with literature data for a 0.1 mm ceramic membrane for a 3
, 3stage process. The results showed that the 4
permeate obtained by 0.02 mm SS membrane contained no residual casein. The average skim milk flux for each stage (68.6, 76.2, and 85.7 L m2 h1) with the SS membrane was higher than that for each stage (54.0, 54.0 and 54.6 L m2 h1) with the ceramic membranes; serum protein removal for each stage with the SS membrane was close to that with ceramic membranes. Overall, the 0.02 mm SS membrane has potential applications in milk protein fractionation. © 2018 Elsevier Ltd. All rights reserved.

1. Introduction Membrane filtration is a pressure-driven fractionation process that has been widely used in dairy processing units. One of the most significant applications is the separation of micellar casein from €n, Sman, & Boom, 2004; serum protein in skim milk (Brans, Schroe Espina, Jaffrin, Ding, & Cancino, 2010; Karasu et al., 2010; McCarthy, Wijayanti, Crowley, O'Mahony, & Fenelon, 2017; Saboya & Maubois, 2000). When skim milk is concentrated using a membrane, the whole micellar casein can be retained and a portion of serum protein can pass through it. To achieve higher micellar casein purity, additional diafiltration steps are necessary (Hurt & Barbano, 2010; Lawrence, Kentish, O'Connor, Barber, & Stevens, 2008; Nelson & Barbano, 2005; Piry et al., 2012). Micellar casein concentrate (MCC), a concentrated liquid colloidal suspension, has a percentage of serum protein removal ranging from 60 to 95% (w/w) based on the total serum protein in starting skim milk (Beckman, Zulewska, Newbold, & Barbano, 2010). MCC, as a functional dairy liquid, has been widely used in

* Corresponding author. Tel.: þ86 10 62819421. E-mail address: [email protected] (J. Lv). 1 Contributed equally to this work. https://doi.org/10.1016/j.idairyj.2018.01.002 0958-6946/© 2018 Elsevier Ltd. All rights reserved.

milk protein standardisation before cheese making, and the lower concentration factor (CF) microfiltration retentate and high CF microfiltration retentate have been successfully applied in Cheddar cheese making and mozzarella cheese production, respectively (Ardisson-Korat & Rizvi, 2004; Govindasamy-Lucey, Jaeggi, Johnson, Wang, & Lucey, 2007; Neocleous, Barbano, & Rudan, 2002). Additionally, MCC also has potential use in high-protein nutritional beverages (Beliciu, Sauer, & Moraru, 2012; Sauer & Moraru, 2012), meal-substitute products, whipped topping, coffee whiteners and body-building foods (Hurt & Barbano, 2010). Ceramic membranes are the most commonly used membrane type in commercial milk protein fractionation processes based on microfiltration. Currently, 0.1 mm ceramic uniform transmembrane pressure (UTP) membranes have been the most effective membranes for serum protein removal and MCC production (Hurt, Zulewska, Newbold, & Barbano, 2010; Saboya & Maubois, 2000). Polymeric spiral-wound (SW) membranes can provide performance comparable with that of ceramic membranes during skim milk microfiltration process when operated at comparable levels of transmembrane pressure (TMP), shear stress and temperature (Beckman et al., 2010; Mercier-Bouchard, Benoit, Doyen, Britten, & Pouliot, 2017). However, while polymeric membranes offer similar performance compared with ceramic membranes, a low TMP is required to facilitate serum

S. Zhang et al. / International Dairy Journal 80 (2018) 26e34

protein transmission and it can be operationally difficult to retain the necessarily low TMP at the industrial scale (Karasu et al., 2010; Lawrence et al., 2008). Stainless steel (SS) membranes combine a rugged tube made of sintered SS powder with patented coating technology, having a TiO2 layer permanently bonded to the porous SS tube. This sintering process creates a smooth and foulant-resistant membrane surface with a nominal pore size of about 0.1 or 0.02 mm. With the TiO2 separation layer coated on its inner surface, an asymmetric structure is created by the gradual increase in pore size from the TiO2 layer to the SS substrate. As the permeate passes through the membrane tubes, it faces decreasing flow resistance as the pore size of each ensuing layer increases. Such an asymmetric structure lowers TMP, avoiding in-depth pore blockages and results in higher filtration flux rates. This inert and highly durable inorganic membrane can withstand elevated temperatures and pressures, concentrated solvents or extreme pH and has a much longer durability (more than 10 years of life span). SS membranes are an alternative when handling difficult industrial streams with high viscosity and solid contents under extreme conditions and has been successfully applied in the reclamation of sodium hydroxide from alkali wastewater in chitin processing (Zhao & Xia, 2009). SS membranes in skim milk processing have not been reported extensively and it is not known whether it can be a potential alternative to ceramic and SW membranes. The objective of this study was to firstly compare the separation performance for micellar casein and serum protein by 0.1 and 0.02 mm pore sized SS membranes during a 4
concentration process. Furthermore, experiments using one 3
concentration stage and two 3
diafiltration stages, in terms of serum protein removal efficiency were performed with the optimal 0.02 mm SS membrane compared with results of 0.1 mm ceramic membrane, data were taken from Hurt et al. (2010). 2. Materials and methods 2.1. Experimental design Fresh raw milk (900 kg) was obtained from Beijing Sanyuan Dairy Ltd. (Beijing, China). In the pilot laboratory of Institute of Food Science and Technology, raw milk was first pasteurised at 72  C with a holding time of 15 s. The cooling stage temperature (outlet temperature) was kept constant at 50  C by adjusting the flow velocity. After pasteurisation, raw milk was immediately

