Comparative assessment of high-intensity ultrasound and hydrodynamic cavitation processing on physico-chemical properties and microbial inactivation of peanut milk

Ultrasonics - Sonochemistry 59 (2019) 104728

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Comparative assessment of high-intensity ultrasound and hydrodynamic cavitation processing on physico-chemical properties and microbial inactivation of peanut milk Akshata R. Salve, Kakoli Pegu, Shalini S. Arya

T

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Food Engineering and Technology Department, Institute of Chemical Technology, NM Parikh Marg, Matunga, Mumbai 400 019, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Non thermal processing Ultrasonication Hydrodynamic cavitation High-temperature short-time Peanut milk Microbial inactivation

Ultra-sonication (US) at varying intensities (200 W, 300 W and 400 W) and hydrodynamic cavitation (HC) at increasing pressures (6 bar, 8 bar and 10 bar) on freshly extracted peanut milk as non-thermal processing of milk for enhanced quality. The effects of US and HC was investigated on physico-chemical properties of peanut milk, microbial inactivation (total plate count and yeasts and molds), microstructure by optical microscopy and particle size, ζ-potential, sedimentation index, rheology and color measurements. The high temperature short time (HTST) treated milk samples have shown 1.53 and 2 log reduction in TPC, yeast and molds respectively with highest protein hydrolysis of 15.7%. Among the non-thermal treatments HC has shown highest log reduction of TPC at around 1.2 for sample treated at 10 bar pressure, whereas the US treatment was most effective for yeast and mold at 400 W with log reduction of 0.9. A non-Newtonian flow behaviour was observed for all peanut milk samples. Viscosity determined by Herschel-Bulkley equation decreased significantly (p > 0.05) after both cavitation treatments. The US was found to be superior to HC and HTST with improved separation index and colour attributes. Therefore, the US and HC appear to be a remarkable non-thermal processing methods for peanut milk and or any dairy or non-dairy beverages.

1. Introduction The Ultra-sonication (US) and hydrodynamic cavitation (HC) are among the upcoming non-thermal food processing technologies, due to its ability of stimulating physical and chemical changes without heat application in foods [1]. HC technology well known for effluent and water treatments, can be explored for food processing applications. There are various modes to generate cavitation, however, acoustic and HC have been of interest in academics and industry due to simplicity of operation, cost effectiveness, chemical-less procedure and feasibility of generating required intensities and conditions of cavitation appropriate for various chemical and physical alterations in a medium [2,3]. The traditional thermal processing has various ill effects on food products, like denaturation of protein, lactose, loss of vitamins and certain bioactive compounds [4]. HC follows the Bernoulli’s principle, is similar to that of US where the generated cavities expand and collapse in the medium as the pressure recovers, generating a shock wave that affect viability of microorganisms and promote physico-chemical changes in the liquid food systems like fruit juices and dairy products during processing [5]. The intensity required for cavitation with higher

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energy efficacy can be acquired to promote desired changes in the peanut milk by governing operating conditions, controlling US intensities and geometric of the reactor (For HC) [6,7]. In milk products, ultrasonication alone as a non-thermal processing technology is not explored, except few reports without heat application [8,9] and use of low temperatures [10,11]. On the other hand, processing under elevated ultrasonic powers and temperatures has been found to induce physical-chemical degradation and sensory unacceptability of milk and milk products [12,13]. However, homogenization [14], emulsification [15], hydrolysis [16], microbial inactivation [17], crystallization of lactose [18] and extraction [19] are the major applications of US in dairy industry which is also thought to be achieved with HC processing. Around the world, the protein of vegetable source is being consumed as a substitute or extender of animal protein particularly, in developing countries like India traditional protein sources such as poultry, meat and dairy products are expensive and unaffordable [20]. The several studies on peanut milk have reported the use of non-defatted or partially defatted peanut kernels for its production [21–23]. Rubico et al. reported that extracts from non-defatted peanuts when exposed to elevated temperatures i.e. 121 °C for 3 sec while

Corresponding author. E-mail address: [email protected] (S.S. Arya).

https://doi.org/10.1016/j.ultsonch.2019.104728 Received 17 June 2019; Received in revised form 2 August 2019; Accepted 6 August 2019 Available online 07 August 2019 1350-4177/ © 2019 Elsevier B.V. All rights reserved.

