Effect of high-pressure microfluidization on nutritional quality of carrot (Daucus carota L.) juice

Abstract

In this study, the effect of high-pressure microfluidization on the colour and nutritional qualities of the orange carrot juice was investigated. The juice was processed at three different pressures (34.47 MPa, 68.95 MPa and 103.42 MPa) with three different passes (1, 2 and 3 passes). After that, total phenolic content (TPC), antioxidant activity, carotenoids, color properties, and total soluble solids content of the processed carrot juice were evaluated. As a result, no specific trends in TPC and antioxidant activity of the juice were observed through the variations of processing conditions. However, microfluidization significantly (p < 0.05) improved the carotenoids content in carrot juice. With increasing number of pass, concentrations of β-carotene and lutein had increased significantly. Similarly, increasing process pressure initially increased carotenoid content significantly (up to 68.95 MPa), further increase pressure to 103.42 MPa did not cause significant changes in carotenoid concentration. Furthermore, color properties such as lightness, redness, yellowness, and chroma value were reduced significantly with the increase of pressure and the number of passes. The results indicated that high-pressure microfluidization could be used as a novel alternative nonthermal technology to heat pasteurization to improve the color and nutritional qualities in orange carrot juice, resulting in a desirable, high-quality juice for consumers.

Introduction

Carrot (Daucus carota L.) is a widely cultivated vegetable that gained popularity due to its attractive nutritional value. It is a rich source of both lipophilic (carotenoids and xanthophyll) and hydrophilic (phenolics) antioxidants. The predominant lipophilic antioxidant in carrot is β-carotene, which has multiple health benefits such as pro-vitamin A activity, prevention of cardiovascular disease and cataract. Apart from β-carotene, lutein is another antioxidant predominantly present in orange and yellow carrot, which plays a crucial role in the prevention of age-related macular degeneration (Alves-Rodrigues and Shao 2004) and reducing the risk of atherosclerosis (Dwyer et al. 2001). The rich amount of hydrophilic phenolic antioxidants in carrot also offers a wide range of health-promoting properties such as anticancer, anti-atherogenic, anti-inflammatory and antimicrobial (Koley et al. 2017).

Carrot is consumed either raw as a salad ingredient or processed into various products. Among different kinds of processed products, carrot juice is one of the preferred products by consumers. From the safety point of view, thermal pasteurization is commonly used in conventional processing techniques to inactivate the inherent enzymes and pathogenic microorganisms. However, heat processing often adversely affects the color, flavor and nutritional compounds of final processed products. Therefore, the processing industry is continually looking for novel nonthermal processing technologies. Several attempts have been made to apply nonthermal processing techniques in producing carrot juice. For example, Park et al. (2002) have reported that combined treatment of high-pressure carbon dioxide and high hydrostatic pressure efficiently inactivates the enzymes' polyphenol oxidase, lipoxygenase, and pectin methylesterase. Similarly, Van Loey and Hendrickx (2004) have reported that high-pressure treatment had increased the antioxidant potentiality of carrot juice. In another study, Quitão-Teixeira et al. (2008) reported high energy pulsed electric fields as an efficient nonthermal technology for inactivation of peroxidase enzyme and color preservation of carrot juice. Researchers also have used high-pressure homogenization techniques which inactivate the E. coli growth in carrot juice (Pathanibul et al. 2009). However, these studies were primarily focusing on the inactivation of enzymes and pathogenic microorganisms, not the quality of carrot juice. Recently, Jabbar et al. (2014) have reported that sonication treatment increased the carotenoids, chlorogenic acid, sugar, and some minerals and enhanced microbial safety of carrot juice, but combined treatment with blanching was recommended to achieve the best results.

Recently, the use of microfluidization as a nonthermal processing technique has increased. Microfluidization is a novel high shear processing technology, which creates micro- or nano-sized particles by utilizing a fixed-geometry interaction chamber to efficiently convert high pressure into the combination of high shear and impact forces, high energy dissipation as well as hydrodynamic cavitation (Panagiotou et al. 2008). Presently, this technology is used in food technology for preparation of nanoemulsion (Jafari et al. 2007) and liposome (Thompson and Singh 2006), for increasing bioaccessibility of carotenoids (Cha et al. 2012), improvement of dietary fiber (Chen et al. 2013), and more recently for improvement of techno-functional properties of pea protein isolates suspensions (Oliete et al. 2017). In addition to this, few attempts have also been made to use the high-pressure microfluidization process as a cold processing technique for fruit juice processing but not yet fully explored in the literature. For example, Karacam et al. (2015) have reported that the microfluidization process increased phenolic content and antioxidant activity and improved the color properties of strawberry juice. In another study, Wu et al. (2016) have reported that the process increased total soluble solids, clarity and improved the color properties of red jujube juice.

