Ultrasound assisted intensified recovery of lactose from whey based on antisolvent crystallization

Accepted Manuscript Ultrasound Assisted Intensified Recovery of Lactose from Whey Based on Antisolvent Crystallization Chinmay N. Gajendragadkar, Parag R. Gogate PII: DOI: Reference:

S1350-4177(16)30281-4 http://dx.doi.org/10.1016/j.ultsonch.2016.08.011 ULTSON 3338

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

16 June 2016 28 July 2016 7 August 2016

Please cite this article as: C.N. Gajendragadkar, P.R. Gogate, Ultrasound Assisted Intensified Recovery of Lactose from Whey Based on Antisolvent Crystallization, Ultrasonics Sonochemistry (2016), doi: http://dx.doi.org/10.1016/ j.ultsonch.2016.08.011

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Ultrasound Assisted Intensified Recovery of Lactose from Whey Based on Antisolvent Crystallization

Chinmay N. Gajendragadkar, Parag R. Gogate*

Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai – 400 019, India.

*Corresponding author Tel.: +91 22 33612024, Fax: +91 22 33611020; E-mail address: [email protected] 1

ABSTRACT The current work deals with understanding the fundamental aspects for intensified recovery of lactose from paneer (cottage cheese) whey using the anti-solvent assisted sonocrystallization. Ultrasonic horn (22 kHz) with varying power levels over the range if 40 W to 120 W has been used for initial experiments at 100 % duty cycle and two different levels of ultrasonic exposure for 10 min and 20 min. Similar experiments were also performed using ultrasonic bath for the same time but with different ultrasonic frequencies (22 kHz and 33 kHz). It was observed that the lactose recovery as well as purity increased with an increase in ultrasonic power for 100% duty cycle for the case of treatment time as 10 min whereas the lactose recovery and purity increased only till an optimum for the 20 min treatment. In the case of ultrasonic bath, lactose purity increased with an increase in the ultrasonic frequency from 22 kHz to 33 kHz though the lactose recovery marginally decreased. Overall, it was observed that the maximum lactose recovery was ~98% obtained using ultrasonic horn while the maximum lactose purity was ~97%. It was also observed that maximum lactose recovery was ~94% obtained using ultrasonic bath while the maximum lactose purity was ~92%. The work has enabled to understand the optimized application of ultrasound so as to maximize both the lactose yield and purity from whey.

Keywords- Lactose; Sonocrystallization; Anti-solvent; Scale-up; Ultrasonic horn; Ultrasonic bath.

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1. INTRODUCTION Whey is the liquid or the permeate formed as a waste product during the manufacture of cheese, paneer (resemling to cottage cheese or farmer’s cheese), chhena or chhana (unripened curd cheese or moist form of paneer obtained from buffalo milk) or other coagulated dairy products [1]. Whey is obtained during the curdling process of manufacturing of cheese on addition of edible acids like citric acid (in the form of lemon juice), acetic acid (in the form of vinegar), lactic acids (from starter bacteria like Lactococcus, Lactobacillus, Leuconostoc etc.) or proteolytic enzymes such as rennet [2]. Typically production of 1 kg of cheese is associated with generation of 9 L of liquid whey which is a substantial amount and constitutes to environmental concerns [3- 6]. The disposal of whey in the water bodies like rivers, lakes etc. or open fields leads to serious risk to the environment as well as constitutes to the loss of vital nutrients like proteins, peptides, vitamins, lipids and minerals present in the whey. The degradation of whey by bacteria causes oxygen depletion in the water and soil since whey has a very high biological oxygen demand (BOD) of 30-50 g/l and chemical oxygen demand (COD) of 60-80 g/l which is mainly due to the presence of lactose. Hence, it is very much necessary to extract or recover both proteins and lactose which can be beneficially used in food or nutrition and pharmaceutical formulations and also help to reduce the environmental impact [2, 5-7].

Lactose recovery is generally based on the use of an

anti-solvent to induce crystallization of lactose from whey. Whey also contains minerals such as calcium, magnesium, phosphorous, potassium, chloride, sodium in the range of 0.1 g/l to 1.5 g/l and other trace elements like zinc, iron, iodine, copper in the range of 0.2-1.5 mg/ml which are the same minerals and trace elements present in the milk itself. Thus, there is a high possibility of these minerals and inorganic salts also to be present in the recovered lactose obtained from the anti-solvent sonocrystallization study, driving the possible utility of the recovered lactose as supplements. 3

The process of obtaining a solid in its pure and crystalline form from the saturated solution is called as crystallization. The process generally involves three steps as supersaturation (by evaporation, cooling or addition of anti-solvent), nucleus formation followed by crystal growth (deposition of particles on nuclei) [8]. The process of crystallization can be intensified based on the use of ultrasound based on the physical effects of ultrasound induced cavitation. Indeed, the use of ultrasound in food and dairy processing industries is a new and upcoming approach with various applications [9]. Use of ultrasound can enhance the nucleation rate and the rate of crystal formation in the crystallization process. The shear forces and turbulence generated due to micro-streaming increases the heat and mass transfer rates uniformly and also prevents the agglomeration of particles. Ultrasound breaks the crystals as they are formed, resulting in additional nuclei formation and uniform small-sized crystal generation. Also the cavitation bubbles itself can act as nucleation sites sometimes avoiding the need of the seeding crystals. Generally speaking, frequency, intensity, power and pulsing are the important parameters that can be controlled to give desired intensification benefits and particle characteristics in the case of sonocrystallization [6, 10]. In the past, sonocrystallization of lactose has been investigated using anti-solvent as ‘ethanol’ with optimization of ultrasonic parameters to yield maximum yield and purity of lactose [11, 12]. Bund and Pandit [11, 12] investigated anti-solvent sonocrystallization using reconstituted lactose solutions (11.5% w/v to 17.5 % w/v) in ultrasonic bath with the volume being 10 ml and total sample volume approximately being 85-90 ml. Similarly, Patel and Murthy [13, 14] performed sonocrystallization studies on reconstituted lactose monohydrate solutions (12% w/v to 18% w/v) using anti-solvent ‘acetone’ and ‘n-propanol’ using ultrasonic bath with total sample volumes of 20 ml.

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Bund and Pandit [15] investigated the anti-solvent crystallization of lactose from the buffalo milk whey to study the effect of anti-solvent concentration, use of seeding and time of ultrasound application as well as understand the effect of initial pH and end pH on the quality of lactose. It was reported that with an increase in anti-solvent concentration from 65% v/v to 85% v/v, lactose recovery increased from 84.8% to 92.6%. In the case of the effect of pH, it was reported that with a decrease in initial pH of 4.1 to pH of 2.2, lactose recovery decreased slightly from 97.4% to 95.9%. The ash content also decreased from 2.62% w/w to 0.81% w/w with pH change from 4.1 to 2.2. A seeding concentration of 1% w/w to 3 % w/w in 1 hour of anti-solvent crystallization increased lactose recovery from 76% to 92.6%. It was also reported that around 80% of the lactose recovery was observed in the 1 st hour with subsequent increase to 90.9% and 95.9% after 3h and 5 h of crystallization time respectively. Lactose purities (97-98%) were reported to be the same at all crystallization time [15]. Bund and Pandit [11] reported that a lactose recovery of 91.5% was obtained in 5 min of sonication using an ultrasonic bath operating at a frequency of 22 kHz with 85% v/v antisolvent ‘ethanol’ concentration from a reconstituted lactose solution (17.5% w/v) at an ambient temperature of 30±2°C. It was reported that higher the initial lactose concentration, higher was the lactose recovery since greater rates of supersaturation are achieved in the concentrated solutions. It was also observed that protein acts as inhibitor to lactose crystallization and hence when the protein concentration (in the form of bovine serum albumin-BSA) increased, the rate of the lactose crystallization was reduced also giving lower recoveries. Further, the lactose recovery could either be increased by increasing sonication time or introducing a long standing time after a short exposure of ultrasound [11]. The effect of the process parameters such as temperature, initial pH, end pH, sonication time, stirring and seeding on the lactose recovery and purity were optimized using L12- orthogonal array method in the subsequent work of Bund and Pandit [12] and it was established that seeding, 5