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Table 1 HC processing parameters. Pressure (bar)

Volumetric flow rate (lpm)

Throat velocity (m/s)

Cavitation number

Cycle passes for 15 min

6 8 10

4.6 6 7.5

3.9 5.1 6.4

0.06 0.05 0.04

69.2 90 112.5

During the US treatment, the samples were kept in chilled water bath to minimize the temperature rise in the sample. The initial temperature of the peanut milk samples was 30 ± 1 °C. At the end of the cavitation treatment, the maximum rise in the temperature of peanut milk was recorded to be 48 ± 3 °C for US processing.

processing, resulted in a whiter milk with superior textural properties as compared to partially defatted peanuts [24]. The use of full fat peanut kernels not only improved the sensory quality but was also more economical due to elimination of defatting process. Even though the shelf life of the peanut milk might extend by pasteurisation at high temperatures but it will be at the expense of nutrients and quality loss. Hence, there is a need of simple and less expensive processing technologies like US and HC for broader applications like in processing of milk, dairy products, fruit juices and other beverages. The world is now inclined towards production of functional food products, along with development of practical and emerging technologies for processing of various products. The aim of this study is to evaluate the effect of the US power (200, 300 and 400 W for 3 min) and HC (6 bar, 8 bar and 10 bar) at varying intensities as compared to the thermal treatment HTST; 72 °C for 15 s i.e. high temperature short time on peanut milk, assessing the physicochemical and microbiological changes to produce a novel food product with good stability.

2.3.2. HC processing The hydrodynamic cavitating reactor was purchased from Zero-d Industries Pvt. Ltd., Mumbai, India. It includes a holding tank of 2 L capacity volume, a positive displacement pump (reciprocating) 0.5 Hp pump, with a variable frequency drive used in the setup with a max pressure of 12 bar and maximum flow rate of about 600 lph. The initial volume used for the treatment during various processings and respective flow rates with cavitation numbers are mentioned in Table 1. The HC was carried out by passing 1 L of freshly extracted peanut milk through a venturi throat diameter of 5 mm at varying inlet pressure i.e. 6 bar, 8 bar and 10 bar for 15 min. The temperature of every sample was measured using a digital thermometer before and after treatment, and the temperature was not controlled while treating samples thus, any rise in the temperature was a reaction of cavitation process. The initial temperature of the peanut milk samples was 30 ± 1 °C. At the end of the cavitation treatment, the maximum rise in the temperature of peanut milk was recorded to be 45 ± 5 °C for HC.

2. Material and methods 2.1. Materials Peanuts (JL-24 variety) were purchased from Agricultural produce market committee (APMC) market (Mumbai, Maharashtra, India). Sodium hydroxide, phenolphthalein, sodium potassium tartrate, copper sulphate pentahydrate, sodium carbonate sodium chloride, tricholoro acetic acid, sodium phosphate monobasic, sodium phosphate dibasic were obtained from SD Fine-Chem Ltd. (Mumbai, India). Total plate count agar and potato dextrose agar were procured from Hi-media (Mumbai, India).

2.3.3. HRST processing One sample was subjected to HTST treatment, where peanut milk was heated at 72 °C for 15 s on a hot plate, the temperature was monitored with the help of thermometer dipped in peanut milk. 2.4. Determination of pH A regularly calibrated pH meter with pH 4 and pH 7 buffer standards, was used to study effect of processing on pH of the peanut milk samples.

2.2. Preparation of peanut milk The traditional procedure for preparation of peanut milk was followed according to a method described in [21] with modification. Whole skin peanut kernels were cleaned manually, weighed and washed in running water. The kernels were then soaked in water for 18 h (1:3 w/v). The water was then discarded and the peanuts were washed thoroughly. The peanuts were ground to a fine paste in an electric grinder and mixed with 6 volumes of water. This slurry was agitated and filtered through a 2-layered muslin cloth. The milk thus obtained was immediately stored in air tight containers at 4 °C.

2.5. Titratable acidity To determine the effect of treatments on titratable acidity of peanut milk, the samples (10 ml) were mixed with 10 ml deionized water and titrated with 0.1 N sodium hydroxide using phenolphthalein as an indicator [26]. 2.6. Total soluble solids (TSS)

2.3. Sample processing Total soluble solids of the samples was determined using a refractometer at room temperature with Brix scale 0–100 according to [27].

2.3.1. US processing The US treatment was performed using a Digital Sonifier, Model 450 (Bransons Ultrasonic Corporation, Danbury, USA) at 20 kHz, producing mechanical vibrations by the convertor when fed with this high frequency electrical energy. The sonicator was set at increasing amplitude corresponding approximately to the power levels of 200 W, 300 W & 400 W and the exposure time was 3 min for each milk sample according to the method of Guimarães et al. [25]. Increasing the Ultra-sonication time beyond 3 min increased the temperature of milk to 50 °C and above due to acoustic cavitation. The US instrument was equipped with a 13 mm diameter probe. 50 ml of samples were treated at different US intensities in glass bottles with probe dipped 3 mm in the sample.