27

centrifuged by a disc bowl centrifuge (Armfield Limited, Ringwood, UK). Approximately 850 kg skim milk was cooled to 4  C and stored at 4  C. Skim milk was divided into three equal batches and from each batch 120 kg skim milk was collected and heated to 50  C on the second day. Two SS membrane systems (Hyflux Ltd, Tuas, Singapore) both having four 1.5 m monochannel tubular modules (inner diameter of the monochannel tube is 18.3 mm) and total membrane surface area of 0.35 m2 were used. The tubular membrane was installed horizontally in the membrane system and fixed by clamps, installed with two different pore size (0.1 and 0.02 mm) SS membranes (Hyflux Ltd, Tuas, Singapore). Both tank were filled with 60 kg skim milk after cleaning, and operated at 50  C to produce a 4
retentate. The TMP was 25 ± 3 kPa. During the whole 4
concentration process for the two membrane systems, the change in permeate flux (L m2 h1) for every 15 min was monitored by recording the permeate weight for 1 min. When the weight of the permeate reached 45 kg, the CF [original skim milk (L) retentate (L)1] of 4 was achieved. Samples from retentates and permeates were collected and stored at 4  C. The comparison experiment between 0.1 and 0.02 mm SS membrane was repeated two times on the following two days using the skim milk taken from two different batches (Fig. 1). 2.2. Cleaning procedure before experiment A cleaning procedure is an essential step before a membrane system is used for skim milk processing. Reverse osmosis water (25 L) at room temperature was poured into the feed tank and the membrane system was flushed for 15 min. Flush water was drained after the flush procedure was finished. The cleaning procedure was started with an alkaline sanitiser (a combination of 2%, w/v, sodium hydroxide solution and 0.5%, w/v, EDTA-Na2 solution) at 70  C for 15 min. Then the membrane system was drained and reverse osmosis water at 50  C was used to flush the membrane up to neutral pH. After the reverse osmosis water was drained, 2.0% (w/v) citric acid solution at 60  C was used to clean the membrane for 15 min. When the acid cleaning was finished, the membrane was drained and equilibrated to neutral pH using reverse osmosis water. The flux, J (L m2 h1), was calculated as:

J ¼ Dm=A
Dt

(1)

on the conditions of TMP of 25 ± 3 kPa based on the permeate weight (m) collected in 1 min (t) over the membrane surface area

Fig. 1. Schematic presentation of the stainless steel membrane system used for micellar casein concentrate production.

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(A). After the pure water flux was calculated, the membrane was drained. The above procedure was applied to both the 0.02 and 0.1 mm SS membrane. Cleaning efficiency was calculated as the starting water flux after milk (L m2 h1)/ending water flux after cleaning (L m2 h1). 2.3. Experiment for serum protein removal efficiency by 0.02 mm stainless steel membrane Comparison of the serum protein removal efficiency between the 0.02 mm SS membrane and the 0.1 mm UTP ceramic membrane (Hurt et al., 2010) was done by a 3
concentration, 3-stage process. Skim milk (60 kg), obtained from one of the three batches of milk, at 50  C was brought into the feed tank and the feed temperature was kept at 50  C during the whole process by adjusting the retentate flow velocity. In stage 1, skim milk was concentrated to a 3
concentration. In stage 2, 40 kg reverse osmosis water at 50  C was first added, then a 3
retentate was obtained. In stage 3, the operation stated in stage 2 was repeated. The diafiltration was performed in a discontinuous mode. Throughout the run, samples from the retentate and the permeate were collected at the end of each stage for chemical analysis and permeate flux

Component A removal ð%Þ ¼ 100


by a muffle ashing method (AOAC, 2000; method 930.30; 33.5.02). TN and NPN content were determined by Kjeldahl methods (AOAC, 2000; methods 991.20: 33.2.11 and 991.21: 33.2.12, respectively). The noncasein nitrogen content (NCN) of raw milk, skim milk and permeate was determined using Kjeldahl (AOAC, 2000; method 998.05; 33.2.64). To accurately determine the NCN content in retentates, the method described by Zhang and Metzger (2011) was used. TP was calculated by subtracting NPN from TN and multiplying by 6.38; CN was calculated by subtracting the NCN from TN and multiplying by 6.38; and SP content was calculated by subtracting NPN from NCN and multiplying by 6.38. All of samples were stored at 4  C and analysed within two days after collection. Table 1 shows the composition of the raw and pasteurised skim milk that was used in the study.