Till now, a limited study is available in the literature about the effect of microfluidization, a slightly different technique of high-pressure homogenization on color and nutritional values of carrot juice. Earlier, Yu and Rupasinghe (2014) studied the effect of high-pressure homogenization along with other processing techniques on carrot juice quality. However, the author did not investigate the effect of high-pressure homogenization alone on total soluble solids or β-carotene. In very recent times, Liu et al. (2019) reported that high-pressure homogenization improved the bioavailability of the total carotenoids. However, the author did not study the effect of this technique on individual carotenoids. Therefore, this study seeks to provide a technical basis for the application of microfluidization technology in carrot juice processing. The objective of the present study was to evaluate the effect of high-pressure microfluidization process at three different pressures (34.47 MPa, 68.95 MPa, and 103.42 MPa) with three different numbers of passes (1, 2 and 3) on the color and nutritional properties i.e. total phenolics, β-carotene, lutein and antioxidant potentiality of the orange carrot juice.

Material and methodology

Juice preparation

The orange carrot (Daucus carota L.) was collected from the local market. After that, the root was brought to the laboratory followed by thoroughly washing with water and peeling with a stainless peeler. After that, the peeled root was passed through the juicer (Inalsa, India). To remove the solid particles, the extracted juice was filtered through a muslin cloth. For pasteurization of the juice samples, the method proposed by Yu and Rupasinghe (2014) was followed with slight modification.: the juice was heated to 90 °C for a holding time of 1 min in a water bath. The temperature was measured using a digital thermometer. After heating the juice was hot filled in pre-sterilized glass bottles, capped with sterilized caps and cooled immediately. The bottles were stored at 4 °C until further analysis. Three replicates were used for all the experiments and fresh, untreated juice was used as a control.

Chemical reagents

All chemical reagents used were of analytical grade. ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] (Pub Chem CID: 5360881), neocuproine (Pub Chem CID: 65237), Trolox (Pub Chem CID: 40634), β-carotene and lutein were purchased from Sigma-Aldrich, Bengaluru, India.

High pressure microfluidization process

250 mL of the juice was placed in a 0.3 L reservoir and processed with a high-pressure Microfluidizer (Model M-110P, Microfluidics Inc., Westwood, MA, USA) equipped with a Z-type interaction chamber (100 µm). The samples were treated at three different pressures (34.47 MPa, 68.95 MPa, and 103.42 MPa) with three different numbers of passes (1, 2 and 3). To prevent the excess heating of juice and the destruction of bioactive compounds, cooling water was circulated through a pump connected to the interaction chamber. The temperature of the juice was measured before and after every pass and a temperature of not more than 49 °C was recorded in the whole experiment (Table 1). After each treatment, the samples were collected in sterile bottles, immediately cooled to 30 °C and stored at 4 °C for further qualitative analysis.

Table 1.

Change in temperature of juice during microfluidization

Processing treatment	Initial temperature (°C)	Final temperature (°C)
Control	22 ± 0.87e	22 ± 0.73f
Pasteurization (90 °C 1 min−1)	21 ± 0.52e	–
MF5000-1	20 ± 0.63f	40.5 ± 0.78e
MF5000-2	40.5 ± 0.67d	42 ± 0.84de
MF5000-3	42 ± 0.51cd	43.5 ± 0.68d
MF10000-1	21.5 ± 0.83e	43 ± 0.71d
MF10000-2	43 ± 0.49c	45.5 ± 0.95b
MF10000-3	45 ± 0.91b	46.5 ± 0.83b
MF15000-1	20 ± 0.68f	45 ± 0.58c
MF15000-2	45 ± 0.54b	48 ± 0.69a
MF15000-3	48 ± 0.73a	49 ± 0.58a

Data are expressed as mean ± standard deviation of triplicate samples

Different superscripts in the same column represent significant differences between samples (p < 0.05)

Determination of total phenolic content (TPC)

The total phenolic content of carrot juice was determined using Folin–Ciocalteu reagent (Singleton et al. 1999). An aliquot of 100 μL carrot juice was mixed with deionized water (2.9 mL), Folin–Ciocalteu reagent (0.5 mL), and 20% Na₂CO₃ (2.0 mL) solution. The mixture was allowed to stand for 90 min. Then the absorption was measured at 760 nm against a reagent blank in UV–vis spectrophotometer (Varian Cary 50, Agilent Technologies, Australia). Results were expressed as gallic acid equivalent (GAE) in milligrams per 100 grams fresh weight (mg GAE/100 g fw).