2.7. Degree of protein hydrolysis The degree of protein hydrolysis (DH) in peanut milk after treatment was determined using TCA method [28] with few modifications. In the first part, 500 µl of hydrolysed samples were mixed with 500 µl of 20% TCA solution. Following the incubation of 30 min at room temperature, the supernatant were centrifuged at 10,000 rpm and the protein content of peanut milk samples was determined by the method of Lowry et al, [29]. DH was determined using following equation: 2

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%Degree of hydrolysis = 100*

soluble protein content in 10% TCA (mg ) total protein (mg )

2.13. Sedimentation index To determine sedimentation index, two 20 ml aliquots of each treatment were immediately transferred to two graduated tubes after processing, capped and were stored at refrigerated condition (4 ± 2 °C). The measurements were recorded after every 24 h until untreated sample showed separation, according to the method described by Silva and Meireles [31], the separation index (SI) was calculated according to the Eq. (5).

(1) 2.8. Microbial inactivation The peanut milk was subjected to microbiological analysis before and after the treatment including one sample treated at 72 °C for 15 s (HTST). The total plate count (TPC) and molds and yeasts were quantified according to the method described by [25]. The estimation of TPC was done on plate count agar and molds and yeast on potato dextrose agar was carried out by spread plate method. The colony forming units (cfu) were counted after incubating at 37 °C for 24 h, and molds and yeasts at 30 °C for 48 h. For the estimation of microbial inactivation, the number of log reductions (γ) was calculated, according to Balasubramanyam et al. [30]. The γ was calculated according to unprocessed sample (N0), which functioned as control, as described in equation

γ = log10 (N0) − log10 (Nf )

HS ⎞ ⎞ *100 SI (%) = ⎛1 − ⎛ ⎝ HT ⎠ ⎠ ⎝ ⎜

⎟

(5)

where, HS is height of the upper phase (mm) and HT is the total height of the beverage (mm) 2.14. Color The Hunter Lab colorimeter (model Color Quest II) and the CIE (Commission internationale de l'éclairage) coordinates: L*a*b* and L*C*H* were used to determine the effect of each treatment on color of peanut milk. To evaluate the difference between color of treated peanut milk samples, the color difference (ΔE*) using each CIE coordinates were calculated, according to the Eqs. (6) and (7).

(2)

where, N0 is the number of microorganisms viable in untreated milk and Nf is the number of microorganisms viable after US and HC processing. 2.9. ζ-potential

* = ΔELab

The ζ-potential was measured to determine the surface charges of the peanut milk processed by US, HC and HTST by a chamber of micro electrophoresis (ZetaSizer Nano-ZS, Malvern Instruments Ltd. Worcestershire, UK). The treated and untreated samples were diluted in deionized water to 50 µl/mL for the estimation in triplicate.

(ΔL*)2 + (Δa*)2 + (Δb*)2

* = ΔELCH

(6)

(ΔL*)2 + (ΔC *)2 + (ΔH *)2

(7)

where, L* represents lightness and darkness, a* red and green, b* blue and yellow, C* chroma, H* hue and δ means the difference between the processed and unprocessed samples.

2.10. Optical microscopy

2.15. Statistical analysis

The microstructure of peanut milk was observed in an optical microscope (AxioScope.A1, CarlZeiss, Germany) before and after each processing, using 100 × objective lens. The software AxioVision Rel. 4.8 (Carl Zeiss, Germany) was used to capture the images.

Statistical analysis of data was performed by ANOVA i.e. analysis of variance using Statistical Package for Social Sciences (SPSS) software for Windows version (16.0). The data was expressed as the mean of triplicate estimations ± standard deviation and at p < 0.05 level, the differences were considered statistically significant. Duncan test was used for pair-wise comparison of outcome variable mean.

2.11. Particle size distribution A Mastersizer 2000 (Malvern Instruments Ltd, Malvern, UK) was used to determine the particle size distribution by the method of laser diffraction, where distilled water was used as dispersant (refractive index = 1.33). The peanut milk was analysed before and after each treatment including the unprocessed milk in triplicate. The d50 was used to evaluate the particle size, which is the diameter of particles at 50% of cumulative volume.

D32 =

∑ ni d3i /∑ ni d2i

3. Result and discussion 3.1. Effect of US and HC on physico-chemical characteristics of peanut milk The effect of increasing intensities of US, HC and HTST on total soluble solids (TSS), titratable acidity and pH of peanut milk are shown in Table 2. US lead to increase in the TSS with the increase in protein content [32]. A similar trend can be observed in this study, where, sonicated sample have showed to have greater TSS from 9.1° Brix

(3)

where ni is the droplets number with diameter di. Table 2 Effect of US and HC processing in physic-chemical properties of peanut milk.

