Ganju 2017 Métodos

Accepted Manuscript
A review on approaches for efficient recovery of whey proteins from dairy industry
effluents
Sparsh Ganju, Parag R. Gogate
PII: S0260-8774(17)30320-5
DOI: 10.1016/j.jfoodeng.2017.07.021
Reference: JFOE 8963
To appear in: Journal of Food Engineering
Received Date: 01 April 2017
Revised Date: 18 July 2017
Accepted Date: 19 July 2017
Please cite this article as: Sparsh Ganju, Parag R. Gogate, A review on approaches for efficient
recovery of whey proteins from dairy industry effluents, Journal of Food Engineering (2017), doi:
10.1016/j.jfoodeng.2017.07.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
1 A review on approaches for efficient recovery of whey proteins

2 from dairy industry effluents
3
8 Sparsh Ganju, Parag R. Gogate*
9
10
11
12
13
14 Chemical Engineering Department,
15 Institute of Chemical Technology,

16 Matunga, Mumbai-40019,
17 India.
18
19
20
21
22
23 *Author to whom correspondence may be addressed
24 E-mail: pr.gogate@ictmumbai.edu.in; Tel: +91 22 33612024 Fax: +91 22 33611020
25
26 ABSTRACT
27 A significant proportion of waste generated by the dairy industry is in the form of
28 whey, which creates a lot of environmental concerns especially in developing countries. The
29 most significant problem is its significant BOD content ranging from 35,000-55,000 mg/L.
30 Considering the vast quantum of whey produced globally (about 145 million tons per year), it
31 is imperative to propose suitable approaches which can be used to recover valuable products
32 such as lactose or proteins and also help in reducing the environmental concerns. The current
33 review provides an in-depth understanding into different available approaches for recovery of
34 whey proteins also highlighting the recent advances. Special emphasis has been made on the
35 critical analysis of the membrane separation methods which are currently accepted as the
36 most viable methods for whey protein recovery. In addition, some novel approaches for whey
37 protein separation have also been discussed. The work also presents the engineering aspects
38 related to the various methods of pre-treatment (increase the shelf life of whey and also help
39 in mitigation of fouling) and post-processing of whey proteins (spray and freeze drying) for
40 effective utilization as valuable products. Discussion has also been provided on the
41 optimization of operating parameters so as to maximize the recovery efficacy and on the
42 reactor configurations with possible potential for scale up. Overall, it appears that there is
43 considerable promise for developing commercial approaches for the recovery of whey
44 proteins conforming to the principle of wealth from waste.
45
46 KEY WORDS
47 Whey proteins; Membrane separations; Pre-treatment; Operating parameters; Spray
48 drying; Value added products
49
50 1. INTRODUCTION
51 The dairy industry generates huge quantities of whey which is largely considered as
52 waste. However, over the past few decades there has been an increased interest in the
53 utilisation of whey for the production of value added products like whey proteins. The current
54 work aims to highlight the recent advances in the field of whey protein separation from whey
55 with a primary emphasis on the use of membrane separation methods. Initially a brief
56 introduction into the generation of whey along with the description of the most important
57 compounds has been presented followed by overview of the membrane based methods.
58 1.1 Whey
59 Milk and dairy products are an integral part of our life. Milk products provide nutrition
60 in the form of energy (carbohydrates present as lactose), nitrogen (from the proteins) and also
61 act as a source of calcium. Whey is a by-product of the dairy industry and produced in
62 significant quantum due to the volume of required milk products especially in countries like
63 India and China. Whey is the liquid remaining after the process of curdling of milk and is
64 primarily obtained as an integral by-product of the process for cheese and casein
65 manufacture. Typically, 96% of whey comes from the manufacture of cheese and the rest is
66 from the process of casein manufacture. Depending on the type of cheese to be manufactured
67 and the enzymes used for manufacture, whey can be classified into two major categories as
68 sweet whey and acidic whey. Sweet whey is produced during the making of rennet type of
69 cheese like cheddar and Swiss cheese whereas acidic whey is a co-product from the process
70 for manufacture of certain acidic dairy products like cottage cheese and strained yogurt.
71 Whey composition and characteristics are also dependent on the source of milk (cow,
72 sheep, etc.), the feed of the milk-producing animal, the processing method used, the time of
73 the year and the stage of lactation. A typical representation of the whey composition is
74 provided in Table 1 (Tsakali et. al., 2010). It is indeed surprising that for a number of years,
75 the whey obtained from the dairy industry was considered as waste (still considered in
76 developing countries) and either disposed directly into the water resources creating
77 significant environmental concerns or used as animal feed (post drying) and partly as a
78 fertilizer. The global utilization of whey is depicted in figure 1 (Tsakali et. al., 2010).
79 Dairy industries, over the last few years, have increasingly resorted to the use of
80 various techniques and technologies to treat the whey and recover the principal components
81 as proteins, lactose and minerals. The processing of whey to yield valuable products helps to
82 reduce environmental pollution and it also provides the dairy industry with an added
83 economic incentive due to the possible sale of these processed products. A range of possible
84 valuable products that can be obtained from whey is shown in figure 2. It is also important to
85 understand that subsequent value addition is also possible from the first stage recovered
86 products.
87 1.2 Whey proteins
88 Whey proteins are a mix of globular proteins that can be isolated from whey and have
89 excellent nutritional properties. The recovered proteins consist of primarily two types, whey
90 protein and casein. Casein is a phosphoprotein and used as a food additive, a binder for safety
91 matches and also as a source of carbohydrates, amino acids, calcium and phosphorous. The
92 quantum of whey protein typically ranges from 6.0 to 10 g/L in the case of sweet whey and
93 6.0 to 8.0 g/L in the case of acid whey (Bozanic et. al., 2014). It is a great source of amino
94 acids and is available in various forms. Whey protein consists of mainly two types, β-
95 lactoglobulin (β-Lg) and α-lactalbumin (α-La). β-Lg is the major whey protein found in the
96 cow and sheep milk. Unlike α-La, it has no clearly defined function except that it is a
97 lipocalin protein. The molecular weight of β-Lg is 18363 Da and its iso-electric point is 5.1.
98 α-La is the second major whey protein and consists of 123 amino acids and 4 disulphide
99 bridges. Its molecular weight is 14178 Da and the iso-electric point is around 4.4 (Lucy et.
100 al., 2008). The milk protein content varies based on the protein source. Cow milk has about
101 18% whey protein of total milk protein content. On the other hand, human milk can have a
102 whey/casein ratio over the range of 70/30 to 80/20 during early lactation, which is reduced to
103 50/50 in late lactation (Lonnerdal, 2003).
104 The various forms of commercially available whey proteins are:
105 a) Whey Protein Concentrate (WPC): It is a processed form of whey protein which has the
106 lowest level of fats and cholesterol as compared to other forms of commercially available
107 whey but a high level of bioactive compounds. It also contains carbohydrates in the form
108 of lactose and has protein content in the range of 65-70% (Levin et. al., 2016). It has a
109 mild to slightly milky taste.
110 b) Whey Protein Isolate (WPI): WPI is whey protein which is further processed to remove
111 fats and lactose. It has lower quantities of bioactive compounds but a higher protein
112 content (>90%). Similar to WPCs, it has a mild to slightly milky taste.
113 c) Whey Protein Hydrolysate (WPH): WPH are pre-digested and partially hydrolysed whey
114 proteins manufactured with an objective of easier metabolism. WPHs have anti-oxidative
115 properties (Pena-Ramos and Xiong, 2001) and a protein content of 70-80% (Sinha et. al.,
116 2007). The added processing steps leads to WPHs being expensive than WPCs and WPIs.
117 d) Native Whey: Native whey is extracted from skimmed milk and may be produced as
118 concentrate or isolate.
119 1.3 Whey protein separation
120 Whey protein separation essentially consists of three stages:
121 a) Pre-treatment: Pre-treatment is required mainly to avoid the problems faced in the
122 separation and drying stages as discussed in the following sections.
123 b) Separation: The separation of whey protein from whey has become a topic of great
124 concern and subsequent research over the past two decades. The quantity of whey
125 produced is almost 9-fold greater than the quantity of cheese produced (Bozanic et al.,
126 2014) and has a detrimental effect on the environment if left untreated. Citing the
127 substantial amount of nutritional value associated with whey, there have been several
128 methods which have been used to separate whey into its various economically-useful
129 components. These separation techniques are primarily based on membrane separation
130 processes with ultrafiltration being the highest contributor in the production of whey
131 proteins. The subsequent section on separation will concentrate not only on these
132 membrane-based methods but will also highlight some of the non-conventional methods
133 of whey protein separation. The major problem faced in the separation of whey proteins
134 is the fouling of membranes. This leads to a reduction in flux and efficiency of whey
135 protein separation and often requires the use of pre-treatment techniques to negate the
136 effects of membrane fouling.
137 c) Drying: Separation of whey proteins is just the first step in making the whey protein
138 commercially viable. For commercial viability, this whey protein needs to be dried in
139 order to reduce transportation costs and increase its shelf life. The most commonly used
140 method of drying is spray drying. A major challenge in the drying of whey proteins is to
141 optimise the application of heat to the separated whey proteins in order to mitigate the
142 possibility of denaturation of the protein. Freeze drying does not pose the threat of
143 denaturation as it is performed under vacuum and hence lower temperatures. The section
144 on drying delineates the process of whey protein drying and also discusses a relatively
145 newer but expensive method of drying- freeze drying.
146 The review will now provide overview of the membrane separation methods including the
147 improved configurations for higher capacity operations. The work also explains the recent
148 advances in all these processing stages including the separation methods. The engineering
149 aspects of the different pre-treatment approaches and ways to improve the membrane
150 operations have also been presented quantifying the degree of improvement that can be
151 obtained.
152
153 2. MEMBRANE SEPARATION METHODS
154 The most revolutionising technology in the dairy sector has unarguably been the membrane
155 separation technology. The membrane separation technology refers (Winston and Sirkar,
156 1992) to the use of semi-permeable membrane filters to concentrate or fractionate a feed
157 stream of liquid giving two product streams of different compositions. The membrane
158 selectively allows certain compounds to pass through while restricting the flow of other
159 compounds through it and this affinity for certain compounds is the main driving mechanism
160 for separation. The compounds which pass through the membrane along with the liquid make
161 up the “permeates” and the liquid which is retained is known as the “retentate”. In general,
162 the dairy industry employs membrane separation techniques at a very large scale (Saxena et
163 al., 2009) as presented in table 4. About 66.67% of the membrane area installed in the dairy
164 sector is used for the treatment and filtration of whey whereas the remaining 33.33% is used
165 for the treatment of milk (Kumar et al., 2013). The two major types of membrane separation
166 techniques employed by the dairy industry for the separation of whey proteins are
167 ultrafiltration and microfiltration.
168 2.1 Ultrafiltration
169 In ultrafiltration, similar to other filtration processes, hydrostatic pressure forces a liquid
170 against a semi-permeable membrane which leads to the retention of suspended solids and
171 solutes having high molecular weights. Water and lower molecular weight solids can pass
172 through the membrane without much hindrance. Ultrafiltration is the most commonly used
173 industrial method for the separation of whey proteins and offers as a unique method for the
174 recovery of whey proteins in their native form. The typical molecular weight cut-off for this
175 process is 10 kDa. Whey protein separation by ultrafiltration is normally performed at
176 temperatures below 55 C and an inlet pressure of 300 kPa with the membrane pore size over
177 the range of 200–250 nm. The application of ultrafiltration can be used to increase the protein
178 content of whey up to 85%. There have been a lot of modifications which have been made to
179 conventional ultrafiltration with an objective to increase the yields of whey protein recovery.
180 The subsequent sections provide a brief discussion on these modifications.
181 2.1.1 Cascaded ultrafiltration
182 In a cascaded ultrafiltration system either the retentate or permeate from one membrane stage
183 is transferred to the next or previous stage as the feed. Some streams may also be recycled to
184 improve the cascade performance. Such an arrangement allows for additional enrichment of
185 whey proteins. Patil et al. (2014) reported the use of a three-stage ultrafiltration cascade for
186 the continuous purification of whey protein isolate. Three different cascade configurations
187 were examined in the work as represented in Figure 3. One of these configurations was a
188 countercurrent configuration with recycling of permeate. In order to establish the optimum
189 performance and efficacy of different membranes, single-stage experiments were also
190 performed under steady state to establish the stable conditions for three-stage cascade
191 operation. It was reported that a relatively constant and optimum performance was achieved
192 using a 2 g/L solution of WPI in a 7.2 pH solution containing 5 mM NaCl at a cross flow
193 velocity of 0.1 m/s. It was also reported that all the three cascade systems gave superior
194 results as compared to a single-stage system and higher degrees of separation were observed
195 if the ratio of product stream flow rate to waste stream flow rate was high. Configuration B
196 was reported to give best results in terms of high recovery with good purity as compared to
197 both cascades A and C. Cascade A was reported to give better results provided optimal trans-
198 membrane pressure is applied but this required a different membrane surface for each stage,
199 which was a limiting factor. It was concluded from the study that if the streams between
200 different cascade stages are integrated appropriately, the separation efficiency can be
201 significantly enhanced as compared to single stage operation.
202 2.1.2 Bio-catalysis
203 An attempt to enhance the size of the proteins during the operation can lead to a reduction in
204 membrane fouling coupled with an increase in membrane flux and whey protein recovery
205 (Barba et al., 2001). Typically peptides as well as α-La and β-Lg pass into the permeate
206 stream though sometimes these proteins get stuck in the membranes and lead to fouling of
207 membranes. Studies have also confirmed that large sized proteins have less influence on the
208 membrane and are also easier to clean as compared to the small sized proteins (Tolkach and
209 Kulozik, 2006, Norazman et al., 2013). These findings have encouraged experimentation on
210 the subject of bio-catalysis with an objective of enhancing the size of the proteins. These
211 methods use catalytic membranes to catalyse the whey proteins during the separation process
212 itself. Of the various enzymes capable of cross-linking proteins, transglutaminase (TG) was
213 reported to be the most useful as it could catalyse α-La, β-Lg and even caseinomacropeptide
214 (Tolkach and Kulozik, 2005) by forming intra and intermolecular crosslinks, resulting in the
215 formation of a high molecular polymer.
216 Wen-Qiong et al. (2017) also reported the use of a transglutaminase (TG) membrane which
217 acted as a catalyst for conversion of the whey proteins into cross-linked whey proteins. In this
218 study, the filtration performance of the membrane was assessed after the catalysis of cheese
219 whey by TG under different conditions and various parameters such as protein rejection rate,
220 whey protein recovery rate, volumetric processing rate, lactose rejection rate and relative
221 permeate flux were considered. It was reported that the whey protein recovery rate increased
222 by about 15% and the lactose rejection rate decreased by 10% with the use of catalytic
223 membranes along with an increase of about 30% in the permeate flux under TG enzyme
224 catalysis. These results were obtained at a pH of 5, enzyme concentration of 40 U/g,