2.5. Calculation of component removal Component removal percentage as an indicator of membrane separation efficiency can be calculated; it is based on the component content in the permeate at the end of each stage and in the original skim milk, and the equation used is (Michalski et al., 2006):

nh i.  o ½Apermeate
ðCF  1Þ ½Askim milk CF

changes were also determined. Permeate samples from each stage were put into the conical flask and photographed with a camera (Nikon, Tokyo, Japan) for colour analysis. After processing, the retentates were collected and the cleaning procedure was repeated. 2.4. Chemical analyses Samples obtained from raw milk, pasteurised skim milk, retentate and permeate of the 4
concentration process by 0.1 and 0.02 mm SS membrane were analysed for total solids, lactose, ash, total protein (TN), true protein (TP), CN and SP contents. TN, TP, CN and SP content were also determined for samples of retentate and permeate collected from the end of each stage of the 3
, 3-stage process by 0.02 mm SS membrane. Total solids content was determined by forced-air oven drying methods (AOAC, 2000; method 990.20; 33.2.44). A colorimetry method as described by Teles, Young, and Stull (1978) was used for the lactose content determination. Fat content determination used an ether extraction method (AOAC, 2000; method 989.05; 33.2.26). Ash content was measured

(2)

2.6. Sodium dodecylsulphate polyacrylamide gel electrophoresis analysis Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to compare the separation performance of 0.1 and 0.02 mm SS membrane. A TriseHCl polyacrylamide running gel (T, 12%; C, 3.4%; pH 8.8), and stacking gel (T, 5%; C, 3.4%; pH 6.8) was prepared for use in electrophoresis (under reducing conditions) (GE Healthcare Bio-Sciences Corporation, New Jersey, USA). For SDS-PAGE analysis, 0.125 mL, 5 mL and 0.885 mL aliquots from 4
retentate, permeate and skim milk samples, respectively, obtained from 0.1 and 0.02 mm SS membranes, were diluted to 10 mL with ultrapure water. Then 10 mL of diluted sample was mixed with 10 mL SDS buffer, heated in a boiling water bath for 5 min and centrifuged using a mini centrifuge. Ten microlitres of supernatant was loaded onto the SDS-PAGE gel. Standard procedures were used for running, staining and destaining the gels. The gels were scanned by AlphaEase®FC gel imaging system and clear profiles were obtained by adjusting the black, white and gamma values using AlphaEase software (Alpha Innotech Corporation, San Leandro, CA, USA).

Table 1 Composition of raw milk, pasteurised skim milk, permeate and retentate from a 4
concentration filtration process with 0.1 and 0.02 mm SS membranes.a Item

Composition (% by weight) TS

Raw milk Pasteurised skim milk 0.02 mm permeate 0.1 mm permeate 0.02 mm retentate 0.1 mm retentate

Lactose a

13.26 9.18b 6.54a 6.78b 18.04a 17.39b

a

4.91 5.55b 5.31a 5.38b 5.24a 5.07b

Ash

TN a

0.67 0.65a 0.52b 0.59a 1.22b 1.10a

NPN a

3.11 2.91b 0.59a 0.73b 11.71a 11.01b

a

0.16 0.17b 0.18a 0.19a 0.27a 0.22b

NCN a

0.72 0.76b 0.40a 0.73b 2.11a 1.24b

TP

Casein a

2.94 2.74b 0.40a 0.54b 11.45a 10.79b

a

2.39 2.15b 0.00b 0.19a 9.78b 9.60a

Serum protein a

0.56 0.59b 0.21a 0.54b 1.84a 1.01b

Casein in TP 81.03a 78.49b 0.20b 47.69a 90.60b 83.90a

a Abbreviations are: TS, total solids; TN, total nitrogen; NCN, non-casein nitrogen; NPN, non-protein nitrogen; TP, true protein [(TN  NPN)
6.38]; casein calculated as (TN  NCN)
6.38; serum proteins calculated as (TP  casein)
6.38. Values are means (n ¼ 3); means in the same column not sharing a common letter are significantly different (P < 0.05). Fat content of raw milk and pasteurised skim milk was determined as 4.59 and 0.07% (by weight), respectively.

S. Zhang et al. / International Dairy Journal 80 (2018) 26e34

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Table 2 Flux changes by pore size 0.1 and 0.02 mm SS membrane before, during and after a 4
concentration process.a Membrane type

Skim milk flux (L m2 h1)

Pure water flux Before milk (L m2 h1)

After milk processing (L m2 h1)

Decline (%)

After cleaning (L m2 h1)

Recovery (%)

0.1 mm 0.02 mm

49.58a 64.28b

520.42a 150.57b

391.36a 105.25b

24.80a 30.00b

517.86a 149.78b

99.51a 99.48a

a Skim milk flux is the average flux during processing skim milk. Values are means (n ¼ 3); means in the same column not sharing a common superscript letter are significantly different (P < 0.05).