2.12. Rheology The effects of processing treatments i.e. US, HC and HTST was studied on the rheological properties of the peanut milk. The rheology assay was performed immediately before and after each treatment. A stress-controlled rheometer (Anton paar MCR-301, Graz, Australia) using 5 cm stainless steel flat-plate geometry and a 50 µm gap, to obtain flow curves. The shear rate was varied between 0 and 1000 s−1 and measurements were made in duplicate at ambient conditions. The flow curves were obtained to fit the Herschel-Bulkley equation (Eq. (4))

σ = σo + k*γ n

(4)

Treatments

TSS (°)

pH

Untreated US 200 W US 300 W US 400 W HC 6 bar HC 8 bar HC 10 bar HTST

9.1 ± 0.0a 11.4 ± 0.2b 12.5 ± 0.1c 12.9 ± 0.1d 9.0 ± 0.1a 9.1 ± 0.1a 9.1 ± 0.1a 14.2 ± 0.1e

6.6 6.7 6.7 6.7 6.7 6.7 6.7 6.7

Titratable acidity

± ± ± ± ± ± ± ±

0.0a’ 0.1b’ 0.0b’ 0.0b’ 0.0b’ 0.0b’ 0.0b’ 0.0b’

1.08 0.21 0.14 0.13 0.26 0.24 0.24 0.26

± ± ± ± ± ± ± ±

0.01d^ 0.01e^ 0.01a^ 0.01a^ 0.01f^ 0.00f^ 0.01f^ 0.07f^

Protein hydrolysis (%) 8.8 ± 0.1c* 11.2 ± 0.7d* 12.4 ± 0.6b* 10.5 ± 0.1e* 10.8 ± 0.1f* 12 ± 0.1a* 11.9 ± 0.0a* 15.7 ± 0.5g*

All the values are Means ± standard deviations (n = 3). Values within a given column vertically followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test.

where, σ is shear stress (mPa), σo is yield shear stress, k is consistency index (mPa.sn), γ̇ is shear rate (s − 1) and n is flow behaviour index. 3

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3.3. ζ-Potential

(untreated) to 11-12° Brix and HTST to be 14° Brix which could be due to water absorption or partial cooking of peanut particles at high temperature, resulting in swelling of this particles, thus showing significant difference among the treatments. Whereas, the HC treated samples presented no significant difference among them. The shock waves generated by subsequent generation and collapse of bubbles enhanced the penetration of the solvent into the distorted peanut cells; thus, leaching protein out from the cells, resulting into a simultaneous increase in protein content and total soluble solids [33]. A slight increase in the pH of processed peanut milk samples was observed as compared to untreated sample. Also the titratable acidity of peanut milk was observed to decrease with increasing intensity of cavitation for both the treatment (US and HC), which could be due to the change in the charge of the particles induced by cavitation further leading to increase in the negative charge of ζ-potential as indicated in Table 2. As ζ-potential becomes more negative, pH increases and ζ-potential becomes more positive as pH decreases [34]. It can be observed in Table 2 that the protein hydrolysis of the peanut milk increased (around 1–2%) in the US and HC treated samples when treated at 400 W, 8 and 10 bar respectively. This may be due to formation of bubbles and collapse which gives thermal, mechanical (turbulences, shear stress and collapse pressure) and chemical (generation of free radicals) effect leading to breakdown of cell walls and thus leaching out of intercellular protein takes place which increase the protein content [32,33]. Also, the protein hydrolysis of treated sample increased by 2–5% in US and 2–3% in HC processing (Table 2).

ζ-Potential gives the electrical potential difference between the mobile dispersion medium and the stationary layer of the dispersion medium attached to the dispersed particle which signifies the physical stability of the product [35]. Higher ζ-potential (over 60 mV) either negative or positive gives excellent stability, below 20 mV has limited stability and further below at 5 mV results in aggregation and coagulation [36]. As observed in Table 3, the ζ-potential of the ultrasonic treatment significantly increased from −27.6 mV (untreated) to −30 mV (US 300 W). This indicates more stability of the treated peanut milk which can also be confirmed by the separation index (Fig. 3). The US intensity of 400 W leads to instability with significantly reduced ζpotential (−11 mV) forming aggregates due to super cavitation. At lower intensities due to increase in the negative charge there might be stronger interparticle electrostatic repulsions, improving the stability of particles by inhibiting the aggregation of particles [32]. Whereas no significant difference in the ζ-potential was observed in the HC and HTST treated sample.