Antioxidant activity

Cupric reducing antioxidant capacity (CUPRAC)

In this method (Apak et al. 2008), 100 μL aliquot of carrot juice was mixed with 1 mL each of 10 ⁻² mol CuCl₂, 7.5 × 10⁻³ mol neocuproine alcoholic solution, 1 mol ammonium acetate (pH 7.0) buffer solution. Finally, 1 mL of water was added to make the final volume to 4.1 mL. The reagent mix was kept at ambient temperature (25 °C) for 30 min. After that, the absorbance was measured using UV–vis spectrophotometer at 450 nm against the reagent blank. Antioxidant capacity was expressed as μmol TE/g fw using molar absorptivity of Trolox 1.67 × 10⁴.

Trolox equivalent antioxidant capacity (TEAC)

The TEAC was measured by the method proposed by Re et al. (1999). The ABTS^·+ free radical (7 mmol) was prepared by mixing 7 mmol ABTS with 2.45 mmol of potassium persulphate, which acted as an oxidizing agent. An aliquot (30 μL) of carrot juice was mixed to 270 μL of ABTS^·+ radical, and the absorbance was measured at 734 nm, immediately after mixing and exactly 10 min after initial mixing. Similar to the method used for DPPH radical, the percentage inhibition of ABTS^·+ radical was calculated and results were expressed as μmol TE/g fw.

Carotenoid extraction and HPLC procedure

The extraction method was adapted from Saha et al. (2015) with little modifications. Briefly, 3 mL juice was partitioned using hexane: acetone (1: 1, v/v) mixture and then shaken vigorously. The organic layer was separated and collected with a second tube. The process of extraction was repeated thrice. The entire organic layers were pooled together, and the solvent was evaporated using a vacuum rotary evaporator (Heidolph, Germany) at 40 °C. The dried mass was dissolved in tertiary butyl methyl ether (MTBE), passed through a 0.45 μm syringe filter and injected in HPLC for analysis. All procedures were carried out at diffused light exposure.

Estimation of carotenoids

Extracts obtained by solvent extraction were analyzed using HPLC for lutein, and β-carotene content determination. The HPLC had following parts: 600 quaternary pump, an auto-injector with 20 μL loop, 2998 photodiode array detector (Waters Corp., Milford, MA, USA) and 5-μm C30 YMC column (size 250 × 4.6 mm) (YMC Co. Ltd., Ireland). 2 mL of sample was injected into HPLC. Before injection, the sample solution was filtered through a 0.4 μm nylon filter. The mobile phase was comprised of an isocratic mixture of MTBE: methanol (80:20, v/v) and it was run at a flow rate of 1 mL per min. Carotenoids were measured at a wavelength of 450 nm. “Empower 2” software was used to determination of the concentration of carotenoids using peak areas.

Color

The color parameter was calculated as the method followed by Koley et al. (2014). A colorimeter (Labscan XE colorimeter, HunterLab, Inc., Reston, VA, USA) was used to measure color coordinates in the L*a*b* color space (CIELAB). The instrument was calibrated with a standard white and black plate. Initially, L* (lightness), a* (redness) and b* (yellowness) were measured. Then hue and chroma were calculated by using the following Eqs. (1, 2):

$$\text{Hue angle}={tan}^{-1}\left(b^{\ast }/a^{\ast }\right)$$ 1

$$\text{Chroma}=\sqrt{a^{\ast 2}+b^{\ast 2}}$$ 2

Fresh carrot juice (L*_ref = 46.16, a*_ref = 20.79, b*_ref = 26.62) was used as the reference. Total color change (ΔE) was calculated from the following formula:

$$\mathrm{\Delta }\text{E}=\sqrt{{\left(L^{\ast }-L_{\mathit{ref}}^{\ast }\right)}^2+{\left(a^{\ast }-a_{\mathit{ref}}^{\ast }\right)}^2+{\left(b^{\ast }-b_{\mathit{ref}}^{\ast }\right)}^2}.$$ 3

Total soluble solids content (TSS) and pH

A refractometer (Atago, Tokyo, Japan) was used to measure the total soluble solids of the carrot juice. Results were expressed as °Brix (20 °C). The pH of the juice was estimated by pH meter (pH Tutor Bench Meter, EU Tech instrument).