2.7. Statistical analysis Data analyses for significant differences were done by analysis of variance (ANOVA) using the Proc GLM procedures of SAS (version 9.1.3 portable, SAS Institute Inc., Cary, NC, USA). To determine if significant differences existed due to difference in pore sizes of the SS membranes, the general linear model (GLM) was dependent variable ¼ membrane type þ replicate þ error. To determine if significant differences existed due to processing stage, the GLM model used was dependent variable ¼ processing stage þ replicate þ error. 3. Results and discussion 3.1. Comparison of the 0.1 and 0.02 mm stainless steel membranes 3.1.1. Comparison of 4
permeates and retentates composition The chemical composition of permeates obtained by 0.1 and 0.02 mm SS membranes is shown in Table 1. Total solids, lactose, total protein, and serum protein content in permeate obtained by the 0.1 mm SS membrane were greater than that in the permeate obtained by the 0.02 mm SS membrane. Due to the difference in membrane pore size, the permeability of component for the 0.1 mm SS membrane was higher than for the 0.02 mm SS membrane under the same operating conditions. The micellar casein content in permeate of 0.1 mm SS membrane was 0.19 ± 0.00%, indicating that a small amount of micellar casein was lost into the permeate, but for the 0.02 mm SS membrane permeate there was no micellar casein detected. SDS-PAGE (Fig. 2) confirmed that micellar casein bands emerged on the permeate sample from the 0.1 mm SS membrane, but did not exist on the permeate sample from the 0.02 mm SS membrane. The permeates obtained using the 0.1 and 0.02 mm SS membranes were significantly different. This difference in bands profile should be a result of the difference in membrane pore size. The micellar casein particle size ranges from 0.02 mm to 0.4 mm, but mainly centres around 0.02e0.1 mm; serum protein particle size ranges from 0.003 to ~0.006 mm. It can be concluded that a smaller portion of micellar casein (particle size < 0.1 mm) will run into permeate if 0.1 mm SS membrane is applied and similar results have been reported by Zulewska, Newbold, and Barbano (2009) using 0.1 mm ceramic graded permeability membrane. When the smaller particle size portion (0.02e0.1 mm) of micellar casein pass through the membrane, membrane fouling increase and the permeability of other components in skim milk should be impacted. Ash content in permeate of 0.1 mm SS membrane was 0.59 ± 0.01% (w/w), which is greater than that in the permeate of the 0.02 mm membrane, which is associated with the micellar casein losses. A portion of ash, especially for the calcium and phosphate, was bonded to the micellar casein, and the micellar casein was lost into the permeate, leading to a little increase of content in the permeate. A comparison of appearance of 4
permeates obtained by 0.1 and 0.02 mm SS membrane was done by photography. Permeate

from the 0.02 mm SS membrane was clarified and its colour was green yellow; however, permeate from the 0.1 mm SS membrane was cloudy and the colour was white. Previous studies reported that the presence of micellar casein in the permeate can lead to cloudiness in appearance (Mercier-Bouchard et al., 2017; Zulewska et al., 2009), so from the perspective of appearance, it can also be concluded that micellar casein was present in the permeate of 0.1 mm SS membrane, but absent from permeate of 0.02 mm SS membrane. This was consistent with the micellar casein content determined by Kjeldahl method in Table 1. From the analysis on permeates obtained from 0.1 and 0.02 mm SS membrane, the 0.02 mm membrane permeate is an optimal choice for protein fractionation of skim milk into a casein-rich retentate and a permeate with serum protein. The total solid, lactose, total protein, and serum protein contents in the retentate of the 0.02 mm SS membrane were greater than those in the retentate of the 0.1 mm SS membrane. The micellar casein and ash content in retentate of the 0.1 mm SS membrane was less than that in the retentate of 0.02 mm SS membrane (Table 1). The content of these components determined in 4
retentate were consistent with that in 4
permeates (Table 1), the more the components run into the permeate, the less the components are retained in the retentate. Another significant difference that existed between the two types of retentate is the micellar casein content as a percentage of TP content (Table 1). The percentage of micellar casein in TP in retentates of 0.1 and 0.02 mm SS membranes were

Fig. 2. SDS-PAGE gel for the proteins in skim milk, retentate and permeate from a 4
concentration of skim milk with 0.1 and 0.02 mm SS membranes. Lanes 3 and 6, skim milk used for 0.02 and 0.1 mm SS membranes, respectively; lanes 2 and 5, 4
concentration retentate separated with 0.02 and 0.1 mm SS membranes, respectively; lanes 1 and 4, permeates collected from the 4
concentration with 0.02 and 0.1 mm SS membranes, respectively; lane M, marker.

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83.90 ± 0.10% and 90.60 ± 0.13%, respectively, which could reflect that the 0.02 mm SS membrane had greater efficiency than the 0.1 mm SS membrane in serum protein removal. Retentate protein profiles for the 0.02 and 0.1 mm SS membrane on the SDS-PAGE gel (Fig. 2) did not show notable differences. Fig. 2 showed that not only a-CN, b-CN and k-CN bands existed in the two types of retentate, but also the a-La and b-Lg bands still existed in the two types of retentate, which indicated that if higher serum protein removal percentage is desired, diafiltration steps should be applied. The serum protein removal percentages as the investigation index for MCC production efficiency were 26.92 ± 0.98% and 68.6 ± 2.00% for 0.1 and 0.02 mm SS membrane during a 4
concentration process. The obvious difference of serum protein removal should contribute to the micellar casein permeation. For 0.1 mm SS membrane, 6.72 ± 0.06% of micellar casein in skim milk passed into permeate and micellar casein particle sizes are approximate to 0.1 mm, so membrane resistance increased and serum protein removal was impacted (Table 3). 3.1.2. Comparison of membrane flux Pure water flux before milk, average skim milk flux during processing, pure water flux after milk processing, and pure water flux after cleaning were all determined (Table 2), and skim milk flux changes were also monitored every 15 min during the 4
concentration process (Fig. 3). Owing to the difference in membrane pore size, pure water flux before milk processing for the 0.1 mm SS membrane (520.4 L m2 h1) was greater than for the 0.2 mm SS membrane (150.6 L m2 h1) at TMP of 25 ± 3 kPa. However, the average skim milk flux during processing for 0.1 mm SS membrane (49.6 L m2 h1) was less than that for the 0.02 mm SS membrane (64.3 L m2 h1) (Fig. 3). This phenomenon may be related to the micellar casein particle size and the membrane pore size. For the 0.1 mm SS membrane, a portion of micellar casein passed through the membrane, since the particle size of micellar casein running to the permeate is close to the membrane pore size, so the membrane resistance will increase greatly and the skim milk flux should be influenced. For the 0.02 mm SS membrane, no micellar casein passes through the membrane, so the membrane resistance could not be higher than in the 0.1 mm SS membrane and the skim milk flux is greater than 0.1 mm SS membrane. In Fig. 3 it can be seen that concentration polarisation resistance increased and the membrane flux decreased for both the 0.1 and 0.02 mm SS membrane during the increase in retentate concentration. The average time consumed by 0.1 mm and 0.02 mm SS membrane for a 4
concentration process was 155 ± 5 min and 120 ± 4 min, respectively, so 0.02 mm SS membrane has higher processing efficiency. Pure water flux after milk processing for 0.02 and 0.1 mm SS membrane was 105.3 and 391.4 L m2 h1, respectively. The water flux decline values of the 0.1 and 0.02 mm SS membranes, which can reflect the degree of membrane fouling, were determined as 24.80 ± 0.26% and 30.00 ± 0.57%, respectively, implying that Table 3 Component removal from starting skim milk for 0.1 and 0.02 mm SS membrane with a 4
concentration process.a Membrane type