initial and end pH were the most influencing process parameters in deciding the lactose recovery and purity. Patel and Murthy [13] reported maximum lactose recovery of 89.2% from 16% w/v reconstituted lactose solution in 4 min of sonication time with 85% v/v acetone as antisolvent carried out on ultrasonic bath of 20 kHz. Lactose recoveries reportedly increased from 45.3% for 70% v/v acetone concentration to 89.2% for the case of 85% v/v acetone concentration. The lactose recovery was also reported to increase from 81% to 85.3% with an increase in initial lactose concentration from 12% w/v to 18% w/v. Also lactose recovery decreased steadily from around 90% at pH 1 to around 80% at pH 4. In another study, sonocrystallization of lactose was performed using anti-solvent npropanol by Patel and Murthy [14]. It was reported that a maximum lactose recovery of 98.5 % from 16% w/v reconstituted lactose solutions was obtained using 95% v/v n-propanol concentration in 4 min of sonication based on use of ultrasonic bath of 20 kHz. It was also reported that at sonication time of 8 min, lactose recovery only marginally increased from 81% to 83.9% with a reduction in pH from 4.2 to 2.9 at 0.4 % w/v protein concentration. Similar to the observations of Bund and Pandit [11], it was reported that an increase in protein concentration resulted in reduced lactose recoveries. Lactose yields increased from 65.2% w/w to 98.5% w/w with an increase in n-propanol concentration from 80% v/v to 95%v/v. It was also reported that lactose recovery increased from approximately 65% to 85% with an increase in lactose concentration from 12% w/v to 18% w/v at pH 4.2 with 85% v/v n-propanol and 0.4 % w/v protein content as opposed to increase in lactose recovery from approximately 60% to 68% with an increase in lactose concentration from 12% w/v to 18% w/v at pH 2.9 with 85% v/v n-propanol and 0.4% w/v protein concentration [14].

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Recently, Zisu et al. [16] investigated the effect of ultrasound on the concentrated whey solutions containing 32±2% lactose using the non-contact approach at a frequency 20 kHz with varying flow rates from 4 L/min to 12 L/min and energy densities over the range of 3-16 J/ml at constant temperature of 30±1°C on a pilot scale. A non-contact sonication cell is an alternative to direct sonication where the chances of erosion of probe and contamination of the product are a problem and hence the approach presented by Zisu et al. [16] is very important considering the possible commercial applications in the food industry. The sonication cell design also allows modular implementation and in-line operation eliminating the need of sonotrodes due to multiple low power transducers being attached to the outer surface of the metallic cell. It was reported that the sonication of concentrated whey solutions increased the rate of crystallization due to rapid formation of lactose crystals in response to ultrasonic cavitation effects. It was also reported that the rate of sonocrystallization was greater than stirring for approximately 3 hours but slowed down between 2 hours and 3 hours from the start of the process as the metastable limit was achieved. To avoid the slowing of the process, a second ultrasonic treatment cycle was introduced at the metastable limit to stimulate further nuclei formation. It was also reported that not only the overall crystal size of the lactose crystals obtained was smaller as compared to lactose crystals obtained after stirring but also the crystal size distribution was also found to be narrower. In yet another recent study, a simultaneous crystallization of mixture of calcium phosphate salts, sodium chloride and sugars (predominantly lactose) from the concentrated saline dairy effluent (at solid loading of 22 wt% and 30 wt% and at temperatures between 10°C and 70°C) has been investigated by Kezia et al. [17]. It was reported that the crystallization induction time was shortened by the use of two short pulses of ultrasound with energy consumption of 3.7 J/kg and 16 J/kg spaced ten minutes apart. It was also reported that the pulsed approach had a major impact on the crystal size and induction time. Increasing 7

the acoustic power too had a large impact in decreasing the induction time. It was proposed that the first pulse of ultrasound generates new nuclei and partly enhances the mass transfer rates whereas the second pulse of ultrasound breaks the growing crystals and thus generates secondary nuclei. Overall it can said that, in the existing literature illustrations, the intensified recovery of lactose has been investigated concentrating on the effect of parameters like pH, antisolvent loading, seeding, temperature, initial lactose concentration, protein concentration and sonication time on the efficacy of anti-solvent sonocrystallization. In comparison to the pilot scale study on sonocrystallization reported by Zisu et al. [16], the current work involves the essential comparison of efficacies of both the contact approach by ultrasonic horn as well as non-contact approach by ultrasonic bath which has not been investigated in details to the best of our knowledge. Also the work focuses on the low concentrated whey actually obtained as a waste sample and investigates the effect of change in frequency, time and power dissipation levels. In the present work, ultrasound has been used for process intensification of antisolvent induced sonocrystallization over the entire treatment time to improve the recovery and purity of lactose from the actual sample of ‘paneer’ (cottage cheese) whey using ultrasonic horn as well as ultrasonic bath with an objective of identifying the effect of type of ultrasonic reactor. The scale of operation is also 5 times higher as compared to the earlier reported investigations with actual volume of the low concentrated whey solution (~20 % w/v) being 50 ml and total volume after addition of anti-solvent being approximately 350-355 ml. The effect of ultrasonic frequency has been also investigated for the first time in addition to understanding the effect of sonication time and power dissipation on the lactose recovery and purity. The parameters like initial lactose concentration (~ 20% w/v) and anti-solvent concentration (85% v/v) were selected on the basis of previous studies for performing antisolvent sonocrystallization study at a large scale of operation. Overall, the important 8

objectives of the present work include understanding the effect of ultrasonic parameters like power dissipation levels, frequency, reactor configuration and time of sonication on the lactose purity as well as extent of recovery from the actual whey solutions procured as dairy industry waste. The effect of ultrasound and the process parameters on the hydrodynamic particle size of lactose has also been established. The presented results would be very useful considering the possible benefits of reducing the biological content in the wastewater as well as yielding valuable products such as lactose which can either be used as supplements or used for further processing such as based on lactose hydrolysis. 2. MATERIALS AND METHODS 2.1 Materials Standard lactose monohydrate, standard glucose, standard galactose, standard lactulose, dinitrosalicylic acid (DNSA), concentrated sulphuric acid (98% pure, LR), glycine, sodium

hydroxide

pellets,

methylamine

hydrochloride,

copper

sulfate,

cysteine

hydrochloride, tryptophan, sodium potassium tartrate and sodium sulfite were obtained from Hi Media Chemicals. Fresh samples of paneer whey were obtained from a local dairy in Mumbai. China Grade Absolute Ethanol (99.9% pure) was used as an anti-solvent. 2.2 Equipments The different equipments used in the work were vertical ultrasonic horn operating at 20 kHz (calorimetric power dissipation of 8.56 W, rated power dissipation of 120 W and having single transducer with 1 cm tip diameter) and rectangular ultrasonic bath with variable frequency mode (dimensions as 22.5 cm × 15.4 cm ×14.7 cm, 4 transducers, calorimetric power dissipation of 7W for 22 kHz operation and 11.6 W for 33 kHz operation, rated power dissipation of 120 W) procured from M/s Dakshin, Mumbai). A glass reactor having capacity