225 temperature of 40 C and a retention time of 60 minutes. A decrease in membrane resistance
226 is also observed after catalysis. The reported results are promising and demonstrate the
227 potential uses of using TG industrially for the efficient separation of whey proteins.
228 2.1.3 High Performance Tangential Flow Ultrafiltration (HPTFF)
229 Conventional ultrafiltration membranes exploit the difference in the size of protein particles
230 to obtain separation into permeate and retentate phases. HPTFF uses the difference in size of
231 the particles as well as the charge characteristics of molecules and this modification can be
232 effectively used to obtain higher separation efficiency. An advantage of using HPTFF is that
233 it can even be used to separate biomolecules with the same molecular weight based on the
234 differences in the charge. It is also important to understand that HPTFF builds on the
235 fundamentals of the already existing ultrafiltration technology and has a well-established
236 industrial infrastructure, though not in the dairy industry applications.
237 Typically at the isoelectric point, proteins have neutral charge which also implies that there
238 are no ionic layers associated with them and their effective volume is the minimum at the
239 isoelectric point. As the pH deviates from the isoelectric point, a net charge develops on the
240 protein and leads to an increase in the effective volume and retention of proteins. The use of
241 charged membranes can hence enhance the separation of proteins. For instance, the use of a
242 positively charged membrane will cause increased retention of positively charged proteins
243 due to an increase in the effective volume caused due to repulsion of like charges. Cheang
244 and Zydney (2003) reported that the use of charged membranes resulted in a 100-fold
245 purification and greater than 90% recovery of β-Lg from a binary mixture of α-La and β-Lg.
246 Arunkumar and Etzel (2013) also demonstrated the use of charged membranes to enhance the
247 separation of α-La and β-Lg. It was reported that both these proteins differ in their isoelectric
248 points (4.4 vs 5.2, respectively) but not so much in their molecular weights (or sizes), which
249 are 14.4 kDa and 18.4 kDa, respectively. Thus, use of positively charged composite
250 regenerated cellulose membrane (MWCO = 300kDa) increased the selectivity of α-La as
251 against β-Lg (when compared with an uncharged membrane). Overall, an increase in the
252 fractionation selectivity of α-La by a factor of five was reported with 87% pure α-La being
253 obtained using the positively charged 300 kDa membrane in a two-stage flow configuration.
254 It was thus established that the addition of a positive charge allowed selective fractionation of
255 dairy proteins which are about 15-20 times smaller than the membrane’s MWCO. It is
256 important to understand that no direct study for whey protein recovery could be seen though
257 there is indeed a great promise.
258 2.2 Advantages of membrane separation techniques
259 Membrane separation methods have the following advantages over other methods of
260 processing like membrane distillation, anion exchange membranes and precipitation:
261 i) Membrane technology is a novel non-thermal and environmentally friendly
262 technology. The non-thermal nature of membrane separation ensures that the
263 temperature change during the process is kept to a minimum, which also helps in
264 the abatement of various temperature related issues like denaturation of proteins.
265 ii) Various selective mechanisms such as ion exchange and solution diffusion can be
266 used to yield higher selectivity in the membrane separation. The material of
267 construction of membranes can also be chosen such that it has a higher affinity for
268 certain proteins. The recent advancement in technology has also been based on the
269 manufacture of synthetic membranes with specific properties as per the industrial
270 needs.
271 iii) The membrane configurations are suitable for easy industrial applications due to
272 compact design and low maintenance. Also, the existing capacity can be increased
273 easily by adding membrane modules in parallel to the existing ones.

274 iv) Specialised knowledge base is not required to handle or operate these membrane
275 modules making the actual use very simple.
276 2.3 Comparison of various membrane separation techniques
277 2.3.1 Performance comparison of ultrafiltration, nanofiltration and reverse osmosis
278 Yorgun et al. (2008) compared the performance of seven membrane separation processes (4
279 nanofiltration modules, 2 reverse osmosis units and 1 ultrafiltration module; all available
280 commercially in spiral-wound configurations) applied for the recovery of whey protein from
281 white cheese whey and curd cheese whey. The testing of each module (configuration details
282 given in table 5) was performed using single stage operation as well as the cascaded
283 operation (operating conditions listed in table 6). The operation was such that no fouling
284 remained in the module after each run and the membranes were reused after each experiment.
285 Before the reuse, the cleaning of membranes was performed using an initial nitric acid
286 solution (Ultrasil 75) followed by alkaline cleaning using Ultrasil 110 which was ultimately
287 followed by a final disinfection step using a sodium hydroxide solution (Ultrasil 125). The
288 cleaning efficiency was gauged by measuring the pure water flux of modules after cleaning.
289 The feed solutions for these experiments were white cheese whey and curd cheese whey
290 which were obtained from a company which generated approximately 78,000 L of whey per
291 cycle of cheese production. The composition of both feed streams have been given in table 7.
292 It was reported that the protein rejection and the permeate flux values for the modules A, B,
293 C and E were promising whereas module D gave a high permeate COD but a relatively poor
294 operational performance. It was reported that a single stage operation of module D was better
295 due to its economic benefit especially in cases where the effluent stream is to be further
296 concentrated or treated. It was also established that the performance of RO system was poor
297 as the transmembrane pressure (TMP) during the experiment was only raised to 8 bar, which
298 is not sufficient for an RO module to deal with highly loaded influents. Typically, a TMP of
299 30-40 bar should be applied for obtaining better results in terms of permeate flux. Further, it
300 was reported that use of cascaded nanofiltration and reverse osmosis membrane led to the
301 separation of protein and lactose at different stages. The nanofiltration stage (which behaved
302 like an ultrafiltration module) concentrated the protein portion whereas the RO stage was able
303 to recover lactose leading to a clean effluent.
304 2.3.2 Application of commercially available ultrafiltration membranes for α-La production
305 An evaluation of commercially available, wide-pore ultrafiltration membranes was performed
306 by Metzger et al. (2011) for the production of α-La enriched whey protein concentrate
307 (WPC). The feed whey was first mechanically clarified and pasteurised at 63 ᵒC for 30
308 minutes and then subjected to microfiltration to obtain a pre-purified feed stream devoid of
309 casein fines, lipid materials, and aggregated proteins. The pilot-scale microfiltration unit used
310 in the work contained a 0.2 μm spiral-wound polyvinylidene fluoride (PVDF) membrane
311 with a filtration area of 5.2 m2. The TMP, temperature and pH of the microfiltration process
312 were 55 kPa, 31 ᵒC and 6.3, respectively. The permeate from the microfiltration process was
313 then used as the feed for the wide-pore ultrafiltration studies performed using 3 membrane
314 types (PVDF50, PVDF100 and PES300) at three TMP (110, 207 and 310 kPa). The three
315 membranes used in the study had MWCO as 50, 100 and 300 kDa respectively. Among the
316 three types of wide-pore membranes used, it was reported that optimal separation of whey
317 protein was obtained using the PVDF50 membrane operated at a TMP of 207 kPa. An α-La
318 purity of 0.63 and α-La:β-Lg ratio of 1.41 was reported to be obtained with an overall flux of
319 49.46 L/m2h. An economic analysis of this process for a hypothetical plant presented in the
320 work indicated the cost of one kilogram of α-La enriched WPC 80 as $17.92/kg, which is
321 significantly lower as compared to the market value. The study clearly established the
322 commercial viability of α-La enriched WPC production.