3.4. Optical microscopy The effect on the microstructures of the peanut milk processed by US (200, 300 and 400 W), HC (6 bar, 8 bar, and 10 bar pressure) and HTST can be seen in Fig. 1. A dispersed phase and a continuous phase are the two main components of milk. In some dairy products, the continuous phase is primarily comprised of water and other soluble substances, which cannot be observed using an optical microscope. Whereas, the dispersed phase is comprised of fat globules, milk proteins and lactose. In the presented study since the milk is prepared from a protein rich plant source (peanuts), the dispersed phase is comprised of fat globules, peanut protein and disrupted peanut cells. Peanuts contain 40–45% fat [37], which was partially extracted while preparation of milk along with protein. Since no prior treatment was given to the milk, the effect of treatments could be assessed by these components. In this study, on characterising peanut milk in optical microscopy, the structures that might be presence of protein micelles which were < 1 µm, fat globules fell in the range of 0.1–2 µm depending on the type and intensity of treatment, disrupted and fragmented peanut cells and other debris of 5–10 µm size. Particles bigger than 20 µm could not be seen at 100X magnification, however were identified and quantified by laser diffraction of particle size analyser. As it can be observed in Fig. 1a, the untreated or unprocessed milk presented fat globules of bigger size, similar observations could be made from HTST sample (Fig. 1.h), where the fat globules were unaffected by the thermal treatment. In sonicated samples, the intensity of 200 W for 3 min (Fig. 1b), slightly reduced the size of fat globules and further significantly reduced with the subsequent treatment of higher intensities of 300 (Fig. 1c) and 400 W (Fig. 1d), indicative of the imparted effect on peanut milk. This effect resulted in more homogenized milk as justified by the particle size distribution results also by the fact that the

3.2. Microbial inactivation Log reduction of HTST pasteurization, US and HC is shown in Table 3. It is seen that HTST sample have 1.53 log reduction in total plate count (TPC) and around 2 log reduction in yeast and mold. The cavitation and sonication have similar log reduction in terms of TPC (1.07–1.19) and very less reduction for yeast and mould (0.3–0.9) (Table 3). The increase in the US intensities and operating pressures of HC lead to an increase in the log reduction of microbes. The sound waves and the constriction in the liquid flow develop cavities which created shock waves and high shear stresses resulting in collapse of the bubble, localised high temperature and pressure which leads to disruption of cell walls of microorganisms [3,25]. As per industry standards the log reduction should be around 5 for effective pasteurization, which is high as compared to the obtained results. However further increase in nominal powers of US and pressure of HC might lead to aggregation, flocculation and log reduction [32]. Therefore, it is suggested that for improved microbial inactivation, cavitation is carried out at higher intensities for longer durations or can be used in combination with other conventional techniques such as thermization. Also, cavitation has good homogenization and emulsion effect as shown in the image given by optical microscope at 100X, it can be used as pretreatment process which will enhance the effect of other thermal processes.

Table 3 Microbial inactivation of total plate count, yeast and molds and ζ-potential after US and HC cavitation treatment. Treatments

AMHB(γ)

Yeast and Molds

ζ-potential (mV)

Outlet temperature (°C)

Untreated US 200 W US 300 W US 400 W HC 6 bar HC 8 bar HC 10 bar HTST

– 1.12 1.12 1.13 1.07 1.12 1.19 1.53

– 0.7 ± 0.1a’ 0.5 ± 0.1ac’ 0.9 ± 0.1ab’ 0.2 ± 0.0d’ 0.6 ± 0.0a’ 0.3 ± 0.0c’ 1.95 ± 0.1e’

−27.6 −28.2 −30.1 −11.0 −27.7 −26.2 −27.9 −27.7

– 44.5 ± 0.06a* 46 ± 1.00d* 47.6 ± 0.91bd* 45.2 ± 0.08bc* 48.7 ± 1.10e* 50.1 ± 0.93e* 72.0 ± 0.4f*

± ± ± ± ± ± ±

0.08a 0.11a 0.10a 0.09a 0.06a 0.05a 0.02b

± ± ± ± ± ± ± ±

a^

1.0 1.0a^ 0.9c^ 0.8b^ 0.5a^ 1.0a^ 0.9a^ 1.0a^

All the values are Means ± standard deviations (n = 3). Values within a given column vertically followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test. 4

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Fig. 1. Optical microscopy images of peanut milk processed by US (200, 300 and 400 W), HC (6 bar, 8 bar, and 10 bar pressure) and HTST. Where a; Unprocessed peanut milk, b; US 200 W, c; US 300 W, d; US 400 W, e; HC 6 bar, f; HC 8 bar, g; HC 10 bar and h; HTST. The scale bar represents 10 μm.

5

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3.6. Rheology

sedimentation index of these samples was better than the untreated and HTST. Hence bigger particles or structures and be effectively reduced by employing US of high nominal powers. A similar trend was observed for HC treated samples, where 6 bar HC (Fig. 1e) treated sample showed hardly reduced fat globules. Whereas for subsequent higher pressures of 8 (Fig. 1f) and 10 bar (Fig. 1g), the fat globule size was significantly reduced, indicative of the intensity of treatment resulting in even more homogenized milk. These results are in agreement with reports of Marchesini et al. [13], where milk somatic cells counts was substantially reduced with increasing US nominal powers and duration. In another study Rojas et al. [38] observed increased US energy inputs resulting in increased destruction of fruit pulp cells. This is attributed to the intense shear rates produced by HC and US treatment, improving the gelling network and intermolecular interactions and forces. In another study by Silva and Meireles [31], ultra-sonication improved the degree of polymerization of inulin in emulsions of annatto seed oil thus increasing its encapsulation ability and emulsion stability.