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of 1 L equipped with open axial impeller and motor (in the case of ultrasonic bath the glass reactor was placed in center with overhead stirring by open axial impeller) has been used for the investigations. UV spectrophotometer (Shimadzu Analytical India Pvt. Ltd.), vacuum dryer (JISL, India), centrifuge (Eltek India Pvt. Ltd.), vacuum distillation unit (Equitron ROTEVA) and temperature-controlled water bath (Neolab Instruments) and glassware like test tubes, beakers, funnel etc. 2.3 Experimental methodology Paneer whey was procured from a local dairy and stored at 8 ± 2° C. The utilization of whey was achieved in maximum of 7 days since the lactose concentration in whey decreases after 7 days. The pH of the whey obtained was 5.2 ± 0.3 and the initial lactose concentration of whey, determined by using dintrosalicylic acid (DNSA) colorimetric test for reducing sugars at 540 nm [18], varied from 4.34% w/v to 6.25% w/v. The initial whey protein content in whey, determined by modified Folin-Lowry colorimetric test at 660 nm, varied from 0.197 % w/v to 0.39% w/v [19]. Whey was centrifuged at 8000 × g acceleration for 35-40 min and subsequently filtered with Whatman Grade 1 filter paper to remove the fats present in the whey. The whey was then subjected to evaporation at 95°C on a hot plate to denature whey proteins to keep the protein concentration below 0.1 % w/v since whey proteins act as inhibitors for lactose crystallization from whey. The evaporation was continued to achieve a lactose concentration ~20% w/v by removing excess water. The final lactose and protein concentration, i.e., after evaporation were observed to be over the range of 17.3% w/v to 21.7% w/v and 0.075% w/v to 0.105% w/v respectively. Absolute ethanol- China Grade (99.9%) was added to 50 ml concentrated whey to achieve effective concentration of 85% v/v in the reactor vessel. The reaction mixture was stirred with an open axial impeller of stainless steel at rpm of 325±25 to keep the contents in the vessel in suspension. Vertical ultrasonic horn with the required power levels at 100% duty cycle was inserted into the reaction system. 10

After the crystallization was complete, obtained lactose powder was vacuum filtered and later vacuum dried at 60° C for 6 hours. The schematic illustration of the experimental set-up of sonocystallization of lactose on ultrasonic horn has been depicted in figure 1. The dried lactose powder was then weighed to determine the percentage lactose recovery [11, 12]. Lactose purity was determined by Nickerson’s lactose assay method by preparing a stock solution of 1 mg/ml [20]. The lactose powder was checked for ash content [21], protein content [22] as well as for the presence of degradation products such as lactulose, glucose and galactose. The presence of proteins in the lactose powder was determined by modified Folin-Lowry method [19, 22]. Ash content was determined by subjecting 1 g of lactose powder in the porcelain dish at 550° C for 20 hours [21]. Lactulose assay was performed using the colorimetric assay described by Zhang et al. [23]. Nickerson’s colorimetric assay was performed for detection of glucose and galactose (monosaccharide subunits of lactose) [20]. In order to conclusively establish the presence of only lactose and absence of any degradation products such as monosaccharides, an ion-pair chromatography analysis was performed on Aminex HPX-87H column (Bio-Rad Laboratories) with 5 mM sulphuric acid as the mobile phase / eluant. The ethanol which was obtained after vacuum filtration was recovered by vacuum distillation (RotaVac) equipment and recycled in the subsequent experiments. Effect of the important parameters such as sonication time (10 min and 20 min) and power dissipation (40W to 120W) of vertical ultrasonic horn (20 kHz) on the lactose recovery was investigated at fixed 100% duty cycle. The increase in temperature for lactose sonocrystallization process for 10 min and 20 min was not more than 5°C (exact values for increase in temperature were between 1.5 and 3°C). Experiments were also carried out on rectangular ultrasonic bath with variable frequency operation mode with 100% duty cycle but with different frequencies to study the effect of ultrasonic frequency (22 kHz and 33 kHz) on 11

lactose recovery and purity. A conventional crystallization process without the use of ultrasound was also carried out for 1 hour which served as ‘control’ for the comparison with the results obtained on ultrasonic exposure and establish the process intensification benefits. 2.4 Analytical techniques 2.4.1 Lactose content / purity: The lactose content in the fresh whey as well as the concentrated whey was determined using UV- spectroscopy dinitrosalicylic acid (DNSA) assay for reducing sugars at 540 nm [18]. For determining initial lactose concentration in the whey, 0.02 ml of whey was diluted upto 1 ml (dilution factor of 50) followed by addition of 1ml of DNSA reagent. The mixture was heated in hot water bath at 95°C for 5 min and then immediately cooled in cold water solution. The solution then was made upto 5 ml and analysed using UV spectrometry analysis at 540 nm. For determining final lactose concentration in the concentrated whey after evaporation, same procedure was followed with 0.008 ml of whey and dilution factor of 125. Lactose purity of the recovered lactose powder after sonocrystallization was performed using colourimetric assay method described by Nickerson et al., [20] since this method is selective only for lactose estimation and showed no sensitivity to other sugars which can be present in the solution after exposure to ultrasound. In this method, methylamine reacts with lactose in the presence of buffer at pH ~ 13, a condition in which glucose and galactose does not interfere in the analysis and produces a red chromophore to be detected at 540 nm by UV spectroscopy [20]. The exact methodology used in the work consisted of initially pipetting 5 ml of water in one test tube (which serves as blank) and sample solution to be analyzed in another test tube. Subsequently, 5 ml of glycine-NaOH buffer (pH- 12.8), 0.5 ml of methylamine solution (5 % w/v) and 0.5 ml of sodium sulphite (1% w/v) were added in each of the test tube. The contents in the test tube were then mixed 12

thoroughly. It is important to use freshly prepared 1% w/v sodium sulfite solution during the analysis. The test tubes in water bath were heated at 65° C for 25 min and then cooled in ice water bath for 2 min to stop the reaction between lactose and methylamine. The absorbance was measured against blank at 540 nm. The standard curve was constructed by preparing working standard lactose solutions at different concentrations of 0.5, 0.75, 1, 1.25, 1.5 mg/ml and analyzing the absorbance. 2.4.2 Whey protein content For whey protein determination [18] in the fresh whey and concentrated whey, 0.04 ml of whey was diluted to 0.2 ml (Dilution factor- 5) which was mixed further with alkaline CuSO4. The solution was incubated for 10 min and then 0.2 ml of Folin- Ciocalteau reagent was added (diluted 1:1 ratio) and incubated for 30 min and subsequently analysed by UV spectroscopy at 660 nm. Whey protein content determination was performed in duplicate. 2.4.3. Lactulose assay The lactose powder was subjected to lactulose assay as per the method described by Zhang et al. [23] so as to determine the presence of lactulose in the recovered lactose samples. This assay was selected for analysis because it can detect the presence of lactulose in lactose powder as low as 5µg. The methodology for assay was as follows - 1 ml of lactulose solution + 75% w/v sulphuric acid was mixed and kept in water bath at 46°C for 5 min. 0.2 ml of Cysteine-HCl tryptophan reagent was added, mixed properly and again kept in water bath at 46°C for 30 min. The test tubes were cooled to room temperature and absorbance was measured at 518 nm for analysing the pink chromophore developed into the solution. The various reaction schemes occurring in the analysis has been depicted in figure 4.