323 Cowan and Ritchie (2007) studied the effects of membrane charge and solution pH on
324 filtration of α-La (14.1 kDa) and β-Lg (18.4 kDa) using commercially available
325 polyethersulfone (PES) membranes. The PES membrane was modified by functionalization
326 achieved by polymerisation of styrene in the membrane pores followed by sulphuric acid
327 treatment of the resulting polystyrene grafts in a membrane containing an open pore structure
328 with charged sulfonated grafted polymer chains. The fluxes across the membranes were
329 found to decrease from 1936 L/ (m2 hr bar) for the raw membrane to 772 L/ (m2 hr bar) for
330 the treated membrane but with higher selectivity. There was a decrease observed in each
331 stage of modification of the raw PES membrane to the final sulphonated polymerised
332 membrane. This decrease in flux can be explained by the fact that fully sulphonated PES is
333 water soluble leading to the swelling of membrane leading to a constriction of flow path. The
334 subsequent polymerisation of styrene partially fills the pores with globular polymer grafts,
335 resulting in decreased permeability. Finally, the sulphonated polymer grafts would extend
336 into the membrane pores further reducing the membrane permeability. The data obtained
337 from this study confirmed that it is possible to increase the selectivity of α-La through a
338 variety of effects such as reduced membrane pore size and enhanced electrostatic repulsion
339 between the charged membrane and the proteins. On an average, the selectivity for the
340 functionalised membrane improved by 5 times as compared to the raw membrane at pH 7.2.
341 This value was also observed to be 50% greater than the selectivity at pH 3.2, indicating an
342 enhancement due to electrostatic repulsion.
343
344 3. FOULING AND PRE-TREATMENT
345 3.1 Fouling
346 The use of membrane based separation methods worldwide to concentrate whey, and/or
347 fractionate and purify proteins at a global scale has also led to a major processing limitation
348 based on the fouling. Membrane fouling is caused due to a combination of several
349 phenomena such as concentration polarisation, pore blocking or cake formation. Mass
350 transfer through a membrane becomes limited due to the formation of deposits on the
351 membrane during the concentration of whey proteins from whey by ultrafiltration or
352 microfiltration.
353 The process of aggregation of whey proteins typically leads to enhanced fouling of
354 membranes. Steinhauer et al. (2015) investigated the impact of whey protein aggregates on
355 the extent of fouling as well as the interaction of native whey proteins and whey protein
356 aggregates. The study was performed in both dead-end lab scale and cross-flow pilot scale
357 filtration modules. It was reported that β-lactoglobulin accelerated membrane fouling during
358 microfiltration and ultrafiltration due to thiol/disulphide reactions. For cross-flow filtration of
359 sweet whey, membrane fouling was reported to be increased by whey protein aggregates up
360 to a certain degree of whey denaturation but above a critical value of 30% protein
361 denaturation, the flux was found to increase again. The trend was explained based on the
362 improved erosion of larger protein aggregates as well as reduced reactivity of heat-treated
363 whey protein aggregates. It was established from the study that heat-induced aggregates of β-
364 lactoglobulin strongly affect membrane fouling due to the formation of a highly reactive
365 deposit.
366 Considering that fouling is a major problem limiting the efficacy of the membrane
367 based separation process, pre-treatment of whey needs to be performed so as to induce
368 selective changes to limit the negative effects that may be caused due to fouling.
369 3.2 Pre-treatment
370 Pre-treatment of whey before separation into its constituent components is a necessity
371 and is carried out predominantly before separation techniques involving membranes like
372 microfiltration, ultrafiltration and reverse osmosis. Pre-treatment of whey before filtration is
373 done to reduce the effects of membrane fouling and the selection process typically is driven
374 by the low heat stability of whey protein solutions. Membrane fouling occurs due to the
375 blocking of pores of the membrane being used which results in a gradual reduction of flux
376 with time and an increase in the pressure drop. At the same time, the sensitivity of whey
377 proteins to heat also affects the shelf life of these proteins causing shorter shelf lives than
378 desired. An ideal pre-treatment system will be one which completely negates the effect of
379 membrane fouling and at the same time also gives the desired preservative properties to
380 processed whey proteins. Pre-treatment methods may also be useful against concentration
381 polarisation.
382 The most popular methods of pre-treatment used in the dairy industry are as follows:
383 i) Chemical pre-treatment
384 ii) Heat (Thermal) pre-treatment
385 iii) Ultrasound pre-treatment
386 iv) Use of turbulence promoters
387 These methods are sometimes also used in combination with an objective of
388 enhancing the efficacy of the protein separation process. The basic understanding into each of
389 these pre-treatments has now been provided.
390 3.2.1 Chemical pre-treatment
391 Chemical pre-treatment is probably the most common type of pre-treatment used for
392 enhancing the efficacy of membrane separation. It involves the addition of chemicals to whey
393 with an objective to remove the components which contribute to membrane fouling and
394 bridging. Proteins have been identified as the primary component of the deposit obtained on
395 membranes during the processing of whey. However, it has also been shown that calcium and
396 phosphates contribute to membrane fouling due to the formation of insoluble calcium salts,
397 which act as bridging agents between membranes and the proteins. The chemical pre-
398 treatment methods are mostly aimed at the removal of calcium and phosphates by the use of
399 precipitation methods or processes.
400 Almecija et al. (2009a) reported the effectiveness of pre-treatment to improve the
401 separation as compared to the untreated whey separation. Acid whey obtained from
402 pasteurized whole milk was initially defatted, the pH brought down to 4.2 and the coagulated
403 casein was separated by centrifugation. Pre-treatment of the whey sample was performed by
404 the use of CaCl2 (Calcium chloride) and by maintaining the pH and temperature at 7.3 and 55
405 ᵒC, respectively. After allowing time for the aggregation of lipid-calcium phosphate particles,
406 the whey was cooled and centrifuged. An analysis of the protein concentration in the treated
407 and untreated whey was performed using a reversed-phase high-performance liquid
408 chromatography (RP-HPLC) and the results obtained have been reproduced in Table 2. It can
409 be seen from the reported data that no significant alterations occurred due to the pre-treatment
410 with all the five proteins giving nearly identical concentrations in both cases. To determine
411 the influence of pre-treatment on the flux enhancement through ultrafiltration membranes, the
412 clarified acid whey and the untreated whey were subjected to ultrafiltration using a 50 kDa
413 tubular ceramic membrane at similar operating conditions (pH = 7.3). It was observed that the
414 permeate flow increased by a factor of almost 3.0-3.5 when whey was pre-treated confirming
415 the efficiency of the pre-treatment method. At a transmembrane pressure of 1.5 bar, the initial
416 fluxes were 18 and 61 L/(m2h) for the untreated and pre-treated whey, respectively which
417 dropped to 7 and 19 L/(m2h) respectively after 4 hours of operation.
418 3.2.2 Heat (Thermal) pre-treatment
419 Thermal pre-treatment focuses on the controlled application of heat energy in order to
420 improve the shelf life and stability of protein solutions and beverages. Thermal pre-treatment
421 is typically used to reduce or stop the aggregation of proteins which may cause the solutions
422 or beverages to become turbid due to the precipitation of protein aggregates. Heat treatment
423 can also help in reducing the membrane fouling by aiding in the removal of calcium
424 phosphate crystals which would otherwise precipitate in membrane pores, which helps in
425 limiting the problem of membrane fouling. Heat pre-treatment also results in an increased
426 permeate flux (Hickey et al., 1980).
427 Whey proteins, mainly β-lactoglobulin, α-lactalbumin, and bovine serum albumin, at
428 neutral pH exist in a globular state when heated at temperatures above 65 °C. The extent of
429 aggregation depends on the temperature and the duration of heat exposure. Coarse protein
430 aggregates cause turbidity and precipitation during storage. Studies have been mostly directed
431 at applying heat treatment to improve heat stability of whey proteins. Baier and McClements
432 (2001) reported that the introduction of co-solutes such as sucrose, glycerol and sorbitol
433 enhanced the heat stability of whey proteins. Additionally, Zhong et al. (2013) reported the
434 use of combined treatment involving microbial transglutaminase (mTGase) and heat
435 treatment to enhance the stability of whey proteins.
436 In an attempt to understand the effects of heat pre-treatment on the separation of whey
437 proteins, Kim et al. (1989) performed a study which showcased the effects of heat pre-
438 treatment on several functional properties of whey protein concentrate. Whey was pre-treated
439 with calcium chloride to final concentration of 1.2 g/L of Ca and the pH adjusted to 7.3 using
440 NaOH followed by rapid heating to 50 ˚C and centrifugation. Two samples of whey (one pre-
441 treated and the other untreated) were subsequently ultrafiltered at 20-25 ˚C with a Pellicon
442 Cassette UF unit (10 kDa MWCO) made of Polysulfone. The trans-membrane pressure was
443 maintained at about 70 kPa. The flux of pre-treated whey was in the range of 95-100 mL min-
444 1 ft-2 for the treated whey as compared to about 45-50 mL min-1 ft-2 for the untreated whey
445 over a 4 hour test period. The decrease in permeate flux was about 20% and 33 % for the pre-
446 treated and untreated whey samples, respectively. The study established that pre-treatment of
447 whey reduced the protein content of whey by about 10% (average of micro-Kjedahl and SE
448 HPLC).
449 3.2.3 Ultrasonic pre-treatment
450 Ultrasonic pre-treatment has been shown to breakdown protein aggregates and also
451 improve the heat stability. Typically use of ultrasonic treatment alone results in the abatement
452 of fouling to only a small extent but the use of ultrasound combined with the heat treatment
453 gives much higher efficacy with reduced pore blockage and cake growth. It is also important
454 to understand that the protein concentration in the permeate remains mostly unchanged in all
455 the cases. Koh et al. (2014) highlighted the potential of whey pre-treatment using ultrasound
456 and a combination of heat and ultrasound. It was reported that the energy required for
457 ultrafiltration was lower in the case of sonicated whey due to the reduction in the viscosity.
458 However, only heat pre-treatment of the whey led to an increase in the viscosity of whey due
459 to the formation of protein aggregates. The viscosity of 5% WPC80 (whey protein
460 concentrate with 80% whey protein) in the native, sonicated, heat pre-treated, and heat pre-
461 treated followed by sonication forms was found to be 3.2 ± 0.1 cP, 3.0 ± 0.1 cP, 10.0 ± 1.3
462 cP, and 3.0 ± 0.1 cP, respectively. A substantial reduction in viscosity was clearly established
463 when heat pre-treatment was followed by ultrasound. The study also showed that further heat
464 treatment does not have a considerable effect on the viscosity. The protein content was found
465 to be greatly affected by the solid concentration of the feed solution (Iametti et al., 1995,
466 Elofsson et al., 1995, Qi et al., 1995).
467 Ultrasonic fields can also be effectively applied in membrane filtration systems for
468 both flux enhancements (Cai et. al., 2010, Chai et. al., 1998, Li et. al., 2011, Simon et. al.,
469 2000) as well as to improve cleaning efficiency (Popovic et. al., 2010, Lamminen et. al.,
470 2006). Application of an acoustic field causes cavitation in the liquid resulting in shear and
471 turbulence which consequently reduces membrane fouling. The use of a low-frequency, low-
472 power ultrasound significantly enhances the permeate flux during ultrafiltration
473 (Muthukumaran et al., 2005 and Muthukumaran et. al., 2007) attributed to the increase in the
474 mass transfer coefficient within the concentration polarisation layer due to localised flow
475 disturbances and turbulence induced by the bubble collapse and acoustic streaming. It must
476 also be noted that higher acoustic power levels can impact structural integrity of the
477 membrane and hence an optimum power dissipation and duration must be selected. It is also
478 important to maintain a proper installation of the ultrasonic equipment to protect the
479 membranes from zones of intense cavitational activity. Typically, application of low power
480 ultrasound (of the order of 2W/L of liquid) has shown to enhance the permeate flux by almost
481 1.2-1.7 times across the full range of operating conditions (Muthukumaran et al., 2005). Also,
482 it was reported that no damage was observed to the membrane based on the measurements of
483 the flux for water over the course of several weeks.
484 Koh et al. (2014) investigated the effect of ultrasound alone as well as combined
485 operation of heat and ultrasound on the membrane fouling. A 20 kHz, 400 W S-450D
486 ultrasound generator was used for this study. The power delivered ultrasonically into the
487 solution was found to be 31 W. The feed solution to the membrane cells was circulated using
488 a positive displacement pump through a set of coils maintained at 5 ᵒC. No significant change
489 in the ultrafiltration flux decline pattern between the native and sonicated solutions was
490 observed. Also, at a higher concentration of 10 wt. % WPC80, a significant difference in the
491 flux reduction was observed for both the feeds. A steeper initial flux decline was observed for
492 the native sample with the permeate protein concentration remaining the same in both the
493 cases. On the other hand, the heat-treated solutions showed a clearer distinction between the
494 flux data of sonicated and unsonicated feeds as compared to data obtained from using only
495 ultrasonic pre-treatment. The only pre-heated feed gave a steeper initial slope for the flux
496 reduction signifying the blockage of pores caused by larger and denser aggregates. In the case
497 of the sonicated and pre-heated feed, the aggregates were attributed to have broken down into
498 smaller size causing lesser blockages. Thus, the superiority of heat treatment in conjunction
499 with ultrasound method is indeed established for pre-treatment of whey.
500 Ultrasonic pre-treatment along with heat pre-treatment can also be effectively used to
501 improve the heat stability of whey proteins (Ashokkumar et al., 2009). Upon the application
502 of ultrasound to a heated solution of denatured and aggregated proteins, a drastic decrease in
503 the protein aggregate size and viscosity is observed. This was attributed to the intensified
504 mass transfer due to the cavitational effects of acoustic streaming and bubble collapse.
505 Alternatively, Chandrapala et al. (2011) attributed the observed effects to the disruptions of
506 the hydrophobic interactions caused by shear forces generated during acoustic cavitation.
507 3.2.4 Turbulence promoters
508 Turbulence promoters such as vibratory shear and rotating disk modules enhance
509 turbulence and shear near the membrane surface. Vibratory shear-enhanced process (VSEP)
510 is a dynamic filtration system which uses a torsionally-vibrating membrane, around a vertical
511 axis at about 60 Hz with a displacement at the periphery of about 3 cm. This generates very
512 high shear rates at the membrane due to the fluid inertia while keeping the inlet flow rates
513 and head loss to a minimum (Al-Akoum et al., 2005). The shear effects lead to the prevention
514 of particle deposition and a subsequent reduction in the fouling. An increase in the permeate
515 flux was reported (Al-Akoum et al., 2002, 2005) based on the use of the turbulence promoters
516 as compared to a standard spiral wound membrane in the microfiltration and ultrafiltration of
517 skim milk. Despite the increase in permeate flux, these systems have limited potential due to
518 the high cost of equipment and difficulty in scale-up due to the membrane area limitations.
519 3.3 Cleaning of fouled membranes
520 More often than not, pre-treatment of feed solutions during ultrafiltration may not be
521 sufficient to completely stop membrane fouling. Membranes often need to be cleaned to
522 remove the detrimental deposits and restore them to their initial permeation properties.
523 Conventionally, membranes are cleaned by using an alkali cleaning step followed by an acid
524 cleaning stage. A sodium hypochlorite or sodium dodecyl sulphate cleaning step may also be
525 used if the alkali/acid cleaning steps are not completely successful (Ogunbiyi et al., 2008,
526 Kazemimoghadam and Mohammadi, 2007, Almecija et al. 2009b, Zapata et al., 2009).
527 The cleaning procedures might be needed as frequently as once a day (Blanpain-Avet
528 et. al., 2009). The cleaning agents can also cause damage to the membranes leading to a
529 reduction in their lifetime and causing morphological modifications. These adverse effects
530 coupled with the negative environmental impact of the generated wastewaters after the
531 cleaning treatment have led to several researchers focussing on the development of non-
532 conventional cleaning methods including ultrasonic cleaning (Muthukumaran et al., 2007),
533 use of saline solutions (Lee and Elimelech, 2007, Corbaton-Baguena et al., 2014) or using
534 electric fields (Corbaton-Baguena et al., 2016). A discussion on these non-conventional
535 cleaning approaches has been presented in the following sections.
536 3.3.1 Cleaning of ultrafiltration membranes using NaCl solution
537 Corbaton-Baguena et al. (2015) investigated the application of NaCl solution for
538 cleaning of three ultrafiltration membranes of different materials and molecular weight cut-
539 offs (MCWOs) for the system of whey protein concentrates at different concentration levels
540 (22.2, 33.3 and 150 g/L). The different membranes used in the study were polyethersulfone
541 (PES) membrane of 5 kDa cut-off, a ceramic ZrO2-TiO2 membrane of 15 kDa cut-off and a
542 permanently hydrophilic polyethersulfone (PESH) membrane of 30 kDa cut-off. The effect of
543 different operating parameters such as salt concentration, temperature and cross-flow velocity
544 on the extent of cleaning was investigated and the dependency of the hydraulic cleaning
545 efficiency (HCE) on the MWCO, membrane material and operating conditions was assessed.
546 It was reported that NaCl solution is an effective cleaning agents as it was able to clean all
547 three membranes effectively for all the WPC solutions used. An increase in the HCE was also
548 reported with an increase in temperature and crossflow velocity. Beyond a critical NaCl
549 concentration (5 mM) for all membranes, the HCE was reported to decrease with an increase
550 in the NaCl concentration attributed to the competition between cleaning and fouling
551 mechanisms and also due to a reduction in the surface tension. Also, lower HCE was
552 observed at higher WPC concentrations and can be attributed to severe fouling.
553 3.3.2 Cleaning of ultrafiltration membranes using electric field
554 The use of electric fields to clean fouled membranes after whey protein separation
555 was demonstrated by Corbaton-Baguena et al. (2016). Two ZrO2-TiO2 (with MWCO as 15
556 and 50 kDa) ultrafiltration membranes fouled with three types of model whey solutions were
557 investigated in the work. The whey model solutions consisted of aqueous solutions of bovine
558 serum albumin (BSA) at 10 g/L concentration, a mixture of BSA (10 g/L) and CaCl2 (1.65
559 g/L) and three WPCs (total protein content of 45%) at different concentrations (22.2, 33.3
560 and 150 g/L). It was reported that the 15 kDa membrane could be restored successfully to its
561 initial permeation properties by the application of electric field with the addition of NaCl
562 solution. However, it was also observed that the 50 kDa membrane could not be cleaned
563 completely due to severe fouling. Also, higher temperatures of the cleaning solution as well
564 as electric potential were reported to yield higher value of HCE. The presence of NaCl at low
565 concentrations (5 mM) favoured membrane cleaning, giving HCE values of 100% at mild
566 temperatures (over the range 37.5-50C) for the 15 kDa membrane. The best operating
567 conditions to clean the 15 kDa membrane were established as 5 mM NaCl concentration,
568 transmembrane pressure of 1 bar, crossflow velocity of 4.2 m/s, temperature of 43.8C and an
569 electric potential of 30 V.
570 Another study (Tarazaga et. al., 2006) demonstrated the ability of electric fields for
571 membrane cleaning to restore the values of initial fluxes. In this study, electro ultrafiltration
572 was performed using laboratory crossflow filtration equipment. A tubular conductive
573 membrane, Carbosep® (formed by a layer of ZrO2-TiO2 over a carbon support) and a
574 coaxially fitted electrode was used with a half wave rectified current. The study used BSA (a
575 component of whey) as the feed solution. After each experimental run, membrane cleaning
576 was performed by flushing distilled water through the system followed by a cycle of base,
577 water, acid and water. Finally, an electric field was applied to give fast and efficient cleaning.
578 It was observed that the initial flux was always restored after the application of electric field,
579 however, the flux then, declined again. This behaviour can be attributed to the severe fouling
580 operating conditions (solution concentration, low tangential flow velocity, etc.). The cleaning
581 time as a function of the applied potential was also determined for the 0.1% w/w BSA. Table
582 3 gives the reported data for the cleaning times for different applied voltages. It was further
583 established that the application of a higher voltage resulted in lower cleaning times.
584 3.3.3 Effect of heat and ultrasonic pre-treatment on membrane cleaning
585 Koh et al. (2014) studied the effect of ultrasonic treatment on membrane cleaning
586 based on the classifications as reversible and irreversible fouling. Reversible fouling was
587 defined as the deposits which can be removed easily by rinsing water through the
588 ultrafiltration membrane system and extent of reversible fouling was assessed through the
589 flux recovered by water flushing. Irreversible fouling, on the other hand, referred to the
590 fouling layer that can only be removed through chemical cleaning and is calculated based on
591 the flux obtained after chemical cleaning relative to the original water flux obtained at the
592 start of the filtration run. It was reported that the extent of reversible and irreversible fouling
593 was similar for both native and sonicated feeds (without heat pre-treatment) attributed to the
594 small differences between the two samples in terms of both particle size and viscosity. The
595 flux was fully recovered after chemical cleaning in these cases with the value of 103%
596 recorded for the sonicated feed. The value above 100% reflects the presence of residual
597 surfactants, which are known to enhance the water flux. When the membrane was soaked in
598 deionised water between runs, it was reported that the absolute water flux in the next run falls
599 back to the original value, signifying the removal of these surfactants during storage.
600 For heat-treated feed filtration, the flux recovered by water flushing (reversible fouling)
601 reduced considerably, reflecting the denaturation of the proteins. Sonication increased the
602 proportion of reversible fouling that can be removed by this approach but was unable to reach
603 the level observed with the native feed solution. After chemical cleaning also, 100% flux
604 recovery for both heat treated solutions was unachievable which may be due to the inability
605 of the cleaning solutions to break down and dissolve larger aggregates. As the combined heat
606 and ultrasonic treated feed has a smaller proportion of large aggregates and a high proportion
607 of fouling materials are removed by water flushing, the flux obtained after cleaning is higher
608 than that of the only pre-heat treated feed (without sonication). It is also important to note
609 that the flux recovery obtained based on chemical cleaning was higher than that obtained by
610 only water flushing.
611
612 4. OTHER METHODS FOR WHEY PROTEIN RECOVERY
613 Several new methods for the recovery of whey proteins have also been developed with
614 an objective of enhancing the recovery potential and we now present discussion on these
615 upcoming methods.
616 4.1 Membrane distillation
617 Membrane distillation is a developing membrane separation process which unlike
618 membrane filtration processes is thermally driven. The heat for this process can be obtained
619 using solar heating or can be based on integration into existing heat paths (Hausmann et al.,
620 2012). The use of a hydrophobic membrane ensures that only water vapour is allowed to pass
621 and the flux is driven by the vapour pressure gradient between the feed and permeate sides.
622 The ability of this process to use waste heat from industrial processes makes it very energy
623 efficient especially for the concentration applications which are the most energy-intensive
624 operations in the dairy industry (Ramirez et al., 2006). As compared to evaporation which
625 may cause thermal degradation of proteins due to high temperatures, membrane distillation
626 offers advantage based on the operation at low temperatures. Hausmann et al. (2013) reported
627 the application of membrane distillation in whey processing establishing that an increase in
628 cross-flow velocity from 0.05 m/s to 0.14 m/s resulted in mitigation of fouling during the
629 membrane distillation of whey. Also, it was reported that the electrical energy used for
630 membrane distillation was lower in addition to higher fluxes and energy efficiency being
631 achieved. Overall, this method of membrane distillation is quite promising as far as whey
632 protein concentration is concerned at least on the laboratory scale but the application of this
633 method on an industrial scale would require further study.
634 4.2 Anion exchange membranes
635 Separations based on ion exchange are driven by the electrostatic interactions between
636 the surface charges on biomolecules, such as proteins, and clusters of charged groups on
637 membranes. A complementary buffer salt is released when the adsorbing biomolecule
638 displaces the counter-ions associated with the surface. The choice of anion exchange
639 membranes should be such that protein denaturation does not occur. Also, the biomolecules
640 adsorbed on the membranes need to be recovered at a later stage. The performance of three
641 strong anion exchange membranes (CIM QA, Q100 and HiTrap Q) was analysed for the
642 separation of major components of whey as α-La, β-Lg and BSA (Kim et. al., 2003). Elution
643 of the adsorbed proteins was performed using NaCl solution. The obtained results established
644 that HiTrap Q was the most effective anion exchange membrane among the three membranes
645 under consideration.
646 4.3 Precipitation

