1. Introduction
A fruit- and vegetable-rich diet has a positive impact on human health and wellbeing due to the presence of functional and bioactive compounds, such as phenolic antioxidants, carotenoids, vitamins, and flavonoids [
1]. The World Health Organization recommends an introduction of at least 400 g of fruit and vegetables per day in adults [
2]. Therefore, their consumption as juices, rather than fresh products, is moving in this direction [
3].
The growing consumer interest in healthier food and drinks is projecting that the global juice market will increase in the next years. Among vegetable-based products, carrot juice is one of the most popular non-alcoholic beverages consumed in northern Europe [
4]. It is a natural source of antioxidants, such as α- and β-carotene, the precursors of vitamin A and polyacetylene, with anti-tumor properties [
4,
5,
6,
7]. Because of its high pH and high sugar content, spoiling and pathogenic microorganisms can easily grow, affecting the shelf life and safety of the product [
8,
9]. For this reason, fresh carrot juice should be consumed one–two days from production [
10]. To extend its shelf life, thermal treatments are commonly applied at the industrial level. However, other than being energetically unsustainable, these treatments may result in undesirable biochemical and nutritional changes, with negative impacts on their sensory properties (for example, pH, taste, and color) [
11]. Another possibility is using chemical preservatives, however, this does not always lead to satisfactory results and are not well accepted by consumers [
12].
The need for more sustainable approaches has favored the development of non-thermal technologies with the aim to preserve functional and sensorial characteristics of products while guaranteeing their microbial stability [
13,
14,
15,
16,
17]. Among them, high-pressure homogenization (HPH) can significantly reduce both naturally occurring or intentionally added microorganisms, improving the safety and shelf life of the products [
16]. Moreover, it induces physical matrix modifications (changes in pH, viscosity, and particle dimension) [
18,
19], increasing [
20]—or not altering—the presence of functional compounds [
21]. This is a very important aspect regarding food differentiation.
Other than physical treatments, addition of natural antimicrobials (essential oils) [
22,
23] or biocontrol cultures [
9,
24] have also been proposed to replace chemical preservatives. From an industrial point of view, use of lactic acid bacteria to ferment vegetable and non-dairy beverages is gaining more and more interest [
25,
26]. In fact, fermentations that apply tailored bacteria represent a fundamental tool to increase safety and shelf life, as well as preserving and increasing the functionality and the sensorial properties of vegetable drinks and juices [
25,
27,
28,
29]. Siroli et al. [
9] described the potential of three nisin-producing
Lactococcus lactis strains (LBG2, 3LC39, and FBG1P) as a tool to stabilize fermented carrot juice and soymilk from a microbiological point of view. In particular, strain LBG2, having a rapid fermentation kinetic (pH reduction) and a high nisin production in carrot juice, exerted a strong anti-listeria activity and improved the sensory profile of pre-heated carrot juice. On the other hand, the bacteriocin nisin, the first authorized to be used as a natural food preservative, is a natural antimicrobial with a wide range of actions [
30,
31]. While numerous studies have shown the antimicrobial properties of nisin deliberately added in vegetable juices, only a few of them have reported the use of biocontrol strains to produce nisin in situ [
9].
Since, to our knowledge, there are no studies exploring the combination of these two sustainable approaches, the main objective of this study was to investigate the combined effect of HPH and biocontrol culture (L. lactis LBG2) on carrot juice quality and stability. The shelf life of the products obtained was evaluated at two different temperatures of storage (4 and 10 °C). In particular, the growth kinetic of the indigenous microbes (total mesophiles, yeasts, and coliforms) and the viability of LBG2 (when applied) were followed over time during the storage period. Moreover, color, volatile molecule profiles, and carotenoids were measured throughout the storage period of the carrot juice.
2. Materials and Methods
2.1. Carrot Juice
Carrot juice was prepared using fresh carrots, as described by Siroli et al. [
9]. Briefly, carrots were steeped in a solution containing 100 ppm of Sodium hypochlorite for 2 min for sanitization [
32]. Then, they were wiped up, sliced, and placed in a domestic extractor (Russel Hobbs). The resulting extract was collected in a sterile flask. All the trials were conducted using three biological replicates (n = 3).
2.2. Selection of the Appropriate High-Pressure Homogenization Treatment (HPH)
The carrot juice, prepared as reported above, was immediately subjected to HPH treatment using a PANDA high-pressure homogenizer (GEA, Parma, Italy), able to reach 220 MPa, and provided with a C and a R-type valve and a thermal exchanger. The valve assembly comprised a ceramic ball-type impact head, a stainless steel large inner diameter impact ring, and a carbide passage head made of tungsten. The homogenizer was previously washed with 1% NaOH water solution, hot water, and finally refrigerated sterilized water. The test was carried out by setting two different juice inlet temperatures: 25 and 50 °C.