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Visualisation reagent used in the work is cysteine hydrochloride - tryptophan reagent. Lactose does not contain fructose as a monosaccharide unit, and hence pure lactose will not show any pink chromophore formation. Standard curve was also plotted with standard lactulose solution (5-25 µg/ml). Lactulose assay was performed because on localized heating possible in the case of ultrasound assisted approach, lactose can get isomerized via a 1,2- enediol intermediate lactulose which then epimerized to either epilactose or galactose, tagatose, saccharinic acids via β- elimination on prolonged heating [23]. 2.4.4. Glucose-galactose assay Glucose and galactose was analysed using UV- spectroscopy detection at 710 nm for the development of blue colour after heating in an ammonium molybdate solution in the presence of phosphate-phthalate buffer at pH of 5.3 in a condition in which lactose does not interfere according to Nickerson et al. [20] who reported standardized colorimetric assay for glucose-galactose determination. The exact methodology followed in the work started with taking 5 ml of phosphate phthalate buffer (pH- 5.3) into the different test tube and adding 1 ml of water which served as blank in one test tube and sample solution in other test tubes. Subsequently, 5 ml of ammonium molybdate (6% w/v) solution was added and mixed thoroughly. The test tubes were heated in hot water bath at 95°C for 15 min and cooled in tap water for 5 min to stop the reaction. The absorbance was measured against the blank at 710 nm. The standard curve was prepared using the glucose – galactose stock solution (5 mg glucose-galactose/ml). The concentrations used for preparing the standard curve were 0.5, 1, 1.5, 2, 2.5, 3 mg glucose-galactose / ml. The detection of glucose and galactose samples by this assay was performed in triplicates. 2.4.5. Whey protein determination in lactose powder 14

Stock solution of 15 mg/ml of lactose powder samples recovered under various experimental conditions was prepared. A sample volume (0.2 ml) was used for the analysis of proteins according to modified Folin-Lowry method [19, 22]. 15 mg/ml was chosen as the concentration of lactose solution since according Huang et al., [22] there was no lactose interference in the case of the modified Folin-Lowry assay method for the measurement of proteins/ peptides till a lactose concentration of 16 mg/ml. 2.4.6 Melting Point determination The melting point determination of lactose powder was performed using the melting point apparatus obtained from Campbell Electronics, Mumbai, India. 2.4.7. Ion- pair chromatography of lactose powder sample Ion-pair chromatography of the recovered lactose powder was also performed using Bio-Rad’s Aminex-HPX 87H column with 5mM of sulphuric acid as the eluent in order to confirm the presence of only lactose as the sugar in the sample and to establish that no degradation of lactose has occurred to its subsequent monosaccharide’s viz. glucose and galactose due to the effects of ultrasound induced cavitation.

3. RESULTS AND DISCUSSION Different sets of experiment were conducted for sonocrystallization of lactose with ultrasonic horn at varying power levels (40W to 120W) and time intervals of 10 min and 20 min. Ultrasonic bath study for sonocrystallization of lactose was also performed at different frequencies of 22 kHz and 33 kHz for 10 min. The obtained results for three sets of experiments have now been discussed in different sections.

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3.1. Effect of varying power levels on the lactose recovery and purity for 10 min treatment using ultrasonic horn The conventional process, i.e., absence of ultrasound, resulted in a lactose recovery of 73.3% with a purity of 78.6% after 1 hour of crystallization process. In the case of sonocrystallization study using ultrasonic horn (20 kHz, 100% duty cycle) for 10 min, it was observed that with an increase in power dissipation from 40 W to 120 W, lactose recovery increased from 74.7 % to 97.6% respectively as per results given in table 1 and figure 2. The purity of the recovered lactose powder also increased from 86.2 % to 97.6 % with an increase in power level from 40 W to 120 W as per data shown in figure 2. The observed increase can be attributed to the cavitation effects in terms of turbulence, greater heat and mass transfer rates leading to an increase in nucleation rate and crystal growth rate. In the case of anti-solvent induced sonocrystallization, the use of ultrasound also ensures uniform mixing of the contents. The cavitational events also favoured due to the lowering of vapour pressure because of addition of anti-solvent resulting in enhanced supersaturation and decrease in solubility of lactose in the system, leading to enhanced nucleation ad ease of formation of crystals. It is hypothecated that every microscopic cavity formed in the liquid takes up minute amounts of the anti-solvent resulting in spontaneous local supersaturation. Thus, every microscopic cavity formed due to ultrasonic cavitation represents itself as possible site of nucleation for the crystallization process [11]. Subsequently, enhanced mass transfer rates due to the cavitation induced microscale liquid streaming and turbulence helps in enhanced recovery of lactose. Hence, the application of ultrasonic horn for 10 min with 100% duty cycle gives the process intensification of antisolvent crystallization of lactose from whey, in terms of enhanced recovery and purity as well as reduction in the treatment time.

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3.2. Effect of varying power levels on the lactose recovery and purity for 20 min treatment using ultrasonic horn In the case of sonocrystallization study using ultrasonic horn (20 kHz, 100% duty cycle) for 20 min, it was observed that with the an increase in power dissipation from 40 W to 120 W, lactose recovery decreased from 98.6% to 75.6% as per data given in table 2 and figure 3. The purity of the recovered lactose powder also decreased from 96.9 % to 88.7 % with a corresponding increase in power from 40 W to 120 W as per data given in table 2 and figure 3. The observed results for 20 min treatment can be attributed to degradation of lactose due to prolonged exposure to cavitation conditions generating high temperature up to 5000 K and high pressure of order of thousand atm. Thus it can be established that use of ultrasound for 20 min leads to moderate decrease in lactose recovery and purity at for an increase in power levels from 40 W to 120 W as compared to 10 min ultrasonic exposure. 3.3. Effect of change in ultrasonic frequency and time on the lactose recovery and purity using ultrasonic bath In the case of sonocrystallization studies using ultrasonic bath for 10 min, it was observed that with an increase in frequency from 22 kHz to 33 kHz, lactose recovery decreased slightly from 94.4 % to 92.2 % but there was large increase in lactose purity from 79.5 % to 91.5 % as per data given in table 3. The decrease in the lactose recovery with an increase in ultrasonic frequency can be attributed to more intense cavitation leading to reduced recovery. In the case of sonocrystallization studies using ultrasonic bath for 20 min, it was observed that with an increase in frequency from 22 kHz to 33 kHz, lactose recovery decreased from 93.9 % to 88.9 % with an increase in lactose purity from 87.5 % to 92.4 % as per data shown in table 3. It is important to understand that the reduction in the lactose 17

recovery was lower in the case of ultrasonic bath as compared to the ultrasonic horn attributed to reduced cavitational intensity in the case of bath. 3.4. Melting point Melting point (mp) is an important parameter, which can be used to distinguish between the formation of α-lactose monohydrate (mp-201–202°C) and β-lactose (mp-253° C) [24]. The melting point of the recovered lactose in this study was observed to be 196°C which is much closer to α-lactose monohydrate. Hence it can be inferred that the lactose powder obtained in the present work is α-lactose monohydrate. 3.5. Ash content Ash content was determined as a part of studying the purity profile of the recovered lactose powder, which is very important requirement considering the possible commercial applications of the recovered lactose. Ash content was determined by weighing the initial weight of lactose powder in porcelain crucible and the final weight obtained after maintaining the muffle furnace at 550°C for 20 hours [21]. There was no particular trend observed in ash content in the lactose powder with varying power levels for 10 min and 20 min ultrasound exposure which can be observed from the results given in table S1. There was also no particular trend observed in ash content of the recovered lactose from ultrasonic bath with varying frequency and time intervals which can be observed in table S1. However, slightly less ash content was observed in the case of lactose powder recovered by ultrasonic probe for 20 min as compared to 10 min. The lower ash content in lactose recovered for the case of 20 min ultrasonic exposure as compared to 10 min continuous ultrasound exposure may be attributed to the enhanced exposure of the contents to the ultrasound effects which can alter the solubility.