647 In this approach, precipitation of whey proteins is achieved by introducing heat or by
648 the addition of specific chemicals which selectively cause the precipitation of whey proteins.
649 The introduction of heat leads to the formation of aggregates which settle down in the
650 solution and can then be easily removed. A drawback of thermal precipitation is the
651 probability that the proteins may get denatured due to the heat, if not controlled properly.
652 Also, heating is an energy-intensive process, which may not be feasible considering the
653 quantum of whey generated in the process. The process of selective precipitation is based on
654 the use of chemicals like acetone, hydrochloric acid and ammonium sulphate. The addition of
655 chemicals in whey may lead to alteration of chemical properties due to changes in ionic
656 strengths and sometimes chemicals may also make the whey unusable for the specific
657 products such as bio-peptides.
658 A study (Yadav et. al., 2014) was conducted to recover the residual soluble protein after
659 yeast cultivation in cheese whey which was continuously fermented with cell recycle system
660 at 40 ˚C. The yeast was separated by centrifugation and residual soluble protein from the
661 fermented whey supernatant was precipitated by heat treatment (100 ˚C, pH 4.5 and 10 min
662 incubation). A maximum soluble protein recovery of 53% was reported to be achieved at pH
663 4.5 with 54% residual COD removal. It was, however, established that particles that can
664 separate by gravity sedimentation were obtained at a pH of 3.5 with a protein recovery of
665 47%. Thus, a reactor scale up study was conducted at pH of 3.5 with agitation, which resulted
666 in a soluble protein recovery of 68% and a residual COD removal of 62%. In addition to this,
667 precipitation of soluble protein was also evaluated using carboxymethylcellulose. The two
668 precipitation methods were then used sequentially (thermal followed by CMC precipitation)
669 to obtain an increase in protein precipitation to 81% of the total soluble protein.
670 4.4 Colloidal Gas Aphrons (CGA)