The US (400, 600 and 200 W), HC (6, 8 and 10 bar) and HTST influenced the viscosity of peanut milk significantly (Fig. 2B) as compared to the untreated milk (5.54 mPa.s). The flow behaviour of peanut milk was of non-Newtonian fluid for all samples. Viscosity was estimated with a Herschel–Bulkley equation. The viscosity was observed to decrease with increasing nominal powers of US, a comparable pattern was observed for the flow index of US treated samples with no significant difference among 200 W and 300 W treated samples which further decreased significantly at 400 W. The consistency and flow behavior index were also greatly influenced by all three treatments (Fig. 2A). The consistency index (K) was found to be highest for the untreated peanut milk (89.75) which reduced greatly to 0.47 for US treated samples however, was further observed to increase significantly with increasing nominal powers of US. For US treatment the consistency index was inversely correlated with viscosity and flow index. In HC treated samples the viscosity and consistency index were reduced greatly as compared to the untreated however, no significant difference was found in the viscosity of samples treated at pressure intensities of 6 and 10 bar which further increased to 1.79 at pressure of 8 bar. The consistency index was also highest (7.21), whereas flow index was had minimum value of 0.619 for peanut milk treated at 8 bar pressure. The HTST treatment greatly reduced the viscosity and consistency index, whereas increased the flow index as compared to untreated milk. The US and HC affects the milk by disrupting the arrangement and breaking the bigger particles into smaller ones. The rheological changes incorporated by the cavitation treatment made peanut milk less viscous, more fluid and less pseudo plastic or shear thinning behaviour. The thermal energy is also known to disrupt the structure of beverages, destabilise the particles and the interactions occurring between them [61], whereas ultrasonic and HC affected the microstructure of particles hence, altering its conformation and size. In another study on rheological properties of extracted soy milk, reported by Fahmi et al. [33], a Newtonian flow behaviour was observed for all soy milk samples. The viscosity of soy milk was estimated with a power-law equation and was found to increase slightly on ultrasound cavitation. The consistency index was unaffected, whereas the flow-behaviour index was found to increase significantly. Gao et al. [41], investigated the effect of US on reconstituted skim milk, and reported reduction in particle size and viscosity after processing at 170–2040 − J mL·1 energy densities. The viscosity and the size of casein micelles in the milk was observed to decrease due to high intensity US and its ability to break the κ-casein by sonication. Also reduction in the size of whey proteins was reported initiated by acoustic cavitation, indicating denaturation of these proteins. However, on the contrary, Martini and Walsh [42] reported increased viscosity after ultrasonic cavitation while investigating its effect on liquid whey at energy density of 270 − J mL·1.

3.5. Particle size analysis Peanut milk is a complex emulsion, hence it’s difficult to identify the type of particles influenced by US and HC processing. Though, the fact that peanut milk was freshly extracted and no prior treatment was given to the milk, the reduction in the size of fat globules observed in optical microscopy could be due to the effect of US and high intensity HC. Also reduction in size of fat globule is a common characteristic of US and HC [39]. The particle average size in Table 4 is represented by the d50 parameter, which was observed to be decreasing after US processing as compared to the untreated, HTST and HC treated peanut milk. There was no significant difference observed in the particle size of untreated peanut milk (0.18 µm) and US of 200 W, however decreasing dramatically on processing at 300 W and 400 W, bringing the particle size (d50) to as small as 0.02 µm at 400 W. A similar trend was observe for HC treated peanut milk where, high intensity HC at 10 bar resulted in d50 of 0.16 µm, significantly lesser than the treated at 6 and 8 bar respectively. This is commonly due to the breakdown of particles caused by the high intensities of ultra-sonocation and hydrodynamic cavitation effect [40]. On the contrary the HTST treated sample showed d50 of 0.37 µm, which is highest among all the samples including untreated, this effect can be attributed to the high temperature of thermal processing, where the particles are swelling by absorbing water from the surrounding matrix, as also observed during cooking of peanuts. For instance, the observed results can be correlated with the separation index as depicted in Fig. 3A and B. it can be observed at high intensities of US and pressure, the particle size is reduced and well distributed forming a matrix of smaller suspended particles taking longer time to settle and show separation, unlike the untreated where the bigger particles tend to separate. Similar observations were reported by [25,38] where particle size was observed to decrease with increasing intensities of US processing.