18

In the present work, the ash content in the recovered lactose by conventional process was observed to be 5.3% w/w. The ash content observed in the lactose recovered from ultrasonic horn exposure (20 kHz) for 10 min with 100% duty cycle varied from 3% w/w to 4.4% w/w while for ultrasonic horn exposure for 20 min with same duty cycle ash content ranged from 2.47% w/w to 3.76% w/w. The ash content in the recovered lactose from ultrasonic bath with frequency of 22 kHz for 10 min and 20 min sonication time for 100% duty cycle were 3.9% w/w and 2.9% w/w while for 33 kHz for 10 min and 20 min sonication time with same duty cycle the values were 3.67% w/w and 3.7 % w/w which can be observed from table S1. Ultrasonic cavitation leads to generation of ‘hot spots’ in the liquid medium with generation of temperatures of ~5000 K and pressures of few thousand atmospheres with the implosion of cavitation microbubbles. This particular phenomenon can be attributed to marginally less ash content in lactose recovered from anti-solvent assisted sonocrystallization than ash content in lactose samples recovered from conventional process without ultrasonic treatment since the solubility of organic or inorganic salts is enhanced due to the ‘hot spots’ generated by ultrasonic cavitation. Marginally lower ash content in the case of ultrasonic horn as compared to the ultrasonic bath can again attributed to the same fact of higher cavitational activity in the case horn. In the earlier studies, Bund and Pandit [15] observed high ash content in recovered lactose from the concentrated whey having pH of 4.6 as compared to pH of 2.8 which was reportedly attributed to the simultaneous precipitation of inorganic salts along with the lactose powder in the presence of anti-solvent ethanol. Adjustment of pH to 2.8 prior to start of crystallization procedure by addition of anti-solvent led to increase in solubility of the inorganic salts (mostly in their chloride from) in the alcoholic solution. The ash content of the lactose powder dropped to almost 30% with pH adjustment from 4.6 to 2.8. It was concluded that overall variation in the lactose recovery was 3-5% with the pH adjustment with lactose 19

purity observed to be around 99% but at the cost of addition of one more step of pH adjustment which will depend upon the economic feasibility of the process and the desired end use of the recovered lactose powder. 3.6. Protein / peptide content in the lactose powder The protein content in the lactose powder with varying power levels for 10 min and 20 min ultrasound exposure was almost constant as per data shown in table S2. The protein/peptide content of the recovered lactose from ultrasonic bath was also marginally affected by varying frequency and time intervals which can be observed in table S2. However, slightly lower protein content was observed in the case of lactose powder recovered by ultrasonic horn exposure (20 kHz) for 20 min as compared to that obtained for 10 min ultrasonic treatment, which can be attributed to the fact that longer ultrasonic exposures resulted in protein and amino acid degradation. It is important to understand the lactose will not be affected by these cavitating conditions. Typically, proteins are very sensitive in terms of temperature, presence of solvent, pH etc. and hence prone to degradation induced by ultrasonic effects. Although the initial protein content in whey solution was 0.1-0.2 % w/v, higher protein content in the recovered lactose can be attributed to the fact that ethanol resulted in precipitation of whey proteins, degraded short chain peptides, amino acids etc. and thus resulted in higher protein content values as established using modified Folin- Lowry method [21]. 3.7. Purity of lactose powder When lactulose assay was carried out in triplicate for lactose powder solutions (20 µg/ml) recovered by ultrasonic exposure for both treatment time as 10 min and 20 min, there

20

was no development of pink colour indicating the absence of lactulose in all the lactose samples. Similarly when the lactose powder was subjected to Nickerson’s assay [20] for the colorimetric determination of glucose and galactose in the powder, there was no development of blue colour indicating the absence of the glucose and galactose in the recovered lactose sample. In order to confirm the presence of only lactose as the sugar present and no degradation occurring to other sugar or mono- or di-saccharide in the recovered sample after sonocrystallization, ion-pair chromatography was performed using Bio-Rad’s Aminex HPX 87H column with 5 mM sulphuric acid as the mobile phase or eluent. A single broad peak was observed with retention time (Rt) of ~7.6 min for recovered lactose samples (figure 5) which was similar to the standard lactose sample (Rt- 7.7 min). Overall it can be said that the lactose is present in the pure form without formation of any of the thermal degradation products as established by these assays. The lactulose assay performed in the present work confirmed that no lactulose was observed. Glucose- galactose assay and ion-pair chromatography analysis also established that no degradation of lactose (disaccharide) into glucose and galactose (corresponding monosachharides) based on the breaking of C-O-C covalent bond occurred in the presence of ultrasound used for recovery. 3.8 Hydrodynamic particle size distribution of recovered lactose Hydrodynamic particle size is the sum of particle diameter in the solvent (in this casewater) and the solvent layer (or in some cases electrical double layer) formed around that particle. When measurements are performed in suspended state, it reflects the actual variation in the particle size of the recovered/ desired sample due to the change in the process

21

parameters. The particle size measured by dispersing the desired sample in a solvent which is fairly insoluble reflects the particle size obtained from a particular type of size reduction technique used. Hydrodynamic particle size can be measured by Differential Light Scattering (DLS) system. In this study, it is utmost important to study the changes in particle size of the recovered lactose due to change in process parameters like power, frequency, reactor configurations and time of sonication. Thus, the recovered lactose was mixed in water as a solvent to determine the hydrodynamic radius using the DLS system. In the case of lactose recovered using the conventional process, the particle size distribution was quite broad as compared to lactose recovered from sonocrystallization which can be observed from figure 6. The hydrodynamic particle size distribution for the lactose recovered by conventional process, i.e., without the use of ultrasound showed a bimodal size distribution pattern with two major peaks at ~ 500 nm as well as ~ 1000 nm. Also high polydispersiblity index (PdI) of around 0.791 for lactose recovered by conventional process as per data shown in table S3-S5. On the other hand, a unimodal hydrodynamic particle size distribution which can be observed from figures 7-8 was obtained for all lactose samples recovered from anti-solvent assisted sonocrystallization which is quite evident from the single major peaks present in the graph. It can be observed from table S3 and figures 7 A to 7 E that with an increase in power levels from 40 W to 120 W for 10 min sonication treatment, Z-Average (d.nm) decreased from 1345 nm to 1219 nm. In the case of 20 min sonication treatment, for power levels from 40 W to 100 W, particle size decreased from 1044 nm to 926.9 nm and increased slightly at 120 W power level at 945 nm as per data represented in table S4 and figures 7A to 7 E. Thus, it can be inferred from the above observations that in the case of ultrasonic horn, with an increase in power levels as well as time of sonication, there is a marked reduction in hydrodynamic particle size which can be attributed to higher intensity of ultrasonic cavitation with longer 22

duration of sonication time and higher power levels. Higher intensity cavitation avoids any particle agglomeration and also leads to particle breakage giving lower size. In the case of ultrasonic bath, the Z-Average (d.nm) obtained with ultrasonic frequency of 22 kHz decreased from 1431 nm to 1219 nm for an increase in time from 10 min and 20 min as per data shown in table S5 and figure 8 A. Similarly for ultrasonic frequency of 33 kHz the Z-Average (d.nm) decreased from 1169 nm to 1159 nm for a similar increase in time from 10 min and 20 min as per data given in table S5 and figure 8 B. The hydrodynamic particle size decreased with an increase in sonication time as well as frequency because of greater quantum of ultrasonic cavitational events with higher sonication time and ultrasonic frequency. If we compare the results obtained using ultrasonic probe and ultrasonic bath, lower hydrodynamic particle size were observed for recovered lactose obtained using ultrasonic probe since greater quantum of cavitational events with higher intensity occur due to the direct contact with the liquid as compared to the indirect sonication via transducers at the bottom in the case of ultrasonic bath. In the previous studies on anti-solvent assisted crystallization of lactose, irrespective of the fact that whether ultrasound was used in the studies or not, determination of hydrodynamic particle size was not reported to assess the variation due to process parameters and hence the current work forms an important contribution. Instead, the recovered lactose particles were analysed using microscopy techniques with 40x magnification. Also recovered lactose was analysed using laser diffractometer, scanning electronic microscopy (SEM) and differential scanning calorimetry (DSC) for the effect of process parameters on the crystal size and morphology using L9- orthogonal system for the case of acetone as anti-solvent. It was reported that sonication time was found to be the most influencing parameter on volume