Microbiological analyses

The microbiological study of fresh carrot juice, microfluidized and pasteurized juices was done immediately after treatment by appropriate serial dilution agar plate technique where 0.1% sterile peptone water was used for dilution. The presence of spoilage causing microorganisms in the untreated juice and those microbes which survived the heat and pressure treatment was investigated. The diluted samples were plated on plate count agar for total colony count and incubated at 30 °C for 72 h. Lactic acid bacteria were counted on de Man–Rogosa–Sharpe nutrient broth (MRS) after incubation at 30 °C for 72 h. Total yeast counts were enumerated with Malt Extract Agar (MEA) medium after incubation at 25 °C for 72 h. The results were presented as a log value of colony-forming units per mL of carrot juice (log10 CFU mL⁻¹) (Hammad et al. 2013).

Statistical analysis

Results are presented as the mean ± standard deviation of three technical replications. Analysis of variance (ANOVA) was performed (p < 0.05) by using SAS JMP10 Pro and means were separated by Tukey’s test with a confidence coefficient of 0.95 (α = 0.05).

Results and discussion

Phenolic content

Phenolic compounds are the primary contributor to the hydrophilic antioxidant potentiality of carrot. In the present study, the TPC values of all carrot juice samples were within the range from 10.77 to 16.24 mg GAE/100 mL (Table 2). Similar values were reported by Koley et al. (2014). A significant difference was observed between the phenolic content of fresh and pasteurized juice, which might be due to the loss of phenolic compounds due to oxidation during heat treatment. Similar to our study, Ma et al. (2013) observed also considerable losses in phenolics in carrot juice pasteurized by thermal means. They suggested that phenolics of carrot juice as affected by pasteurization process could be the consequence of thermal degradation (oxidation or breakdown) and consumption in the mallard reaction pathway. In addition to this, the isomerization reaction might be one of the causes of the reduction of phenolic content during pasteurization process. No clear trends in phenolic content of juice were observed due to changes in pressure or the number of pass upon the microfluidization process. Karacam et al. (2015) reported similar results at a low processing pressure of 60 MPa that no significant changes were observed in TPC of strawberry juice after microfluidization treatment of up to 5 passes. However, in their study, the microfluidization process increased TPC at a higher pressure of 100 MPa. The difference might be due to the difference in inherent phenolic content, the nature of phenolic compounds and relative abundance of the phenolic degrading enzyme.

Table 2.

Effect of microfluidization on total phenolic content (TPC) and antioxidant activities of carrot juice

Processing treatment	TPC	TEAC	CUPRAC	β-carotene	Lutein
Control	15.61 ± 0.41ab	1.06 ± 0.04abc	2.71 ± 0.18ab	12.98 ± 0.88g	5.03 ± 0.35i
Pasteurization (90 °C 1 min−1)	13.99 ± 0.26cde	1.01 ± 0.10bc	2.53 ± 0.16abcd	46.79 ± 1.57d	9.99 ± 0.45fg
MF5000-1	10.77 ± 0.77f	1.00 ± 0.06bc	2.20 ± 0.19cd	24.98 ± 1.26f	7.78 ± 0.37h
MF5000-2	14.46 ± 0.57bc	1.10 ± 0.02a	2.60 ± 0.12abc	50.42 ± 2.05cd	13.21 ± 0.58cd
MF5000-3	12.79 ± 0.45de	1.24 ± 0.08ab	2.63 ± 0.17abc	71.74 ± 2.95b	15.67 ± 0.65b
MF10000-1	14.18 ± 0.44bcd	1.26 ± 0.07a	2.68 ± 0.12ab	34.37 ± 2.01e	7.62 ± 0.37h
MF10000-2	16.24 ± 0.69a	1.08 ± 0.08abc	2.86 ± 0.14a	57.31 ± 2.92c	12.60 ± 0.59de
MF10000-3	14.91 ± 0.55abc	1.09 ± 0.09ab	2.50 ± 0.16abcd	81.80 ± 3.96a	18.01 ± 0.68a
MF15000-1	10.77 ± 0.57f	0.90 ± 0.07bc	2.13 ± 0.11d	37.91 ± 2.03e	8.90 ± 0.30gh
MF15000-2	12.59 ± 0.39e	1.09 ± 0.11abc	2.41 ± 0.20abcd	52.01 ± 3.05cd	11.21 ± 0.59ef
MF15000-3	13.89 ± 0.33cde	0.89 ± 0.04c	2.27 ± 0.19bcd	66.62 ± 3.00b	14.48 ± 0.58bc