Removal (%) Casein protein

Serum protein

Lactose

Ash

0.1 mm 0.02 mm

6.72a 0.04b

26.92a 68.60b

71.72a 72.71a

68.48a 59.66b

a The theoretical permeable component removal (assumptions: serum protein, lactose and ash no rejection; casein protein completely rejected) for both membranes is 75%. Values are means (n ¼ 3); means in the same column not sharing a common superscript letter are significantly different (P < 0.05).

Fig. 3. Mean (n ¼ 3) flux for 0.1 (C) and 0.02 mm (-) SS membranes during a 4
concentration process at 50  C.

membrane fouling for 0.02 mm SS membrane was more serious. However, after cleaning with alkaline and acid solutions, the pure water flux recovery values for 0.02 and 0.1 mm SS membrane were the same, at 99.5%. Beckman et al. (2010) compared starting water flux before milk (36.6 L m2 h1), fouled water flux (7.1 L m2 h1) and the final water flux after cleaning (35.9 L m2 h1), when polymeric SW membrane was used for a 3
concentration process. Based on the data provided by Beckman et al. (2010), the water flux decline and water flux recovery value for polymeric SW membrane were 80.6% and 98.1%, respectively. Total flux of SW membrane decline reached 62.0% after initial concentration to a CF of 3.0
and 2 sequential diafiltration steps (Mercier-Bouchard et al., 2017). Apparently, the SS membrane suffered a lower degree of membrane fouling and achieved a higher membrane flux recovery. This may be related to the special structure and membrane material for the SS membrane. Choosing an appropriate membrane pore size is important in an industrial milk fractionation. For instance, for some industrial MF applications, it will be of great importance to obtain a permeate purely containing whey proteins with the absence of caseins. This study showed that permeates with 0.02 mm SS membranes contained serum protein with negligible amounts of micellar casein, whereas the permeate with the 0.1 mm SS membrane contained both serum protein and micellar casein. These results are different from those of Jørgensen et al. (2016) who reported that caseins permeated through a ceramic membrane with pore size 0.2 mm, whereas a ceramic membrane with a pore size of 0.05 and 0.1 mm was able to retain almost all caseins. Zulewska and Barbano (2013) studied the separation performance of 0.3 mm SW membranes, but important fouling of the membrane elements by micellar casein micelles occurred whereas the much smaller serum protein contributed to a minor amount of membrane pore constriction. Lawrence et al. (2008) also evaluated the performance of SW membranes for micellar casein and serum protein separation in milk, using 0.3 and 0.5 mm pore diameters. Casein rejection increased with the TMP, from 96% to almost 100%, whereas serum protein transmission decreased from 22% to 1%, as the TMP increased from 50 to 258 kPa. The difference of membrane material can result in the selectivity difference for casein and serum proteins and this should be important reason.

S. Zhang et al. / International Dairy Journal 80 (2018) 26e34

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was higher than 54.0, 54.0, and 54.6 L m2 h1, respectively, for ceramic membranes reported by Hurt et al. (2010). Jørgensen et al. (2016) obtained a permeate flux for skim milk of 59 and 44 L m2 h1, respectively, using ceramic membrane with pore size of 0.1 and 0.05 mm. The average flux for each stage with a polymeric SW membrane was 14.4, 22.2, and 32.6 L m2 h1, respectively (Beckman et al., 2010). Obviously, the 0.02 mm SS membrane exhibited a higher skim milk flux, and can be recommended as a potential alternative for skim milk fractionation.