671 Colloidal gas aphrons are defined as the surfactant stabilized gas microbubbles
672 created by the stirring of a surfactant at high speeds (around 8000 rpm). CGA can be applied
673 for the separation of whey proteins based on principle of flotation (Amiri and Valsarai, 2004).
674 In this approach, a surfactant is typically added into a container which contains whey (Jauregi
675 et. al., 2000). When a stirrer is introduced into this mixture, the formation of aphrons is
676 achieved which leads to the separation of whey proteins by the process of flotation.
677 Amiri and Valsarai (2004) reported that aphron flotation may be successful in the
678 separation of whey protein as opposed to conventional flotation (due to the submicron size of
679 whey proteins). An anionic surfactant, sodium lauryl sulphate (MW = 283.38) was used for
680 aphron generation and two different liquid wastes- acidic caseinate waste (includes whey
681 protein without fat) and Gouda-cheese processing whey were used in the investigation. The
682 effect of disc position (at constant RPM) on protein separation was extensively studied and it
683 was reported that the best separation efficiency occurred when the solid metal disc was about
684 30% lower than the depth of the solution. Each experimental run was performed at a pH of
685 6.3 and samples were collected after 10 minutes for analysis of foam and whey protein
686 content. The reported experiments also confirmed that a gradual addition of surfactant leads
687 to a higher separation efficiency as compared to a method where all the surfactant has been
688 added at once. The increased efficiency was attributed to the existence of surfactant around
689 the spinning disc throughout the operation due to the gradual addition. The presence of
690 surfactant resulted in decreased surface tension and higher mass transfer rates into the
691 solution. The CGA bubble also became more stable due to the formation of smaller bubbles.
692 4.5 Molecular imprinting
693 Molecular imprinting technology is described as a method of making a molecular lock
694 to match a molecular key (Chen et al. 2016). It is a technique for making molecularly
695 imprinted polymers with tailor-made building sites corresponding to the template molecules
696 in shape, size and functional groups. A typical molecular imprinting synthesis protocol
697 contains a template, a functional monomer (forms pre-polymerisation complex with the
698 template by providing functional groups), a cross-linker (fixes functional monomers around
699 template molecules forming a rigid polymer even after the removal of the template), a
700 polymerisation initiator and a solvent (porogen).
701 A study by Soleimani et. al. (2012) described the synthesis of molecularly imprinted
702 polymers (MIPs) using bovine albumin as a template, 2-VP (2-vinylpyridine) as a functional
703 monomer, EGDMA (Ethylene glycol dimethacrylate) as a cross-linker, AIBN
704 (Azobisisobutyronitrile) as an initiator by radical polymerisation and methyl alcohol as the
705 porogen. It was reported that the maximum adsorption of bovine albumin by the fabricated
706 MIPs was 24 mg/g and in actual experiments for real samples, a recovery of around 80% was
707 achieved.
708 The method of artificial recognition by the use of molecularly imprinted polymers can be
709 used to selectively capture whey proteins from whey. It is important to understand that the
710 efficiency of this process for the purpose of whey protein separation has not been widely
711 reported. The economic implications of this technique are also not very clear and further
712 research into this method is definitely required.
713
714 5. DRYING OF WHEY PROTEINS
715 Once the whey proteins have been isolated using the separation processes (mostly
716 ultrafiltration), they need to be dried to get rid of the moisture content and yield a
717 commercially required powder form. Conversion into powder form also increases the
718 physical and microbiological stability of whey proteins and allows for a reduction in transport
719 and storage costs. Generally speaking, drying is achieved using two major approaches of
720 spray drying and freeze drying.