Data are expressed as mean ± standard deviation of triplicate samples

Different superscripts in the same column represent significant differences between samples (p < 0.05)

TPC was expressed in mg GAE/100 mL; Antioxidant activity for TEAC and CUPRAC was expressed in the common unit of µmol Trolox Equivalent/mL; β-carotene and lutein was expressed in µg mL⁻¹

Antioxidant activity

As there is no single versatile method that can assess the ‘total antioxidant capacity’ of food accurately and quantitatively (Koley et al. 2017), therefore, one in vitro electron transfer (ET) based assays, CUPRAC was used. Additionally, another in vitro method that is based on the hydrogen atom transfer (HAT) and ET, TEAC was also used. All antioxidant activity results are given in Table 2. In the CUPRAC assay, the mean antioxidant activity of fresh juice was 2.71 µmolTE/mL. No significant differences were observed between fresh, pasteurized and microfluidized juice except few samples (MF at 34.47 MPa with 1 pass and MF at 103.42 MPa with 1 pass). In the TEAC assay, the mean antioxidant activity of the fresh juice was 1.06 µmol TE/mL. Again, no significant differences were observed between fresh, pasteurized and microfluidized juice in antioxidant activity. Among the two methods, the value in the CUPRAC assay was the highest. In addition to this, the highest correlation has been observed between TPC and CUPRAC (r = 0.688). Thus CUPRAC methods might be the most suitable method for the determination of the antioxidant activity of carrot juice. Other authors have also reported CUPRAC method as the best method due to its rapid reaction kinetics (Apak et al. 2008). Thus, from present experiments, it can be concluded that the microfluidization process does not affect the hydrophilic antioxidant potentiality of carrot juice which might be due to its minimal effect on total phenolic content. Similar to the present study, Suárez-Jacobo et al. (2011) reported that high-pressure homogenization technique did not change antioxidant capacities in apple juice measured by FRAP and DPPH assays. Likewise, Velázquez-Estrada et al. (2013) have reported that high-pressure homogenization did not affect the antioxidant activities of orange juice using the TEAC assay. On the contrary, Karacam et al. (2015) have reported that microfluidization treatment at higher pressure (100 Mpa) improved the antioxidant activity of strawberry juice. The difference might be due to the nature and amount of cell wall-bound hydrophilic antioxidants and the amount of pressure applied, which can inactivate the phenol degrading enzyme in the case of strawberry juice.

Carotenoids

Carotenoids are the primary lipophilic antioxidant in the carrot that performs many health-promoting functions in the human body. Carotenoid content in carrot juice was assessed using HPLC for fresh, pasteurized and microfluidized samples to assess the influence of processing conditions on carotenoids. The major carotenoids in carrot juice identified using HPLC were β-carotene and lutein (Fig. 1). Beta-carotene was the abundant form of carotene in carrot juice. About 72% of the total carotene in fresh carrot juice was β-carotene, and the rest was lutein (28%). Table 2 shows measured β-carotene and lutein content in fresh, pasteurized and microfluidized juice under different pressures (34.47 MPa, 68.95 MPa, and 103.42 MPa) and the number of the passes (1, 2 and 3). Pasteurization caused a significant increase in both carotenes, which is possibly due to increased mobility of carotenoids from cell-matrix after heat treatment. Generally, crystalline chromoplasts were found in tissues of raw carrots juice; high-temperature pasteurization might result in the formation of yellow-colored lipid droplets containing dissolved carotene. In this context, Purcell et al. (1969) hypothesized that upon heating carotenes are solubilized by cellular lipids which are released after the thermal breakdown of cell structure. The lipid content of carrots is about 0.2–0.5 mg kg⁻¹. The amount of lipids may suffice at least for partial dissolution of carotenes (Marx et al. 2003). Therefore, high temperature-induced dissolution of carotenoids followed by their release in solution after the thermal breakdown of cell structure may be the reason for higher carotenoids concentration of pasteurized juice.

Fig. 1.