23

median diameter of lactose crystals. It was also reported that elongated and rod/needle shape crystals were observed for sonicated samples whereas tomhawk shaped crystals were observed for the case of conventional approach based on stirring [25]. Bund and Pandit [15] also analysed the crystal size distribution of lactose recovered from buffalo milk whey by anti-solvent crystallization using microscopy method with photographs being captured at 40x magnification. It was reported that average projected area (µm2) and average particle diameter (µm) decreased with a decrease in crystallization time. The average projected area decreased from 25.4 µm2 to 14.92 µm2 for a decrease in crystallization time from 5 h to 1 h while the average particle diameter decreased from 5.33 µm to 4.19 µm. In the sonocrystallization study of lactose recovered from reconstituted lactose solutions with anti-solvent ‘ethanol’, Bund and Pandit [11] investigated the effect of various parameters such as lactose concentration, sonication time, protein concentration and pH on the particle size of crystals and CSD of recovered lactose. It was reported using lactose solution with higher initial lactose content, minimum protein content and pH of 4.2, more uniform and narrow CSD is obtained. Also, for the higher rate of crystallization, better CSD with lower crystal size diameter was obtained. Also, for the higher rates of crystallization observed with reconstituted lactose solutions with higher initial lactose content, needle shaped lactose crystals were observed in the case of 15.5% w/v and 17.5% w/v lactose solutions in the case of ultrasound assisted approach as against the ‘tomhawk’ shape obtained in conventional approach [11]. In the sonocrystallization study of lactose recovered from reconstituted lactose solutions with anti-solvent acetone, Patel and Murthy [13] reported that with an increase in initial lactose concentration from 12% w/v to 16% w/v, the average projected area of lactose

24

crystals decreased from 8.28 µm2 to 5.77 µm2. It was also reported that with an increase in the lactose concentration from 12% w/v to 16% w/v, the average crystal diameter decreased from 2.94 µm to 2.48 µm. In the sonocrystallization study of lactose recovered from reconstituted lactose solutions from anti-solvent propanol, Patel and Murthy [14] reported that with an increase in sonication time from 2 min to 8 min, the average projected area of recovered lactose crystals decreased from 10.44 µm2 to 5.92 µm2. The spread of CSD was also observed to decrease with an increase in sonication time which is evident from the decrease in the average crystal diameter from ~3.64 µm to ~2.74 µm with an increase in sonication time from 2 min to 8 min [14]. In the present work, hydrodynamic particle size of the lactose powder recovered from low concentrated whey by anti-solvent assisted sonocrystallization has been determined which is more dependent on the change in the ultrasonic process parameters like power, frequency, time of sonication and reactor configurations. In the previous studies by Bund and Pandit [11, 15] and Patel and Murthy [13, 14, 25], the actual particle size of the lactose powder recovered from low concentrated whey or reconstituted lactose solutions by antisolvent assisted sonocrystallization has been studied by microscopy, SEM, laser diffractometer, DSC techniques which is more dependent on the selection of particle size reduction technique being used.

4 CONCLUSIONS The overall objectives of the present work were to scale up the lactose recovery from low concentrated whey containing ~20 % w/v of lactose as well as to obtain maximum yield

25

and purity of the lactose sample by optimising the ultrasonic parameters such as power, frequency, time of sonication and reactor configurations. Following important guidelines can be established based on the findings of the present work: 1. Presence of ultrasound in anti-solvent assisted sonocrystallization increased the lactose recovery as well as its purity within a short span of time. 2. Lactose recovery as well as purity increased with an increase in ultrasonic power levels from 40 W to 120 W for 10 min. of ultrasonic horn exposure. 3. Lactose recovery as well as purity decreased with an increase in ultrasonic power from 40 W to 120 W for 20 min for ultrasonic horn exposure due to degradation of lactose by prolonged exposure to the ultrasonic cavitation. 4. In the case of ultrasonic bath, lactose purity increased with an increase in ultrasonic frequency 22 kHz to 33 kHz at the expense of slight decrease in lactose recovery. 5. Lactose recovery from whey (scale-up approach on ultrasonic horn) was increased to ~ 98% from ~ 92% observed in the prior studies on lactose recovery from reconstituted lactose solutions (laboratory approach on ultrasonic bath) while maintaining the lactose purity almost constant at 98-99%. 6. In the case of ultrasonic probe assisted anti-solvent crystallization of lactose, with an increase in power levels from 40 W to 120 W as well as time of sonication from 10 min and 20 min, there was a marked reduction in hydrodynamic particle size since the intensity of cavitation increased with longer duration of sonication time and higher power levels. 7. In the case of ultrasonic bath assisted anti-solvent sonocrystallization of lactose, the hydrodynamic particle size decreased with an increase in sonication time from 10 min to 20

26

min as well as with an increase in frequency from 22 kHz to 33 kHz because of greater quantum of cavitational events with higher sonication time and ultrasonic frequency. Overall, it has been established that parameters such as time of sonication, ultrasonic power and frequency plays a major role in deciding the lactose yield, purity as well as hydrodynamic particle size distribution. It is also demonstrated that ultrasound intensifies the recovery at large scale operation as compared to the previous reported results.

ACKNOWLEDGEMENT Authors would like to acknowledge the funding of Department of Science and Technology, Government of India under the MOFPI scheme for the research work in the area of intensification of recovery of valuable products from whey using ultrasound. One of the authors, Chinmay N. Gajendragadkar would also like to extend acknowledgement to Mr. Shravan Sreenivasan and Mr. Mahesh Tambe for giving valuable inputs and helping with the analytical techniques of ion pair chromatography and particle size analysis using Differential Laser Scattering (DLS) technique.

5. REFERENCES [1] N. Goyal, D.N. Gandhi, Comparative analysis of Indian paneer and cheese whey for electrolyte whey drink, World J. Dairy Food Sci. 4 (1) (2009) 70–72. [2] P.S. Panesar, J.F. Kennedy, D.N. Gandhi, K. Bunko, Bioutilisation of whey for lactic acid production, Food Chem. 105 (2007) 1–14.