Table 4 Particle size and rheological parameters of US, HC and HTST treated peanut milk. Treatment

d50 (µm)

Untreated US 200 W US 300 W US 400 W HC 6 bar HC 8 bar HC 10 bar HTST

0.29 0.22 0.21 0.02 0.25 0.20 0.16 0.37

± ± ± ± ± ± ± ±

0.05a 0.03ab 0.02b 0.00c 0.04ab 0.03ab 0.02e 0.05d

Viscosity at 100 s-1 (mPa.s) 5.54 2.39 2.42 1.84 1.73 1.79 1.73 1.96

± ± ± ± ± ± ± ±

0.02a’ 0.01e’ 0.02e’ 0.00f’ 0.01b’ 0.01c’ 0.01b’ 0.01d’

K (mPa.s)

n (–)

89.75 ± 0.1a^ 0.47 ± 0.02e^ 0.62 ± 0.02b^ 2.02 ± 0.09c^ 0.62 ± 0.02b^ 7.21 ± 0.04f^ 2.25 ± 0.03 d^ 3.75 ± 0.05g^

0.338 0.939 0.950 0.761 0.955 0.619 0.820 0.845

R2 ± ± ± ± ± ± ± ±

0.007a* 0.011b* 0.008b* 0.006c* 0.01b* 0.09d* 0.01 e* 0.01f*

0.99 0.99 0.99 0.99 0.99 0.99 0.99 0.99

All the values are Means ± standard deviations (n = 3). Values within a given column vertically followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test. 6

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Fig. 2. Graphical representation of the influence of US, HC and HTST treatments on flow behaviour (A) and Viscosity (B) of processed peanut milk. Table 5 Effect of US, HC and HTST treatments on colour of peanut milk. Treatments

L*

Untreated US 200 W US 300 W US 400 W HC 6 bar HC 8 bar HC 10 bar HTST

80.5 80.5 78.2 81.2 78.5 79.7 78.8 78.2

a* ± ± ± ± ± ± ± ±

0.1a 0.6a 0.4b 0.3a 0.6b 0.3ab 0.2b 0.2b

−0.4 −0.5 −1.1 −0.6 −0.5 −0.6 −0.6 −1.1

b* ± ± ± ± ± ± ± ±

0.0a 0.1b 0.0c 0.0b 0.1b 0.0b 0.0b 0.0c

5.8 5.7 3.7 5.1 3.8 3.6 4.8 3.7

C* ± ± ± ± ± ± ± ±

0.1a 0.4a 0.2b 0.1c 0.3b 0.2b 0.1d 0.1b

5.8 5.7 3.8 5.1 3.9 3.7 4.8 3.8

ΔE*lab

H* ± ± ± ± ± ± ± ±

0.1a 0.4a 0.2 d 0.1b 0.3d 0.2d 0.1c 0.1d

178.5 178.5 178.7 178.6 178.6 178.6 178.6 178.7

± ± ± ± ± ± ± ±

0.0a 0.0a 0.0c 0.0b 0.0b 0.0b 0.0b 0.0c

– 0.7 3.2 1.2 3.0 2.4 1.9 3.2

± ± ± ± ± ± ±

ΔE*LCH

0.1a 0.1b 0.3c 0.4b 0.0d 0.1e 0.1b

– 0.7 3.0 1.2 3.0 2.4 1.9 3.0

± ± ± ± ± ± ±

0.1a 0.1b 0.3c 0.4b 0.1d 0.1e 0.1b

All the values are Means ± standard deviations (n = 3). Values within a given column vertically followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test. 7

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e

f

g 8

h (caption on next page)

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Fig. 3. Sedimentation index of peanut milk after 48 h of low temperature storage (6 ± 2 °C): (A) Sedimentation index of peanut milk where, a’, b’, c’ and d’ represent statistical difference and (B) in sequence photos representing the separation of phases of all treatments. Where a; Unprocessed peanut milk, b; US 200 W, c; US 300 W, d; US 400 W, e; HC 6 bar, f; HC 8 bar, g; HC 10 bar and h; HTST.

phase separation. According to the Fig. 3A, the untreated was the most unstable milk, followed by HC 8 bar, HC 6 bar and HTST. Moreover, the increasing intensities of sonication did not influence the stability of peanut milk, this findings are similar with the results recorded by Guimarães et al. [25] where intensification of ultrasound homogenization did not affect the separation index of whey protein beverage added with inulin. The enhancement of sedimentation index in the US treated and high pressure HC is due multiple reasons like the decrease caused in the particles size, facilitating the intermolecular reactions. Also the denaturation of peanut proteins promoted the unfolding of peanut protein molecules disclosing their active sites and increasing the surface hydrophobicity of molecules [12]. In another study by Silva and Meireles [31], inulin and polysaccharides like gelan gum was used to enhance stability of annatto seed oil emulsions, however the intensification of ultrasound homogenization did not improve the sedimentation index. The reports of Shanmugam and Ashokkumar [40] explains the emulsification capacity of cavitation, where stability was delivered to the flaxseed oil emulsion droplets in milk with just 20% of moderately denatured whey proteins, after processing at 633–1689 J mL−1 for pasteurizing skim milk. However, in the presented study, all the processed and unprocessed samples showed a small separation of particles in a form of a band in the bottom of each tube (Fig. 3B) which could be due to the settling of bigger particles or agglomerates of peanut. There are few studies assessing the efficacy of cavitation on stability of beverages and even lesser in non-dairy milks like soy and peanut milk. However comparison can be done with a highpressure homogenization (HPH), which is another emerging technology for processing of beverages and milk, where the high shear forces increases water holding capacity of particles, formation of hydrogen bonds and van der Waals interactions between particles to form homogeneous networks [44].