HPLC chromatogram carrot juice and photodiode array spectra of the main carotenoids in carrot Peaks: 1, lutein; 2, β-carotene

It was interesting to observe that variations of microfluidization process pressure and the number of passes have a significant effect on carotenoid concentration (Table 2). For all tested pressures, concentrations of both β-carotene and lutein increased with the increasing number of pass. However, comparing the results of increasing process pressure, both carotenoid contents increased initially up to the pressure of 68.95 MPa. Further increasing pressure to 103.32 MPa did not improve concentrations of both carotenoids. All juice samples processed through microfluidization had a significant increase in both carotenoid contents compare to fresh juice. Furthermore, after three, in some cases two, passes, both carotenoid contents were significantly higher than pasteurized juice. The optimum results were obtained with processing through microfluidization at 68.95 MPa for 3 passes. Available β-carotene concentrations increased 530% (13–82 µg mL⁻¹) from fresh juice and 74% (47–82 µg mL⁻¹) from pasteurized juice, while lutein concentrations increased 260% (5–18 µg mL⁻¹) and 80% (10–18 µg mL⁻¹), respectively. The increased release of carotenoids in microfluidized juice might be explained by the inherent nature of carrot carotenoids. Carrot root carotenoids occur in chromoplast as pure pigment crystal surrounded by the membrane to form a carotene body, whereas in other crops such as pepper, pumpkin, mango, papaya carotenoids are carried in carotenoid-carrying lipid droplets of globules chromoplast (Arscott and Tanumihardjo 2010; Schweiggert et al. 2012).

Due to localization and their crystal structure, carotenoids in fresh and pasteurized juice may not be entirely solubilized in solvents. During the microfluidization process, the high shear and impact forces might help to disrupt the membranes of the carotene body and break the pigment crystal, thus allowed the release of cellular materials including pigments such as β-carotene and lutein. Similar to the present finding, high-pressure microfluidization was found to increase the detectable lycopene levels in ketchup samples (Mert 2012). In another study, Cha et al. (2012) reported that although the microfluidization process had no significant effect on carotenoids content in C. ellipsoidea, the process had increased the bioaccessibility of carotenoids. Since carotenoids played a vital role in human nutrition and carrot is one of the best sources for carotenoids (Arscott and Tanumihardjo 2010), the microfluidization process could be one of the novel techniques to improve the bio-accessibility of carrot juice.

Color parameter

Color is often considered as an essential nutritional indicator since it is often associated with the number and amount of pigments present in the food product. In the present experiment, pasteurization treatment reduces the lightness of the carrot juice (Table 3). Prominent changes had also been observed on lightness with changes in the number of pass for the microfluidization process: the increasing number of pass decreased the lightness values. The lightness dropped from an initial value of 46.16–43.13 units signifying an increase in the color strength. However, no significant changes in lightness were observed with the changes of processing pressure. Similar to lightness, redness, yellowness, and chroma values were significantly reduced with increasing the number of passes, but not with increasing pressure (Table 3). Again, most microfluidized juice samples and samples obtained with a higher number of passes showed significant changes in the L*a*b* color space compared to fresh and pasteurized carrot juice, respectively. Contrary to the decrease in the L*a*b* color value, the hue value increased after microfluidization. All result indicated that carrot juice treated with microfluidization is more turbid, less saturated in red and yellow, and less intense in color. Similar to the present experiment, Karacam et al. (2015) reported that the microfluidization process decreased the lightness of the strawberry juice. The increase in the redness and yellowness after microfluidization in their study was likely due to the presence of different pigments with different fiber matrix in strawberry. However, in the present studies, reduction in the values of L*, a*, b*, and chroma with the increasing number of pass might be due to an increase in turbidity of carrot juice. Turbidity might be occurred due to repeated fragmentation of soluble fiber (Zhang et al. 2016). Besides this, microfluidization might enhance the transformation of all trans carotenoids into cis isomers, enhance their oxidation or modifies the interaction of non-pigmented compounds with pigmented carotenoids (Zepka et al. 2009). Moreover, there are possibilities of production of other carotenoids which ultimately influenced the color properties. Therefore, in the future, more detail studies are essential to identify pigmented and non pigmented carotenoids after microfluidization. Cserhalmi et al. (2006) proposed the classification based on the color difference (ΔE) as not noticeable (0–0.5), slightly noticeable (0.5–1.5), noticeable (1.5–3.0), well visible (3.0–6.0) and great (6.0–12.0). Based on their classification, pasteurized juice and microfluidized juice at lower pressure or number of pass showed noticeable variation in color. However, juice processed through the microfluidization at higher pressure or number of pass showed a well visible difference in color.