721 5.1 Spray drying
722 Spray-drying is an efficient process of removing water especially applied for the
723 various dairy products like whey proteins and milk (to form milk powder). The important
724 advantage offered by spray drying is that water can be removed from the concentrated whey
725 protein at the lowest temperature and in the shortest time so as to minimize heat damage and
726 heating costs. Spray drying is typically based on generation of very fine droplets using a
727 nozzle or a rotary atomizer into a hot dry air stream typically at 180-220 °C (Gharsallaoui et
728 al., 2007).
729 Typically in the spray drying operation, the concentrated whey protein enters the spray-
730 dryer chamber and exits at the bottom as a powder. A proportion of this powder is carried
731 away by the outlet warm air due to its lightness. Several cyclones are placed outside the dryer
732 in order to remove dry products from humid air through vortex separation which reduces the
733 loss of dry powder. Finally, drying and cooling occurs in a fluidised bed where the powder
734 from the end of the chamber and the cyclones mix together. The method has several
735 advantages including rapid drying, large throughput and continuous operation but has a few
736 disadvantages as well including denaturation of proteins due to the high temperatures and
737 higher costs of operation.
738 A recent modification in the spray drying is based on replacing the conventional
739 nozzle by the ultrasonic nozzles for atomisation. Ultrasonic nozzles are more precise, reliable
740 and give controlled distributions as compared to other nozzles. They also have the ability to
741 handle low flow rates, do not clog easily and give optimized efficiency by avoiding overspray
742 and disposal resulting in the lower feed material consumption. The basic principle behind the
743 working of these nozzles is based on the passage of ultrasound in the form of alternate crests
744 and troughs into a liquid which is subsequently set into vibratory motion on a smooth surface
745 (Gajendragadkar and Gogate, 2016). An increase in the vibration amplitude causes an
746 increase in the amplitude of the crests and troughs, ultimately leading to the collapse of the
747 waves (at critical amplitude) and the atomization of the liquid resulting in the formation of
748 tiny droplets. A major advantage of using ultrasonic spray drying is the formation of low
749 velocity flows from the nozzles allowing the size of the chamber to be reduced significantly
750 also leading to an overall favourable economics of drying (Ashokkumar et al., 2009).
751 5.2 Freeze drying
752 Freeze drying is generally used to produce high-quality dried products which are very
753 sensitive to heat (Sabarez, 2016) based on the utilization of temperatures below the freezing
754 point of the liquid. Freeze drying is also known as lyophilisation and is operated under
755 vacuum. In this technique, the component to be dried is frozen first and then exposed to heat
756 under vacuum which causes sublimation of the frozen liquid. The major approaches for
757 heating are conduction by bringing the frozen materials in contact with the heated shelf and
758 also exposed to radiation from the upper and lower shelves. Convection is largely absent in
759 the case of freeze drying operated under vacuum. As freeze drying is performed at low
760 temperatures and under vacuum (absence of oxygen), most deterioration reactions are
761 retarded to a great extent. The approach also leads to great retention of properties in the
762 components to be dried. Hence, freeze drying is considered to be the best method for the
763 production of high quality dried products. However, the need to freeze the components to be
764 dried and maintain vacuum make this process expensive and also lead to higher production
765 costs. The throughputs of such dryers are also limited and hence the applicability for recovery
766 of whey proteins really needs to be looked at.
767 Spray-freeze-drying (SFD) involves the spraying of a solution into a cold medium, and
768 freeze-drying the resultant frozen particles by contacting them with a cold, dry gas stream in
769 a fluidized bed (usually at atmospheric pressure). The process ensures much faster drying
770 than usually possible by conventional freeze-drying (Anandharamakrishnan et. al., 2010). A
771 study by Anandharamakrishnan et al. (2010 investigated spray-freeze-drying at sub-
772 atmospheric pressures (0.1 bar), which can allow high drying rates but using much less cold
773 gas. The study demonstrated the effectiveness of the SFD technique at sub-atmospheric
774 pressures for a whey protein isolate solution containing 20% w/w solids at gas inlet drying
775 temperatures ranging from -10 ˚C to -30 ˚C. The results of this study yielded a highly porous
776 powder with very little loss of solubility for β-Lg and α-La. The moisture content was
777 reduced to 8.1 % (wet basis) after freeze-drying at -10 ˚C for only 1 hour while drying for
778 100 minutes at 30 ˚C could only produce a wetter product (14% wet basis), which clearly
779 demonstrated efficacy of the SFD process at sub-atmospheric pressures. However, scale-up
780 of this process poses a challenge due to the requirement of a shallow fluidized bed meaning
781 that for commercial quantities a very large surface area will be required.
782
783 6. CONCLUSIONS
784 Whey is a major by-product of the dairy industry which has severe biological and
785 environmental implications. Whey also contains a significant nutritional value which can be
786 harnessed in positive manner. The discussions presented in this work addressed the need and
787 merits of processing whey. Guidelines on various methods of pre-treatments which can be
788 applied to increase the shelf life of whey and also help in mitigation of fouling in the
789 membrane separations have been also presented followed by the discussion on the recent
790 trends in the separation of whey proteins. It has been established based on the overview
791 presented that the most economic and feasible method for the separation of whey proteins is
792 ultrafiltration. Some of the recent modifications in terms of the use of cascades and bio-
793 activated membranes have further enhanced the separation efficiencies. Use of combined heat
794 and ultrasonic pretreatment can significantly enhance the membrane separation efficacy both
795 in terms of enhanced flux and also allowing reuse of the cleaned membranes. The current
796 review also allowed to provide a brief insight into recently reported but not so pervasive
797 methods of whey protein concentration and also separation. It is imperative that these novel
798 methods such as molecular imprinting are tested adequately to assess their technical as well
799 as economic viability if they are to be considered as an alternative to large scale whey protein
800 manufacture. Analysis of methods for post-processing of concentrated whey proteins which
801 include techniques such as spray and freeze drying has also been finally presented. Keeping
802 in mind the current trends and recent advances in the field of whey protein recovery, it would
803 not be wrong to say that the future of whey protein separation using membrane-aided
804 methods will greatly depend on the discovery of newer methods to combat membrane
805 fouling. Additionally, the advent of newer materials (with high affinity for whey proteins) for
806 the construction of membranes would also play an important role in the development of the
807 whey processing industry.
808
809 Acknowledgement:
810 Authors would like to acknowledge the funding of Department of Science and Technology,
811 Government of India under the MOFPI scheme for the research work in the area of
812 intensification of recovery of valuable products from whey using ultrasound.
813
814 7. REFERENCES
815 Al-Akoum, O., Ding, L.H., Jaffrin, M.Y., (2005). Concentration of total milk proteins by
816 high shear ultrafiltration in a vibrating membrane module, Journal of Membrane Science,
817 247, 211-220
818 Al-Akoum, O., Ding, L.H., Jaffrin, M.Y., (2002). Microfiltration and ultrafiltration of UHT
819 skim milk with a vibrating membrane module, Separation and Purification Technology, 28,
820 219-234
821 Almecija, M.C., Guadix, A., Martinez-Ferez, A., Gonzalez-Tello, P., Guadix, E. M., (2009a).
822 A flux enhancing pretreatment for the ultrafiltration of acid whey, Desalination, 245, 737-
823 742
824 Almecija, M.C., Martinez-Ferez, A., Guadix, A., Paez, M.P., Guadix, E.M., (2009b).
825 Influence of the cleaning temperature on the permeability of ceramic membranes,
826 Desalination, 245, 708-713
827 Amiri, M. C., Valsarai, K. T., (2004). Effect of gas transfer on separation on whey protein
828 with aphron flotation, Separation and Purification Technology, 35, 161-167
829 Anandharamakrishnan, C., Rielly, C. D., Stapley, A. G. F., (2010). Spray-freeze-drying of