27

[3] P. Jelen, Whey Processing, Encyclopedia of Dairy Sciences, vol. 4, Academic Press, London, 2000. [4] F.V. Kosikowski, Whey utilization and whey products, J. Dairy Sci. 62 (1979) 1149– 1160. [5] G.M.I. Siso, The biotechnological utilization of cheese whey: a review, Bioresour. Technol. 57 (1996) 1–11. [6] C.N. Gajendragadkar, P.R. Gogate, Intensified recovery of valuable products from whey by use of ultrasound in processing steps – A review, Ultrason. Sonochem. 32 (2016), 102118. [7] A.J. Mawson, Bioconversions for whey utilization and waste abatement, Bioresour. Technol. 47 (1994) 195–203. [8] C.V.S. Subrahmanyam, J.T. Setty, S. Suresh, V.K. Devi, Crystallization, in: Pharmaceutical Engineering-Principles and Practices, Vallabh Prakashan, Delhi, 2001, pp. 361–381. [9] M. Ashokkumar, R. Bhaskaracharya, S. Kentish, J. Lee, M. Palmer, B. Zisu, The ultrasonic processing of dairy products – an overview, Dairy Sci. Technol. 90 (2009) 147– 168. [10] G. Ruecroft, D. Hipkiss, T. Ly, N. Maxted, P.W. Cains, Sonocrystallization: the use of ultrasound for improved industrial crystallization, Org. Process Res. Dev. 9 (2005) 923–932. [11] R.K. Bund, A.B. Pandit, Sonocrystallization: effect on lactose recovery and crystal habit, Ultrason. Sonochem. 14 (2006) 143–152.

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[12] R.K. Bund, A.B. Pandit, Rapid lactose recovery from paneer whey using sonocrystallization: a process optimization, Chem. Eng. Process. 46 (2007) 846–850. [13] S.R. Patel, Z.V.P. Murthy, Ultrasound assisted crystallization for the recovery of lactose in an anti-solvent acetone, Cryst. Res. Technol. 44 (8) (2009) 889–896. [14] S.R. Patel, Z.V.P. Murthy, Anti-solvent sonocrystallization of lactose, Chem. Process Eng. 32 (4) (2011) 379–389. [15] R.K. Bund, A.B. Pandit, Rapid lactose recovery from buffalo whey by use of ‘antisolvent, ethanol’, J. Food Eng. 82 (2007) 333–341. [16] B. Zisu, M. Sciberras, V. Jayasena, M. Weeks, M. Palmer, T. D. Dincer, Sonocrystallization of lactose in concentrated whey, Ultrason. Sonochem. 21 (2014) 21172121. [17] K. Kezia, J. Lee, B. Zisu, M. Weeks, G. Chen, S. Gras, S. Kentish, Crystallisation of minerals from concentrated saline dairy effluent, Water Res. 101 (2016) 300-308. [16] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (3) (1959) 426-428. [17] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193 (1) (1951) 265–275. [18] T.A. Nickerson, I.F. Vujicic, A.Y. Lin, Colourimetric estimation of lactose and its hydrolytic products, J. Dairy Sci., 59 (3) (1975) 386-389. [19] AOAC. Official Methods of analysis (14th ed.). Washington, DC: Association of Official Analytical Chemists, (1984) pp. 281.

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[20] Y.W. Huang, R.T. Marshal, M.E. Anderson, C. Charoen, Automated modified Lowry protein analysis of milk, J. Food Sci. 41 (5) (1976), 1219-1221. [21] Z. Zhang, H. Wang, R. Yang, X. Jiang, A novel spectrophotometric method for quantitative determination of lactulose in food industries, Int. J. Food Sci. Technol., 45 (2010), 258–264. [22] B. Elvers, S. Hawkins, G. Schulz, Ullmann’s Encyclopedia of Industrial Chemistry, VCH Publishing, Germany, vol. A,15,(1990) pp. 107–114. [23] S.R. Patel, Z.V.P. Murthy, Effect of process parameters on crystal size and morphology of lactose in ultrasound-assisted crystallization, Cryst. Res. Technol. 46 (3) (2011) 243–248.

30

LIST OF TABLES Table 1- Results of anti-solvent assisted sonocrystallization of lactose using ultrasonic horn (22 kHz and 100% duty cycle) for 10 min. Table- 2- Results of anti-solvent assisted sonocrystallization of lactose using ultrasonic horn (22 kHz, 100% duty cycle) for 20 min. Table 3- Results of anti-solvent assisted sonocrystallization of lactose using ultrasonic bath (100% duty cycle) for 10 min and 20 min.

31

LIST OF FIGURES Figure 1- Schematic diagram of experimental set-up for ultrasonic probe assisted anti-solvent crystallization of lactose from low concentrated whey. Figure 2 – Effect of varying ultrasonic power levels in the case of ultrasonic horn (20 kHz) at 100% duty cycle and 10 min treatment on A] Lactose recovery B] Lactose purity. Figure 3- Effect of varying ultrasonic power levels in the case of ultrasonic horn (20 kHz) at 100% duty cycle and 20 min treatment on A] Lactose recovery B] Lactose purity. Figure 4- A schematic reaction pathway depicting the reactions involved in lactulose assay. Figure 5- Ion pair chromatography peaks analysis for lactose standard and lactose recovered from anti-solvent assisted sonocrystallization. Figure 6- Hydrodynamic particle size distribution of lactose recovered using conventional process. Figure 7- Hydrodynamic particle size distribution of lactose recovered using ultrasonic horn (20 kHz) treatment for 10 min and 20 min (100% duty cycle) A] 40 W B] 60 W C] 80 W D] 100 W E] 120 W Figure 8- Hydrodynamic particle size distribution of lactose recovered using ultrasonic bath treatment for 10 min and 20 min with 100% duty cycle at 22 kHz and 33 kHz. A] 22 kHz B] 33 kHz

32

Table 1- Results of anti-solvent assisted sonocrystallization of lactose using ultrasonic horn (22 kHz and 100% duty cycle) for 10 min. Experiment

Power

pH of Initial lactose Lactose

Amount

%

%

name

(W)

whey

of

recovery

lactose

of lactose

purity

concentration of (%w/v)

concentratio

whey n of whey lactose after

powder

evaporation

(g)

(%w/v)

Conventional

-

5.47

6.09

19.5

7.15

73.3

78.6

40

5.32

5.88

18.1

6.75

74.7

86.2

60

5.28

5.25

19.8

8.12

82.0

88.2

80

5.17

4.96

18.75

8.37

89.3

92.7

100

5.22

5.31

20.2

9.35

92.6

96.4

120

5.36

5.46

21.6

10.5

97.6

97.6

process for 60 min Ultrasonic probe (1)

Ultrasonic probe (2)

Ultrasonic probe (3) Ultrasonic probe (4) Ultrasonic probe (5)

33

Table- 2- Results of anti-solvent assisted sonocrystallization of lactose using ultrasonic horn (22 kHz, 100% duty cycle) for 20 min.

Experiment

Power

pH of Initial lactose Lactose

Amount

%

%

name

(W)

whey

of

recovery

purity

concentration of (%w/v)

Conventional process

concentration

whey of whey after lactose evaporation

powder

(%w/v)

(g)

lactose

of lactose

-

5.47

6.09

19.5

7.15

73.3

78.6

40

5.41

6.25

21.73

10.7

98.6

96.9

60

5.35

3.64

20.02

9.15

91.5

93. 8

80

5.36

4.90

19.75

8.67

87.8

90.6

100

5.14

4.58

19.36

8.13

84.0

89.2

120

5.17

4.34

18.93

7.16

75.6

88.7

(60

min)

Ultrasonic probe (1)

Ultrasonic probe (2) Ultrasonic probe (3) Ultrasonic probe (4)

Ultrasonic probe (5)

34

Table 3- Results of anti-solvent assisted sonocrystallization of lactose using ultrasonic bath (100% duty cycle) for 10 min and 20 min. Experiment

pH of Initial lactose Lactose

Amount

%

%

name

whey

of

lactose

lactose

recovery

purity

concentration

concentration

of whey (% of whey after lactose w/v)

evaporation

(g)

(%w/v) Conventional

5.47

6.09

19.5

7.15

73.3

78.6

4.91

5.14

17.44

8.23

94.4

79.5

4.91

5.14

17.44

8.19

93.9

87. 5

5.42

5.34

17.25

7.95

92.2

91.5

5.42

5.34

17.25

7.67

88.9

92.4

process at 60 min Ultrasonic bath

at

22

kHz,

7

W

power* & 10 min Ultrasonic bath

at

22

kHz,

7

W

power & 20 min Ultrasonic bath

at

33

kHz, 11.6 W power & 10 min Ultrasonic bath

at

33

kHz, 11.6 W power & 20 min

* The actual ultrasonic power dissipation in the reaction vessel during the reaction was carried out by calorimetric study.