Table 3.

Effect of microfluidization on color properties of carrot juice

Processing treatment	Lightness	Redness	Yellowness	Hue	Chroma	ΔE
Control	46.16 ± 0.14a	20.79 ± 0.14a	26.62 ± 0.27a	52.01 ± 0.41f	33.77 ± 0.17a	–
Pasteurization (90 °C 1 min−1)	44.88 ± 0.11c	19.47 ± 0.11b	25.16 ± 0.09bcd	52.27 ± 0.26ef	31.81 ± 0.02b	2.38 ± 0.40de
MF5000-1	45.84 ± 0.16ab	19.31 ± 0.16b	25.55 ± 0.24bcd	52.92 ± 0.28ef	32.02 ± 0.23b	1.89 ± 0.10e
MF5000-2	45.32 ± 0.19abc	19.15 ± 0.19b	25.40 ± 0.18bcd	52.99 ± 0.35e	31.81 ± 0.17b	2.25 ± 0.10de
MF5000-3	44.92 ± 0.16bc	18.04 ± 0.16de	25.04 ± 0.19cd	54.23 ± 0.30d	30.86 ± 0.18de	3.43 ± 0.09c
MF10000-1	45.16 ± 0.12bc	18.54 ± 0.12c	25.76 ± 0.12b	54.25 ± 0.13cd	31.74 ± 0.15b	2.62 ± 0.13d
MF10000-2	44.59 ± 0.03cd	17.89 ± 0.03e	25.46 ± 0.35bcd	54.91 ± 0.32bcd	31.12 ± 0.30cd	3.51 ± 0.13c
MF10000-3	43.82 ± 0.22de	16.79 ± 0.22g	24.91 ± 0.27de	56.02 ± 0.60a	30.04 ± 0.15fg	4.96 ± 0.13b
MF15000-1	45.27 ± 0.06abc	18.37 ± 0.06cd	25.59 ± 0.23bc	54.33 ± 0.17cd	31.50 ± 0.22bc	2.79 ± 0.08d
MF15000-2	44.37 ± 0.26cd	17.35 ± 0.26f	24.95 ± 0.26cde	55.19 ± 0.12abc	30.39 ± 0.36ef	4.47 ± 0.31b
MF15000-3	43.13 ± 0.07e	16.62 ± 0.07g	24.34 ± 0.25e	55.66 ± 0.33ab	29.47 ± 0.19g	5.64 ± 0.18a

Data are expressed as mean ± standard deviation of triplicate samples

Different superscripts in the same column represent significant differences between samples (p < 0.05)

Total soluble solids and pH

No noticeable difference was observed in the pH value of fresh and microfluidized juice (Table 4). Similarly, no difference was observed in TSS between fresh, pasteurized and most of the microfluidized juice sample. However, TSS has significantly increased in juice microfluidized at higher pressure, i.e. 68.95 MPa and 103.42 MPa, with three passes. Soluble solids include soluble sugar, fiber, and proteins which might not be affected by pasteurization and microfluidization at lower pressure. On the other hand, the microfluidization process at a higher pressure increased the rate and efficiency of rupturing the cell walls and helped release more soluble solids. Karacam et al. (2015) reported similar results of a slight increase of soluble solids in strawberry juice when subjected to the microfluidization process at 60 MPa. However, Suárez-Jacobo et al. (2011) have reported no changes in TSS in apple juice after high-pressure homogenization treatment. The difference in results may be attributed to the original soluble solid contents in different samples and the amount of pressure and number of pass required to rupture the cell walls of various plant samples.

Table 4.