830 whey proteins at sub-atmospheric pressures, Dairy Science and Technology, 90, 321-334
831 Arunkumar, A., Etzel, Mark R., (2013). Fractionation of α-lactalbumin from β-lactoglobulin
832 using positively charged tangential flow ultrafiltration membranes, Separation and
833 Purification Technology, 105, 121-128
834 Ashokkumar, M., Bhaskaracharya, R., Kentish, S., Lee, J., Palmer, M., Zisu, B., (2009). The
835 ultrasonic processing of dairy products- an overview, Dairy Science and Technology, 90,
836 147-168
837 Ashokkumar, M., Lee, J., Zisu, B., Bhaskarcharya, R., Palmer, M., Kentish, S., (2009).
838 Sonication increases the heat stability of whey proteins, Journal of Dairy Science, 92, 5353-
839 5356
840 Baier, S.K., McClements, D.J., (2001). Impact of preferential interactions on thermal stability
841 and gelation of bovine serum albumin in aqueous sucrose solutions, Journal of Agricultural
842 Food Chemistry, 49, 2600-2608
843 Barba, D., Beolchini, F., Cifoni, D., Vegilo, F., (2001). Whey protein concentrate production
844 in a pilot scale two-stage diafiltration process, Separation Science and Technology, 36(4),
845 587-603
846 Blanpain-Avet, P., Migdal, J.F., Benezech, T., (2009). Chemical cleansing of a tubular
847 ceramic microfiltration membrane fouled with a whey protein concentrate suspension-
848 characterisation of hydraulic and chemical cleanliness, Journal of Membrane Science, 337,
849 153-157
850 Bozanic, R., Barukcic, I., Lisak, K., Jakopovic, Tratnik, L., (2014). Possibilities of whey
851 utilisation, Austin Journal of Nutrition and Food Sciences, 2, 1036-1042
852 Cai, M., Zhao, S., Liang H., (2010). Mechanisms for enhancement of ultrafiltration and
853 membrane cleaning by different ultrasonic frequencies, Desalination, 263, 133-138
854 Chai, X., Kobayashi, T., Fujii, N., (1998). Ultrasound effect on cross-flow filtration of
855 polyacrylonitrile ultrafiltration membranes, Journal of Membrane Science, 148, 129-135
856 Chandrapala, J., Zisu, B., Palmer, M., Kentish, S., Ashokkumar, M., (2011). Effects of
857 ultrasound on the thermal and structural characteristics of proteins in reconstituted whey
858 protein concentrate, Ultrasonics Sonochemistry, 18, 951-957
859 Cheang, B., Zydney, A. L., (2003). Separation of α-lactalbumin and β-lactoglobulin using
860 membrane ultrafiltration, Biotechnology and Bioengineering, 83, 201-209
861 Chen, L., Wang, X., Lu, W., Wu, X., Li, J., (2016). Molecular imprinting: perspectives and
862 applications, Chemical Society Reviews, 45, 2137-2211
863 Corbaton-Baguena, M. J., Alvarez-Blanco, S., Vincent-Vela, M.-C., (2014). Salt cleaning of
864 ultrafiltration membranes fouled by whey model solutions, Separation and Purification
865 Technology, 132, 226-233
866 Corbaton-Baguena, M. J., Alvarez-Blanco, S., Vincent-Vela, M.-C., Lora-Garcia, J., (2015).
867 Utilisation of NaCl solutions to clean ultrafiltration membranes fouled by whey protein
868 concentrates, Separation and Purification Technology, 150, 95-101
869 Corbaton-Baguena, M. J., Alvarez-Blanco, S., Vincent-Vela, M.-C., Ortega-Navarro, E.,

870 Perez-Herranz, V., (2016). Application of electric fields to clean ultrafiltration membranes
871 fouled with whey model solutions, Separation and Purification Technology, 159, 92-99
872 Cowan, S., Ritchie, S., (2007). Modified Polyethersulfone (PES) Ultrafiltration Membranes
873 for Enhanced Filtration of Whey Proteins, Separation Science and Technology, 42(11), 2405-
874 2418
875 Elofsson, E. M., Dejmek, P., Paulsson, M. A., (1996). Heat-induced aggregation of β-
876 lactoglobulin studied by dynamic light scattering, International Dairy Journal, 6, 343-357
877 Gajendragadkar, C. N., Gogate, P. R., (2016). Intensified recovery of valuable products from
878 whey by use of ultrasound in processing steps- A review, Ultrasonics Sonochemistry, 32,
879 102-118
880 Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R., (2007). Applications of
881 spray-drying in microencapsulation of food ingredients: an overview, Food Research
882 International, 40(9), 1107-1121
883 Hausmann, A., Sanciolo, P., Vasiljevic, T., Weeks, M., Duke, M., (2012). Integration of
884 membrane distillation into heat paths of industrial processes, Chemical Engineering Journal,
885 211-212, 378-387
886 Hausmann, A., Sanciolo, P., Vasiljevic, T., Kulozik, U., Duke, M., (2013). Performance
887 assessment of membrane distillation for skim milk and whey processing, Journal of Dairy
888 Science, 97, 56-71
889 Hickey, M. W., Hill, R. D., Smith, B. R., (1980). Investigations into the ultrafiltration and
890 reverse osmosis of wheys 1. The effects of certain pretreatments, New Zealand Journal of
891 Dairy Science and Technology, 15, 109-121
892 Ho, W. S., Sirkar, K. K., Membrane Handbook, Springer US (1992)
893 Iametti, S., Cairoli, S., Gregori, B. De, Bonomi, F., (1995). Modifications of high-order
894 structures upon heating of beta-lactoglobulin: dependence on the protein concentration,
895 Journal of Agricultural Food and Chemistry, 43, 53-53
896 Jauregi, P., Mitchell, G. R., Varley, J., (2000). Colloidal Gas Aphrons (CGA): Dispersion and
897 Structural Features, AIChE Journal, 46(1), 24-36
898 Kazemimoghadam, M., Mohammadi, T., (2007). Chemical cleaning of ultrafiltration

899 membranes in the milk industry, Desalination, 204, 213-218
900 Kim, J. I., Choi, D. Y., Row, K. H., (2003). Separation of Whey Proteins by Anion-Exchange
901 Membranes, Korean Journal of Chemical Engineering, 20(3), 538-541
902 Kim, S. H., Morr, C. V., Seo, A., Surak, J. G., (1989). Effect of Whey Pretreatment on
903 Composition and Functional Properties of Whey Protein Concentrate, Journal of Food
904 Science, 54, 25-29
905 Koh, Li Ling A., Nguyen, Hanh Thi Hong, Chandrapala, J., Zisu, B., Ashokkumar, M.,
906 Kentish, S. E., (2014). The use of ultrasonic feed pre-treatment to reduce membrane fouling
907 in whey ultrafiltration, Journal of Membrane Science, 453, 230-239
908 Kumar, P., Sharma, N., Ranjan, R., Kumar, S., Bhat, Z. F., Jeong, D. K., (2013). Perspective
909 of Membrane Technology in Dairy Industry: A Review, Asian-Australian Journal of Animal
910 Science, 26, 1347-1358
911 Lamminen, M. O., Walker, H. W., Weavers, L. K., (2006). Effect of fouling conditions and
912 cake layer structure on the ultrasonic cleaning of ceramic membranes, Separation Science
913 and Technology, 41, 3569-3584
914 Lee, S., Elimelech, M., (2007). Salt cleaning of organic-fouled reverse osmosis membranes,
915 Water Research, 41, 1134-1142
916 Levin, M. A., Burrington, K. J., Hartel, R. W., (2016). Composition and functionality of
917 whey protein phospholipid concentrate and delactosed permeate, Journal of Dairy Science,
918 99, 1-11
919 Li, X., Yu, J., Nnanna, A. G. A., (2011). Fouling mitigation for hollow-fiber UF membrane
920 by sonication, Desalination, 281, 23-29
921 Lonnerdal, B., (2003). Nutritonal and physiologic significance of human milk proteins, The
922 American Journal of Clinical Nutrition, 77, 1537S–1543S
923 Lucy, C. A., MacDonald, A. M., Gulcev, M. D., (2008). Non-covalent capillary coatings for
924 protein separations in capillary electrophoresis, Journal of Chromatography A, 1184, 81–105
925 Metzger, L. E., Marella, C., Muthukumarappan, K., (2011). Evaluation of commercially
926 available, wide-pore ultrafiltration membranes for production of α-lactalbumin-enriched
927 whey protein concentrate, Journal of Dairy Science, 94, 1164-1175
928 Muthukumaran, S., Kentish, S. E., Stevens, G.W., Ashokkumar, M, Mawson, R., (2007). The
929 application of ultrasound to dairy ultrafiltration: The influence of operating conditions,
930 Journal of Food Engineering, 81, 364-373
931 Muthukumaran, S., Kentish, S. E., Stevens, G.W., Ashokkumar, M., (2005). Mechanisms for
932 the ultrasonic enhancement of dairy whey ultrafiltration, Journal of Membrane Science, 258,
933 106-114
934 Norazman, N., Wu, W., Li, H., Wasinger, V., Zhang, H., Chen, V., (2013). Evaluation of
935 chemical cleaning of UF membranes fouled with whey protein isolates via analysis of
936 residual protein components on membranes surfaces, Separation and Purification
937 Technology, 103, 241-250
938 Ogunbiyi, O.O., Miles, N.J., Hilal, N., (2008). The effects of performance and cleaning
939 cycles of new tubular ceramic microfiltration membrane fouled with a model yeast
940 suspension, Desalination, 220, 273-289
941 Patil, N. V., Janssen, Anja E. M., Boom, Remko M., (2014). Separation of Whey Proteins
942 using Cascaded Ultrafiltration, Separation Science and Technology, 49, 2280-2288
943 Pena-Ramos, E. A., Xiong, Y. L., (2001). Antioxidative activity of whey protein hydrolysates
944 in a liposomal system, Journal of Dairy Science, 84, 2577-2583
945 Popovic, S., Djuric, M., Milanovic, S., Tekic, M. N., Lukic, N., (2010). Application of an
946 ultrasound field in chemical cleaning of ceramic tubular membrane fouled with whey
947 proteins, Journal of Food Engineering, 101, 296-302
948 Qi, X. L., Brwonlow, S., Holt, C., Sellers, P., (1995). Thermal denaturation of β-
949 lactoglobulin: effect of protein concentration at pH 6.75 and 8.05, Biochimica et Biophysica
950 Acta, Protein Structure and Molecular Enzymology, 1248, 43-49
951 Ramirez, C.A., M. Patel, K. Blok, (2006). From fluid milk to milk powder: Energy use and
952 energy efficiency in the European dairy industry, Energy, 31, 1984-2004
953 Sabarez, H., Drying of Food Materials, CSIRO Food and Nutrition, Elsevier, Werribee
954 Australia (2016)
955 Saxena, A., Tripathi, B.P., Kumar, M., Shahi, V.K., (2009). Membrane-based techniques for
956 the separation and purification of proteins: An overview, Advances in Colloid and Interface
957 Science, 145, 1-22
958 Simon, A., Gondrexon, N., Taha, S., Cabon, J., Dorange, G., (2000). Low-frequency
959 ultrasound to improve dead-end ultrafiltration performance, Separation Science and
960 Technology, 35, 2619-2637
961 Sinha, R., Radha, C., Prakash, J., Kaul, P., (2007). Whey protein hydrolysate: Functional
962 properties, nutritional quality and utilization in beverage formulation, Food Chemistry, 101,
963 1484-1491
964 Soleimani, M., Ghaderi, S., Ghahraman, A., Soleimani, S., (2010). Synthesis of molecularly
965 imprinted polymer as a sorbent for solid phase extraction of bovine albumin from whey, milk,
966 urine and serum, Microchemical Journal, 100, 1-7
967 Steinhauer, T., Marx, M., Bogendorfer, K., Kulozik, U., (2015). Membrane fouling during
968 ultra- and microfiltration of whey and whey proteins at different environmental conditions:
969 The role of aggregated whey proteins as fouling initiators, Journal of Membrane Science,
970 489, 20-27
971 Tarazaga, C. C., Campderros, M. E., Padilla, A. P., (2006). Physical cleaning by means of
972 electric field in the ultrafiltration of a biological solution, Journal of Membrane Science, 278,
973 219-224
974 Tolkach, A., Kulozik, U., (2005). Fractionation of whey proteins and caseinomacropeptide by
975 means of enzymatic crosslinking and membrane separation techniques, Journal of Food
976 Engineering, 67(1-2), 13-20
977 Tolkach, A., Kulozik, U., (2006). Transport of whey proteins through 0.1 mm ceramic
978 membrane: Phenomena, modelling and consequences for concentration or diafiltration
979 processes, Desalination, 199(1-3), 340-341
980 Tsakali, E., Pretrotos, K., Allessandro, A. D’, Goulas, P., (2010). A review on whey
981 composition and the methods used for its utilisation for food and pharmaceutical products,
982 FOODSIM 2010, CIMO Research Centre, Braganca, Portugal
983 Wen-Qiong, W., Len-Wei, Z., Xue, H., Yi, Y., (2017). Cheese whey protein recovery by
984 ultrafiltration through transglutaminase (TG) catalysis whey protein cross-linking, Food
985 Chemistry, 215, 31-40
986 Yadav, J. S. S., Yan, S., More, T. T., Tyagi, R. D., Surampalli, R. Y., (2014). Recovery of
987 residual soluble protein by two-step precipitation process with concomitant COD reduction
988 from the yeast-cultivated sheese whey, Bioprocess and Biosystems Engineering, 37(9), 1825-
989 1837
990 Yorgun, M. S., Balcioglu, I. Akmehmet, Saygin, O., (2008). Performance comparison of
991 ultrafiltration, nanofiltration and reverse osmosis on whey treatment, Desalination, 229, 204-
992 216
993 Zapata, J.E., Almecija, M.C., Martinez-Ferez, A., Guadix, A., Hernandez, A., Calvo, J.I.,
994 Guadix, E.M., (2009). Analysis of cleaning protocols in ceramic membranes by liquid-liquid
995 displacement porosimetry, Desalination, 245, 541-545
996 Zhong, Q., Wang, W., Hu, Z., Ikeda, S., (2013). Sequential preheating and transglutaminase
997 pretreatments improve stability of whey protein isolate at pH 7.0 during thermal sterilisation,
998 Food Hydrocolloids, 31, 306-316
999
1000 Table 1: Whey composition (Tsakali et. al., 2010)
Whey Type
S. No. Constituent Unit
Sweet Whey Acid Whey
1 Water % 93-94 94-95
2 Dry Matter % 5-6 5-6
3 Lactose % 4.5-5 3.8-4.3
4 Lactic Acid % Traces Up to 0.8
5 Total Protein % 0.8-1.0 0.8-1.0
6 Whey Protein % 0.6-0.65 0.6-0.65
7 Citric Acid % 0.1 0.1
8 Minerals % 0.5-0.7 0.5-0.7
9 pH - 6.2-6.4 4.6-5.0
10 SH Value - About 4 20-25
1001
1002 Table 2: Comparison of chemically pre-treated and unpretreated whey (Almecija et al.,
1003 2009a)
Protein Pre-treated Whey Unpretreated whey