35

Figure 1- Schematic diagram of experimental set-up for ultrasonic probe assisted anti-solvent crystallization of lactose from low concentrated whey.

Lactose recovery and purity (%)

120 100 80 60 40

Lactose recovery Lactose purity

20 0 0

20

40

60

80

100

120

140

Ultrasonic Power (Watts)

Figure 2- Effect of varying ultrasonic power levels in the case of ultrasonic horn (20 kHz) at 100% duty cycle and 10 min treatment on A] Lactose recovery B] Lactose purity.

36

Lactose reccovery and purity (%)

120 100 80 60

Lactose recovery

40

Lactose purity 20 0 0

20

40

60 80 Ultrasonic power (W)

100

120

140

Figure 3- Effect of varying ultrasonic power levels in the case of ultrasonic horn (20 kHz) at 100% duty cycle and 20 min treatment on A] Lactose recovery B] Lactose purity.

Figure 4- A schematic reaction pathway depicting the reactions involved in lactulose assay.

37

Lactose standard peak (concentration- 0.2 mg/ml, Rt- 7.7 min)

Recovered lactose (concentration- 1mg/ml; Rt- 7.6 min) Figure 5- Ion pair chromatography analysis for lactose standard and lactose recovered from anti-solvent assisted sonocrystallization.

38

Figure 6- Hydrodynamic particle size distribution of lactose recovered using conventional process.

39

A] 40 W

10 min.

20 min.

B] 60 W

10 min.

20 min.

40

C] 80 W

10 min.

20 min.

D] 100 W

10 min.

20 min.

41

E] 120 W

10 min.

20 min.

Figure 7- Hydrodynamic particle size distribution of lactose recovered using ultrasonic horn (20 kHz) treatment for 10 min and 20 min (100% duty cycle)

42

A] 22 kHz

10 min.

20 min.

10 min.

20 min.

B] 33 kHz

Figure 8- Hydrodynamic particle size distribution of lactose recovered using ultrasonic bath treatment for 10 min and 20 min with 100% duty cycle at 22 kHz and 33 kHz.

43

SUPPLEMENTARY INFORMATION SECTION Table S1- Ash content in lactose powder obtained using ultrasonic horn and ultrasonic bath with varying process parameters. Table S2- Protein content in lactose powder obtained using ultrasonic horn and ultrasonic bath with varying process parameters. Table S3- Average particle size (Z-Average) hydrodynamic radius (d.nm) and poly dispersibility index (PdI) reflecting hydrodynamic particle size of recovered lactose at different power levels (W) for 10 min and 100% duty cycle in ultrasonic horn (20 kHz). Table S4- Average particle size (Z-Average) hydrodynamic radius (d.nm) and poly dispersibility index (PdI) reflecting hydrodynamic particle size of recovered lactose at different power levels (W) for 20 min and 100% duty cycle in ultrasonic horn (20 kHz). Table S5- Average particle size (Z-Average) hydrodynamic radius (d.nm) and poly dispersibility index (PdI) reflecting hydrodynamic particle size of recovered lactose at different frequencies (kHz) for 10 min and 20 min, 100% duty cycle in ultrasonic bath.

44

SUPPLEMENTARY INFORMATION SECTION Table S1- Ash content in lactose powder obtained using ultrasonic horn and ultrasonic bath with varying process parameters. Conventional process- Lactose ash content- 5.26 % w/w Sr. no

Reactor configurations and process parameters

1.

Ultrasonic horn ( 20 kHz, 10 min, 100% duty

Ash content (% w/w)

cycle) with power levels (in Watts) a.

40

3.85

b.

60

3.01

c.

80

3.62

d.

100

4.39

e.

120

4.34

2.

Ultrasonic horn (20 kHz, 20 min, 100% duty cycle) with power levels (in Watts)

a.

40

3.69

b.

60

2.79

c.

80

2.93

d.

100

3.76

e.

120

2.47

3.

Ultrasonic bath

a.

22 kHz, 10 min sonication

3.94

b.

22 kHz, 20 min sonication

2.89

c.

33 kHz, 10 min sonication

3.67

d.

33 kHz, 20 min sonication

3.73

45

Table- S2- Protein content in lactose powder obtained using ultrasonic horn and ultrasonic bath with varying process parameters. Conventional process protein content - 3.944% w/w Sr. no

Reactor configurations and process parameters

Protein/peptide content (% w/w)

1.

Ultrasonic horn ( 20 kHz, 10 min, 100% duty cycle) with power levels (W) as-

a.

40

2.87

b.

60

2.37

c.

80

2.94

d.

100

4.06

e.

120

3.16

2.

Ultrasonic horn (20 kHz, 20 min, 100% duty cycle) with power levels (W) as-

a.

40

1.91

b.

60

1.89

c.

80

2.19

d.

100

2.16

e.

120

2.09

3.

Ultrasonic bath

a.

22 kHz, 10 min sonication

2.27

b.

22 kHz, 20 min sonication

3.03

c.

33 kHz, 10 min sonication

3.57

d.

33 kHz, 20 min sonication

2.93

46

Table S3 Average particle size (Z-Average) hydrodynamic radius (d.nm) and poly dispersibility index (PdI) reflecting hydrodynamic particle size of recovered lactose at different power levels (W) for 10 min and 100% duty cycle in ultrasonic horn (20 kHz). Sr.

Recovered lactose powder at power Z-Average (d.nm.)

PdI

no.

levels (W) for 10 min

1.

Conventional process

1325

0.79

2.

40

1345

0.40

3.

60

1327

0.04

4.

80

1291

0.19

5.

100

1252

0.43

6.

120

1219

0.45

Table S4- Average particle size (Z-Average) hydrodynamic radius (d.nm) and poly dispersibility index (PdI) reflecting hydrodynamic particle size of recovered lactose at different power levels (W) for 20 min and 100% duty cycle in ultrasonic horn (20 kHz). Sr.

Recovered lactose powder at power Z-Average (d.nm.)

PdI

no.

levels (W) for 20 min

1.

Conventional process

1325

0.79

2.

40

1044

0.04

3.

60

1043

0.04

4.

80

988

0.55

5.

100

926.9

0.29

6.

120

945

0.22

47

Table S5- Average particle size (Z-Average) hydrodynamic radius (d.nm) and poly dispersibility index (PdI) reflecting hydrodynamic particle size of recovered lactose at different frequencies (kHz) for 10 min and 20 min, 100% duty cycle in ultrasonic bath. Sr.

Recovered

lactose

powder

from Z-Average (d.nm.)

PdI

no.

ultrasonic bath

1.

Conventional process

1325

0.79

2.

22 kHz, 10 min

1431

0.44

3.

22 kHz, 20 min

1219

0.11

4.

33 kHz, 10 min

1169

0.1

5.

33 kHz, 20 min

1159

0.02

48

HIGHLIGHTS:


Understanding effect of reactor configuration on intensified recovery of lactose
Understanding the effect of operating parameters on yield and purity of lactose
Establishing the process intensification benefits for the use of ultrasound
Establishing a sustainable approach for valuable products with environmental benefits

49