3.7. Color Table 5, shows the effect of US, HC and HTST processing on the colour of peanut milk by analysing parameters such as L* (lightness or luminescence), a* (redness, green- red), b* (yellowness; blue- yellow), C* (Chroma), H* (hue), ΔE*Lab (color difference due to L*a*b*) and ΔE*LCH (color difference due to L*C*H*). On analysing L* value, no significant difference was observed among the US and untreated milk, except for one treated at power intensity of 300 W, which was slightly darker among other US treated samples. The HC treated samples were darker as compared to untreated, however HTST and all HC treated samples were statistically similar. The a* value was observed to increase significantly at varying processing methods as compared to the untreated. However no significant difference was observed among the increasing intensities of US and increasing pressure of HC, whereas at 300 W the milk samples were more towards green (−1.1) similar with that of HTST. The b* and C* value on contrary was observed to decrease with increasing intensities making samples appear duller than the untreated. For H*, was observed slightly increased values in US, HC and HTST samples as compared to unprocessed, showing that all processing treatments falls counter clock wise in the L*C*H* color space, signifying that the treated samples on comparing with untreated had a reduced amount of redness. The ΔE* parameter is suggestive of the overall effect each treatment had on peanut milk compared to unprocessed milk. According to the ΔE*lab values and ΔE*LCH, samples treated at US 300 W, HC 6 bar and HTST were statistically similar to each other but different from other samples. Nonetheless, the obtained values L*a*b* (< 3.2) and L*C*H*(< 3.0) were slightly higher mainly in US 300 W, HC 6 bar and HTST samples, making the color detectable to the human eye (Fig. 3). Also the parameters like Chroma (color intensity) and hue angle are important in relating the color changes in peanut milk which is otherwise unremarkable. The lightness or luminescence (L* value) for any milk is primarily associated to the existence of casein micelles, in our case peanut protein and fat globules [43], which showed slight difference, suggestive of the influence of US, HC and HTST treatments on these components. The differences in a*, b*, C* and H* might be due to the other peanut components like polyphenols, flavonoids, skin pigments etc. which present in considerable amounts in peanuts [37]. According to Rojas et al. [38], the ultrasonic processing of any plant material has an unpredictable response, with respect to the cell rupture, pigment stability and release of intercellular substances. The obtained results for colour analysis are in line with the findings of [25] where the effect of ultra-sonocation and HTST processing was studied on the prebiotic whey beverage. However there are no reports of effect on colour by HC processing of any milk.

4. Conclusion The studied non-thermal processing technologies i.e. high intensity US and HC for processing of peanut milk are interesting, as there is scope to improve microbial inactivation capacity at high energy densities and durations, additionally they impart better disruption of peanut cells, increases hydrolysed protein content of milk, better sedimentation index avoiding the phase separation and improved microstructure with smaller particle and fat globule size. The flow behaviour of peanut milk was found to be of non-Newtonian fluid with decreased viscosity and greater consistency. Hydrodynamic cavitation method should be considered and explored beyond effluent treatments and extractions, for food and beverage processing. However, advance studies with respect to safety of HC treated food for consumption must be evaluated, also the effect of US and HC on nutritional composition and bioactive profile of peanut milk must be studied before processing on a commercial level.

3.8. Sedimentation index The effect of HTST, US and HC processing at different intensities and pressure on sedimentation index of peanut milk is presented in Fig. 3A and B. All the treated and untreated samples were stable after 24 h of cold storage so were stored for 24 h more to monitor the separation. After 48 h, as shown in the Fig. 3B, a phase separation was observed for untreated (a), HC 6 bar (e), HC 8 bar (f) and HTST (h) treated samples indicating their instability. In Fig. 3A, a graphical representation of the separation index of various treatments has been attempted, where bar graphs for separation index of US 200 W, 300 W, 400 W and HC 10 bar are absent as there was no separation observed. This has been supported by Fig. 3 B where, the pictures of the tubes showing separation of the phases. All sonicated peanut milk samples (b, c, and d) and HC 10 bar (g) processed were stable and presented no

Acknowledgments Authors acknowledge the University Grants Commission, New Delhi, Government of India, for providing financial support under UGCBSR Fellowship Award No. F.4-1/2006(BSR)/5-62/2007(BSR). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ultsonch.2019.104728. 9

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