3.2. MCC production by 0.02 mm stainless steel membrane during a 3
, 3-stage process 3.2.1. Skim milk flux changes Mean (n ¼ 3) flux changes for each stage during one 3
concentration stage and two 3
diafiltration stages are shown in Fig. 4. Skim milk flux during each stage declined gradually as processing time increased and skim milk flux increased with increasing stages. Decline of the skim milk flux during each stage is mainly due to the increasing retentate micellar casein content and caused the aggravation of concentration polarisation and fouling. The increase in skim milk flux, after the retentates of the previous stages were diluted with reverse osmosis water, should have contributed to the reduction of micellar casein concentration and reflected that the membrane fouling of the previous stage was not severe. The trend in the membrane flux change was different from that obtained by Beckman et al. (2010). The tendency described by Beckman et al. (2010) exhibited an initial decline due to the transition of water to milk and then a steady flux was obtained. The steady flux may be related to the membrane module used, membrane pore size and membrane fouling. The average (n ¼ 3) total time used for stages 1, 2, and 3 were 100 ± 2, 90 ± 3 and 80 ± 2 min, respectively. The average (n ¼ 3) skim milk flux for each stage was calculated based on the average total time and permeate weight for each stage. Average (n ¼ 3) flux of 0.02 mm SS membrane for stages 1, 2, and 3 were 68.6, 76.2, and 85.7 L m2 h1, respectively, which

3.2.2. Composition changes of permeates and retentates during concentration and diafiltration The concentration of TN, NPN, NCN, TP, and SP in permeate (Table 4) decreased with increasing stages (P < 0.05). This can be attributed to the total amount reduction of each component in stage 1 and stage 2 and the prior dilution by reverse osmosis water in stage 2 and stage 3. The decrease in serum protein concentration with increasing stages was also confirmed by SDS-PAGE (Fig. 5). It was apparent that the area and the optical density values of serum protein bands became smaller with stages increased, meaning that the concentration of serum protein content was reducing gradually with stage increased. The serum protein contents in the permeate were 0.54%, 0.191%, and 0.09% for stages 1, 2, and 3, respectively. The serum protein content in permeate produced by Hurt et al. (2010) using 0.1 mm UTP ceramic membrane were 0.58%, 0.25%, and 0.14%, respectively, for the 3 stages. The higher serum protein content in permeate obtained by 0.1 mm UTP ceramic membrane can be explained partly by the reason that TP content in the permeate was used as the serum protein content in the permeate regardless of the influence of minor amount of micellar casein in permeate (Hurt & Barbano, 2010), so the serum protein content is a little higher than actual values. Another contribution to the lower serum protein content in permeate produced by the 0.02 mm SS membrane is the impact of membrane rejection coefficient. SS membrane with a permanent layer of TiO2 coated on the porous SS tube may be different from the ceramic membrane with a permanent layer of Al2O3 with respect to serum protein rejection coefficient. The micellar casein concentration in the permeate of the 3 stages (Table 4) was almost zero, and this is consistent with the SDS-PAGE result, because there no micellar casein bands existed in the permeate samples of the 3 stages. The colour of the permeates from the 3 stages exhibited large differences. The green-yellow colour gradually faded away as the processing stages increased. The permeate became more and more clarified and the permeate of the last stage seemed like the reverse osmosis water. This phenomenon indicated that the main colour rendering component Vitamin B can be mainly removed by the first two stages. Due to the removal of colour rendering component and increasing of micellar casein as a percentage of TP in retentate

Fig. 4. Mean (n ¼ 3) flux changes during stage 1 (-), stage 2 (C) and stage 3 (:) when a pore size 0.02 mm SS membrane was used for one 3
concentration stage and two 3
diafiltration stages.

Table 4 Composition of permeates and retentates from a 3
concentration and two 3
diafiltration process by 0.02 mm SS membrane.a Filtration component and stage

Composition (% by weight) TN

Permeate e Stage 1 Permeate e Stage 2 Permeate e Stage 3 Retentate e Stage 1 Retentate e Stage 2 Retentate e Stage 3

NPN a

0.69 0.30b 0.15c 7.16a 6.76b 6.48c

a

0.15 0.10b 0.05c 0.20a 0.11b 0.07c

NCN a

0.69 0.29b 0.14c 1.00a 0.61b 0.41c

TP

Casein a

0.54 0.19b 0.10c 6.96a 6.65b 6.40c

a

0.00 0.00a 0.01a 6.17a 6.14a 6.07a

Serum proteins a

0.54 0.19b 0.09c 0.80a 0.50b 0.34c

Casein in TP 0.00a 0.00a 0.00a 88.55a 92.42b 94.72c

a Abbreviations are: TN, total nitrogen; NCN, non-casein nitrogen; NPN, non-protein nitrogen; TP, true protein [(TN  NPN)
6.38]; casein calculated as (TN  NCN)
6.38; serum proteins calculated as (TP  casein)
6.38. Values are means (n ¼ 3); means in the same column not sharing a common superscript letter are significantly different (P < 0.05).

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S. Zhang et al. / International Dairy Journal 80 (2018) 26e34

significant differences, micellar casein bands on the gel did not change significantly, which is consistent with the determinations (6.17 ± 0.03%, 6.14 ± 0.07% and 6.08 ± 0.07%) by Kjeldahl method. The micellar casein content did not change but the serum protein content reduced, so micellar casein as a percentage of TP increased (Table 4) with increasing stages. After the 3
, 3-stage process, the percentage of micellar casein in TP is 94.72%, which was closer to 95.59% obtained by the ceramic membrane (Hurt et al., 2010) and it may imply that the SS membrane has same efficiency with 0.1 mm ceramic membrane.

Fig. 5. SDS-PAGE gel of retentates and permeates obtained from the end of each stage of process during a 3
concentration stage and two 3
diafiltration stages process using a 0.02 mm SS membrane. Lanes 1, 2 and 3, retentate collected after stage 1, stage 2, and stage 3, respectively; lanes 4, 5and 6, permeate collected after stage 1, stage 2, and stage 3, respectively; lane M, marker.