Effect of microfluidization on total soluble solids (TSS) and pH value of carrot juice

Processing treatment	TSS (°Brix)	pH
Control	7.10 ± 0.09c	6.43 ± 0.03bc
Pasteurization (90 °C 1 min−1)	7.13 ± 0.13bc	6.55 ± 0.02a
MF5000-1	7.21 ± 0.06abc	6.39 ± 0.04c
MF5000-2	7.27 ± 0.09abc	6.41 ± 0.02c
MF5000-3	7.17 ± 0.07abc	6.43 ± 0.02bc
MF10000-1	7.30 ± 0.09abc	6.43 ± 0.03bc
MF10000-2	7.23 ± 0.08abc	6.46 ± 0.04bc
MF10000-3	7.40 ± 0.08ab	6.50 ± 0.04ab
MF15000-1	7.13 ± 0.13abc	6.48 ± 0.03abc
MF15000-2	7.20 ± 0.11abc	6.46 ± 0.02bc
MF15000-3	7.43 ± 0.12a	6.46 ± 0.03bc

Data are expressed as mean ± standard deviation of triplicate samples

Different superscripts in the same column represent significant differences between samples (p < 0.05)

Microbiological analyses

The effect of microfluidization on the microbiological qualities of fresh carrot juice is shown in Table 5. The initial log viable count of the total aerobic bacteria was 5.8 CFU mL⁻¹. The log initial populations of lactic acid bacteria and yeasts were 4.6 and 3.9 CFU mL⁻¹, respectively. Results revealed a significant reduction in the initial microbial counts after pasteurization and microfluidization. Also, the reduction was remarkably accelerated by higher microfluidization pressure and increased passes (Table 5). Pasteurization of fresh carrot juice significantly eliminated the microbial load below the detectable limit whereas reduction through microfluidization was dependent on pressure and number of passes. Microfluidization pressure of 103.42 MPa could eliminate all microorganisms contaminating fresh carrot juice. However, different passes were required for different microbes. In general, all the microbial counts in microfluidized samples were significantly lower than those of untreated ones.

Table 5.

Effect of pasteurization and micro fluidization on the microbial counts (log₁₀ CFU mL⁻¹) contaminating carrot juice

Treatment	Total colony count	Lactic acid bacteria	Yeast
Control	5.8	4.6	3.9
Pasteurization (90 °C 1 min−1)	ND	ND	ND
MF5000-1	5.1	3.9	3.0
MF5000-2	4.7	3.2	2.6
MF5000-3	3.9	2.7	1.9
MF10000-1	3.7	2.0	1.1
MF10000-2	3.0	1.8	ND
MF10000-3	2.6	1.0	ND
MF15000-1	1.9	ND	ND
MF15000-2	1.4	ND	ND
MF15000-3	ND	ND	ND

The data obtained in our study is in agreement with Wuytack et al. (2002), and Diels and Michiels (2006). These authors supported the hypothesis that in a multi-pass treatment the effect of each pass is additive; therefore, each homogenization pass causes the same reduction of the microbial load. The effectiveness of the multiple pass approach in reducing the microbial load is also elaborated by Maresca et al. (2011) who has studied the effect of high-pressure homogenization on the microbial load of different fruit juices. In their study, they showed a significant reduction in microbial load at comparatively lower pressure after multiple passes of juice through the homogenizer. On the contrary, Patrignani et al. (2009) showed a non-additive trend for multiple pass processes at a given pressure level in their study on apricot and carrot juices. They described the type of food matrix, its pH, composition, and viscosity as the main factors affecting the efficiency of pressure treatment. Further, the level of reduction depends on the type of microbes present in the matrix, pressure treatment and storage temperature of the product. The effect of pressure treatment on cell number is explained by different postulates where mechanical destruction of the cell integrity through cavitation and free radical oxidation mainly leads to the inactivation of microbes (Diels and Michiels 2006).

Conclusion

The carrot juice was processed by high-pressure microfluidization, and various quality parameters were analyzed after processing. The microfluidization process did not affect TPC and antioxidant potentiality of carrot juice. A high correlation between TPC and antioxidant capacity (CUPRAC) was detected for processed samples. However, the microfluidization process significantly improved the content of lipophilic antioxidants, β-carotene, and lutein, in produced carrot juice. CIE L*a*b* color values significantly changed for both pressures of 68.95 MPa and 103.42 MPa with increasing pass numbers. A slight increase in TSS in carrot juice was observed. Results suggested that a high-pressure microfluidization process can be a novel alternative non-thermal processing technology for carrot juice production. The optimum processing pressure of 68.95 MPa with three passes is recommended for producing high-quality carrot juice with desired properties. The positive effect on β-carotene, lutein, and TSS is might be results of high shear force and impact during the process of microfluidization. The process might disrupt the cellular membrane, membrane of carotene body, break the pigment crystal and thus allowed the release of cellular materials including pigments and other solutes. A more detail study must be carried out to understand the mechanism of the increase of carotenoids through microfluidization.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.