α-Lactalbumin (g/L) 1.03 0.99
β-Lactoglobulin (g/L) 3.09 3.07
Bovine Serum Albumin, BSA (g/L) 0.10 0.12
Lactoferrin (g/L) 0.41 0.39
Ig-G (g/L) 0.04 0.04
1004
1005 Table 3: Time required for membrane cleaning under different applied voltages
1006 (Tarazaga et. al., 2006)
Time required for cleaning (min) Applied Voltage (V)

2 45
4 30
8 22
12 18
15 15
18 13
1007
1008 Table 4: Membrane areas under operation (Saxena et. al., 2009)
S. No. Membrane Type Membrane area under

operation (m2)
1 Ultrafiltration 400000
2 Nanofiltration 300000
3 Reverse Osmosis 100000
4 Microfiltration 50000
1009
1010 Table 5: Specifications of membrane modules used in terms of material type and
1011 module dimensions for evaluation of membrane efficiency (Yorgun et al., 2008)
Module Module Cartridge

Module/
Supplier Material/ Pmax (bar)/ Tmax (ᵒC) area length diameter
Type
(m2) (mm) (mm)
A/NF Nadir GmbH Polyethersulfone/40/55 7.5 838 108.5
B/NF Nadir GmbH Polyethersulfone/40/55 7.5 1016 101.5
C/NF Trisep Corp. Polyamide-urea/41/45 7.5 1016 102
D/NF Filmtec Polysulfone/54.8/50 5.5 984.3 96
E/UF Nadir GmbH Polyethersulfone/40/55 14.4 972 200
F/RO Trisep Corp. Polyamide-urea/41/45 5.5 1016 102
G/RO Trisep Corp. Polyamide-urea/41/45 5.5 1016 102
1012
1013
1014 Table 6: Operational parameters (transmembrane pressure and pure water flux) of
1015 membrane modules used to determine membrane efficiency (Yorgun et al., 2008)
Type of Pure water flux

Name TMP (bar)
membrane (L/m2h)
A 5 430
B 8 250
Nanofiltration
C 8 220
D 8 210
Ultrafiltration E 3 510
Reverse Osmosis F, G 12 180-200
1016
1017 Table 7: Composition of curd cheese whey and white cheese whey (Yorgun et al., 2008)
Components Curd cheese whey White cheese whey
Fat content, % 0 0.2
COD, g/L Approx. 100 Approx. 110
Protein, g/L 1.42 2.30
Lactose, g/L 43.92 36.52
Minerals, g/L 6.1 6.5
1018
1019 Figure Captions

1020 Figure 1: Distribution of global whey production and consumption (Tsakali et. al., 2010)
1021 Figure 2: Possible uses of whey (Bozanic et. al., 2014)
1022 Figure 3: Cascade configurations for ultrafiltration (Patil et. al., 2014)
1023
Disposed as Feed, Fertilizer Whey Powder and Lactose

or Waste (60 Million tons) (49 million tons)
Global Whey
Production
(145 million tons)
Industrially utilised WPC and WPI
(85 million tons) (30 million tons)
Other Industrial Products

(6 million tons)
1024
1025 Figure 1: Distribution of global whey production and consumption (Tsakali et. al., 2010)
1026
1027
1028 Figure 2: Possible uses of whey (Bozanic et. al., 2014)
1029
1030
1031 Figure 3: Cascade configurations for ultrafiltration (Patil et. al., 2014)
ACCEPTED MANUSCRIPT
Research highlights
 Critical analysis of intensification approaches for recovery of proteins from whey
 Discussion on operating strategies and improved designs for better efficacy
 Combined pretreatment schemes based on heat and ultrasound can be very effective
 Considerable promise established for recovery of proteins leading to environmental benefits
 Post processing of recovered proteins can give value addition

Ganju 2017 Métodos

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Ganju 2017 Métodos

Uploaded by

Copyright:

Available Formats

Accepted Manuscript

Sparsh Ganju, Parag R. Gogate

Reference: JFOE 8963

To appear in: Journal of Food Engineering

Received Date: 01 April 2017

Revised Date: 18 July 2017

Accepted Date: 19 July 2017

1 A review on approaches for efficient recovery of whey proteins

8 Sparsh Ganju, Parag R. Gogate*

14 Chemical Engineering Department,

15 Institute of Chemical Technology,

27 A significant proportion of waste generated by the dairy industry is in the form of

41 optimization of operating parameters so as to maximize the recovery efficacy and on the

44 proteins conforming to the principle of wealth from waste.

47 Whey proteins; Membrane separations; Pre-treatment; Operating parameters; Spray

48 drying; Value added products

87 1.2 Whey proteins

103 50/50 in late lactation (Lonnerdal, 2003).

104 The various forms of commercially available whey proteins are:

109 mild to slightly milky taste.

118 concentrate or isolate.

119 1.3 Whey protein separation

120 Whey protein separation essentially consists of three stages:

122 separation and drying stages as discussed in the following sections.

136 effects of membrane fouling.

145 newer but expensive method of drying- freeze drying.

153 2. MEMBRANE SEPARATION METHODS

167 ultrafiltration and microfiltration.

168 2.1 Ultrafiltration

175 process is 10 kDa. Whey protein separation by ultrafiltration is normally performed at

180 The subsequent sections provide a brief discussion on these modifications.

181 2.1.1 Cascaded ultrafiltration

201 significantly enhanced as compared to single stage operation.

202 2.1.2 Bio-catalysis

215 formation of a high molecular polymer.

224 catalysis. These results were obtained at a pH of 5, enzyme concentration of 40 U/g,

225 temperature of 40 C and a retention time of 60 minutes. A decrease in membrane resistance

228 2.1.3 High Performance Tangential Flow Ultrafiltration (HPTFF)

236 industrial infrastructure, though not in the dairy industry applications.

257 there is indeed a great promise.

258 2.2 Advantages of membrane separation techniques

261 i) Membrane technology is a novel non-thermal and environmentally friendly

273 easily by adding membrane modules in parallel to the existing ones.

275 modules making the actual use very simple.

276 2.3 Comparison of various membrane separation techniques

277 2.3.1 Performance comparison of ultrafiltration, nanofiltration and reverse osmosis

303 to recover lactose leading to a clean effluent.

305 An evaluation of commercially available, wide-pore ultrafiltration membranes was performed

322 commercial viability of α-La enriched WPC production.

342 enhancement due to electrostatic repulsion.

344 3. FOULING AND PRE-TREATMENT

345 3.1 Fouling

367 based separation process, pre-treatment of whey needs to be performed so as to induce

369 3.2 Pre-treatment

383 i) Chemical pre-treatment

384 ii) Heat (Thermal) pre-treatment

385 iii) Ultrasound pre-treatment

386 iv) Use of turbulence promoters

389 these pre-treatments has now been provided.

390 3.2.1 Chemical pre-treatment

399 precipitation methods or processes.

417 dropped to 7 and 19 L/(m2h) respectively after 4 hours of operation.

418 3.2.2 Heat (Thermal) pre-treatment