(Table 4) for the 3 stages, it was observed that the retentate was becoming increasingly white and this result was consistent with the findings of Hurt et al. (2010) for ceramic membrane retentates. As stages increased, TP, NPN, NCN, TP, and SP content in the retentate decreased (Table 4). The TP content in the retentate for each stage were 6.69%, 6.65%, and 6.40%, respectively, which was less than the 8.67%, 8.61%, and 9.08%, respectively, obtained by Hurt et al. (2010) using a ceramic membrane. The apparent difference can be primarily attributed to the difference of TP content in skim milk used by the two studies. The TP content in skim milk used for SS membrane and ceramic study were 2.74% and 3.16%, respectively. Hurt and Barbano (2010) demonstrated that TP concentration of skim milk directly affected the TP content of the retentate and MCC yield. It can be concluded that milk protein standardisation before membrane filtration is an essential step for MCC yield. The serum protein content of retentates for each stage (0.80%, 0.50% and 0.34%) in our study is similar to that of the ceramic membrane retentates (0.78%, 0.49% and 0.40%) and this similarity in content is attributed to the similarity in serum protein content in skim milk used in the studies (0.59% and 0.56% for the present study and the ceramic study, respectively). Karasu et al. (2010) found that serum protein rejection was dependent on transmembrane pressure and temperature in polymeric SW membranes, but not in ceramic membranes. The findings in this study suggest that SS membranes are a better alternative to ceramic membranes than polymeric SW membranes. The decrease in serum protein content in the retentates was also confirmed by the SDS-PAGE profile (Fig. 5). It was observed that the serum protein bands area gradually became smaller and their optical density value also decreased gradually. Although the serum protein bands on SDS-PAGE profile have

3.2.3. Serum protein removal efficiency analysis The actual serum protein removal efficiency has been shown to vary considerably among different membrane types: 98.3% UTP ceramic membrane (Hurt et al., 2010) and 70.3% SW membrane (Beckman et al., 2010). The mean (n ¼ 3) serum protein reduction percentage for each stage by the SS membrane was calculated based on the concentration of serum protein in permeate and original skim milk and shown in Table 5. Based on the model developed by Hurt and Barbano (2010), the serum protein reductions for each stage were, sequentially, 68%, 22% and 7%, if the CF and diafiltration factor were both balanced to 3. After the 3
, 3-stage process finished, 97% serum protein-reduced MCC can be produced. The actual serum protein reduction percentage for each stage by 0.02 mm SS membrane were 60.66, 21.41 and 10.32%, respectively (Table 5). When the 3
concentration, 3stage process finished, 92.38% serum protein-reduced MCC was manufactured. The serum protein removal percentages (60.66% and 21.41%) for stage 1 and stage 2 by 0.02 mm SS membrane were lower than the theoretical values (68% and 22%), but for stage 3, serum protein removal percentage (10.32%) by 0.02 mm SS membrane was higher than the theoretical value (7%). The higher serum protein reduction percentage for the third stage by 0.02 mm SS membrane than theoretical value may be related to the membrane rejection. Membrane rejection may be ascribed to either the inherent characteristics of the membrane or membrane foulant resistance. Hurt and Barbano (2010) indicated that if a membrane rejected serum protein, the serum protein removal percentage of the first stage may be impacted, but the serum protein removal percentage of the following stages may be improved. Based on serum protein removal rate for stage 3 (57.56% of the starting serum protein amount of stage 3), it can be deduced that if an additional 3
stage was added, a total serum protein reduction of 96.77% could be achieved and a serum protein reduction above 95% MCC could be obtained. The serum protein reduction percentages for each stage by ceramic membrane were 64.80%, 22.90% and 10.50%, respectively, which was closer to the theoretical values. After a 3
, 3-stage process by 0.1 mm UTP ceramic membrane, serum protein reduction above 95% was achieved (Hurt et al., 2010), so it is apparent that the 0.02 mm SS membrane exhibited the same efficiency with the ceramic membrane for MCC production. Measuring serum protein removal efficiency using serum protein removal percentages at different processing stages is one

Table 5 Comparison of the serum protein (SP) reduction for each stage during a 3
, 3-stage process using 0.02 mm SS membrane and ceramic UTP membrane.a Processing stage

Stage 1 Stage 2 Stage 3

Theoretical SP removal

SP reduction by SS membrane

SP reduction by ceramic membrane

By stage

Cumulative

By stage

Cumulative

By stage

Cumulative

68 22 7

68 90 97

60.66a 21.41b 10.32c

60.66a 82.07b 92.38c

64.80a 22.90b 10.50c

64.80a 87.80b 98.30c

a Values (%) are means (n ¼ 3); means in the same column not sharing a common superscript letter are significantly different (P < 0.05). Data for SP reduction calculated based on the concentration of SP in permeate and original skim milk; data for SP reduction by ceramic membrane taken from Hurt et al. (2010).

S. Zhang et al. / International Dairy Journal 80 (2018) 26e34 Table 6 Comparison of serum proteins (SP) removed for each stage by stainless steel (SS) membrane and ceramic UTP membrane.a Stage of processing

SP removed by membrane (L m2 h1) SS

Concentration 1st diafiltration stage 2nd diafiltration stage

Ceramic a

0.37 0.14b 0.08c

0.30a 0.11b 0.06c

a Values (in kg) are means (n ¼ 3); means in the same column not sharing a common superscript letter are different (P < 0.05). Data for ceramic membrane taken from Hurt et al. (2010).

considered method, but not the most effective. To compare serum protein removal efficiency of different membranes exactly, the membrane flux must be considered and the most effective expression for serum protein removal is the volume (L) of serum protein removed per square metre of membrane surface area per hour within each stage. The serum protein removal rate by 0.02 mm SS membrane and 0.1 mm UTP ceramic membrane for each stage are shown in Table 5. It is interesting to note that although the serum protein removal percentage by 0.02 mm SS membrane for each stage was lower than by ceramic membranes, the serum protein removal rate for each stage (0.37, 0.14, and 0.08 L m2 h1) by 0.02 mm SS membrane was higher than that of the ceramic membrane (0.30, 0.11, and 0.06 L m2 h1) (Table 6). This should contribute to the higher average skim milk flux (68.6, 76.2, and 85.7 L m2 h1) for each stage by 0.02 mm SS membrane, whereas the average skim milk flux for each stage by 0.1 mm UTP ceramic membrane was only 54.0, 54.0, and 54.6 L m2 h1. The 0.02 mm SS membrane is an ultrafiltration membrane in reality, but so far, the membrane extensively used for MCC production is the 0.1 mm microfiltration ceramic membrane and the pore size of polymeric SW membrane applied for MCC production is 0.3 mm (Beckman et al., 2010). Another difference between the 0.02 mm SS membrane and the 0.1 mm ceramic membrane was in the membrane systems employed. The membrane system used for 0.1 mm ceramic membrane is the UTP system, while the 0.02 mm SS membrane is not. Zulewska et al. (2009) demonstrated that the UTP system is more efficient than graded permeability (GP) system, so if the UTP system is used for 0.02 mm SS membrane, higher serum protein reduction efficiency can be expected. The 0.02 mm UTP SS membrane system could potentially exhibit higher serum protein removal rate than UTP ceramic membrane and could be an alternative for UTP ceramic membrane for MCC production. 4. Conclusion The 0.02 mm SS membrane exhibited superior performance for MCC production than the 0.1 mm SS membrane. The average skim milk flux and serum protein removal rate by the 0.020 mm SS membrane was higher than that of the 0.1 mm SS membrane. The 0.02 mm SS membrane, compared with the 0.1 mm UTP ceramic membranes (Hurt et al., 2010), had potential efficiency advantages for MCC production during a 3
, 3-stage process. The average skim milk flux for each stage with the 0.02 mm SS membrane was higher than with the 0.1 mm UTP ceramic membranes. Serum protein removal for each stage with the 0.02 mm SS membrane was close to that with 0.1 mm UTP ceramic membranes. The membrane filtration system used for the 0.02 mm SS membrane was the main constraint in the present study for serum protein reduction efficiency compared with 0.1 mm UTP ceramic

33

membrane, and for improved performance, it is recommended that the UTP system be applied with the 0.02 mm SS membrane in future work. The stages required for 95% serum protein reduction MCC can be reduced and the serum protein removal rate would be expected to be higher. Additionally, the permeate of the 0.02 mm SS membrane containing no micellar casein, is the ideal feed for SPC production, therefore, future work can also focus on the serum protein reclamation. Acknowledgements This research was funded by National Natural Science Foundation of China (31371808), the Special Fund for Agro-scientific Research in the Public Interest (201303085) and Beijing Innovation Team of Technology System in Dairy Industry. References AOAC. (2000). Official methods of analysis. Association of Official Analytical Chemists. Gaithersburg, MD, USA: AOAC International. Ardisson-Korat, A. V., & Rizvi, S. S. H. (2004). Vatless manufacturing of low-moisture part-skim mozzarella cheese from highly concentrated skim milk microfiltration retentates. Journal of Dairy Science, 87, 3601e3613. Beckman, S. L., Zulewska, J., Newbold, M., & Barbano, D. M. (2010). Production efficiency of micellar casein concentrate using polymeric spiral-wound microfiltration membranes. Journal of Dairy Science, 93, 4506e4517. Beliciu, C. M., Sauer, A., & Moraru, C. I. (2012). The effect of commercial sterilization regimens on micellar casein concentrates. Journal of Dairy Science, 95, 5510e5526. €n, C. G. P. H., Van der Sman, R. G. M., & Boom, R. M. (2004). Brans, G., Schroe Membrane fractionation of milk: State of the art and challenges. Journal of Membrane Science, 243, 263e272. Espina, V., Jaffrin, M. Y., Ding, L. H., & Cancino, B. (2010). Fractionation of pasteurized skim milk proteins by dynamic filtration. Food Research International, 43, 1335e1346. Govindasamy-Lucey, S., Jaeggi, J. J., Johnson, M. E., Wang, T., & Lucey, J. A. (2007). Use of cold microfiltration retentates produced with polymeric membranes for standardization of milks for manufacture of pizza cheese. Journal of Dairy Science, 90, 4552e4568. Hurt, E. E., & Barbano, D. M. (2010). Processing factors that influence casein and serum protein separation by microfiltration. Journal of Dairy Science, 93, 4928e4941. Hurt, E. E., Zulewska, J., Newbold, M. W., & Barbano, D. M. (2010). Micellar casein concentrate production with a 3
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