Two liters of product, at the two different temperatures, were then subjected to different HPH treatments: 0.1 MPa and 150 MPa for 1, 3, and 5 passes. The HPH passes were carried out in the presence of a thermal exchanger to avoid temperature increase caused by homogenization treatment. The samples subjected to the different treatments were then collected in sterile containers and the total microbial load was determined immediately after the treatment. Decimal dilutions were distributed in plate count agar (PCA) plates (Oxoid, Milan, Italy) and colonies were counted after 48 h incubation at 30 °C.
2.3. Shelf-Life Assessment of Carrot Juice Considering the Combination of HPH Treatment and Biocontrol Agent
In these trials, non-thermal treatments such as HPH and fermentation with a nisin-producing Lactococcus lactis strain (LBG2) were combined to stabilize carrot juice. Samples were divided into three groups: (i) control; (ii) HPH; (iii) HPH plus LBG2. For the carotenoid measurements, pasteurized (72 °C for 15 min) carrot juice was also considered.
2.3.1. HPH Treatment
Two liters of raw organic carrot juice was subjected to HPH treatment at 0.1 MPa (used as control) or 150 MPa for 3 passes (selected based on the previous trial). All the HPH treatments were performed according to the methodology reported above. The inlet temperature of the juice was 25 °C and the HPH passes were carried out in the presence of a thermal exchanger. The controls and treated samples were collected in sterilized glass bottles prior to the shelf-life study.
2.3.2. Fermentation Agent
The biocontrol
L. lactis LBG2 belonged to the Culture Collection of the Department of Agricultural and Food Sciences, University of Bologna. This strain, isolated from cow milk, is a nisin Z-producer [
9]. The strain was also previously characterized for fermentative potential in milk, soymilk, and carrot juice, and for nisin production, antimicrobial activity, and modification of the volatile molecules of milk, soymilk, and carrot juice [
9]. The strain was preliminarily grown in M17 broth (Oxoid, Milan, Italy) for 24 h at 30 °C, then refreshed two times in M17 broth for 24 h at 20 °C before the fermentation trials. The proper samples were inoculated with the biocontrol at a concentration of 10
6 cfu/mL and then left to ferment for 7 h at 30 °C prior to the shelf-life assessment.
2.4. Shelf-Life Assessment
All the samples were stored at two different temperatures, 4 and 10 °C, and followed for 12 and 7 days, respectively. According to the temperature applied, aliquots were collected over time (2, 6, 9, and 12 or 1, 2, 5, and 7 days, respectively) for microbiological, sensorial, and chemical analyses.
2.5. Microbiological Analyses, pH, Nisin Concentration
Cell loads of yeasts, total coliforms, total mesophiles, and lattococci (or L. lactis LBG2) were determined by plate counting on yeast peptone dextrose (YPD) (Oxoid, Milan, Italy), violet red bile lactose agar (VRBA) (Oxoid, Milan, Italy), PCA, and M17, respectively. Decimal dilutions of the samples, performed in Ringer solution [0.9% (w/v) NaCl], were inoculated in Petri dishes incubated 48 h at 30 °C for YPD, M17, PCA, and 24 h at 37 °C for VRBA. The pH was measured by using a pH-meter Basic 20 (Crison Instruments, Barcelona, Spain).
Nisin activity determination was performed by the agar well diffusion method as described by Siroli et al. [
9].
2.6. Color Analysis
Color was measured by a Minolta
® CR-400 colorimeter (Milan, Italy) using the CIELab scale and Illuminant D65. The instrument was calibrated with a white tile (
L* 98.03,
a* −0.23,
b* 2.05) before the measurements. Results were expressed as
L*,
a*, and
b*.
ΔE (total color difference) was calculated according to the following Formula:
2.7. Volatile Molecule Profiles
The volatile molecule profiles were detected with GC/MS/SPME technique, as described by Siroli et al. [
9]. Briefly, the samples were analyzed immediately after the treatments and after the different storage periods until reaching the shelf life. A CAR/PDMS, 75 μm fiber (SUPELCO, Bellafonte, PA, USA) was used to perform the solid-phase microextraction (SPME). The samples (5 mL) were placed in vials and incubated for 10 min at 45 °C. Then the fiber was exposed to the vial headspace for 30 min at 45 °C. The volatile molecules adsorbed were desorbed in the gas chromatograph (GC) injector port in splitless mode at 250 °C for 10 min. The headspace of the volatile compounds was analyzed using Gas-Chromatography (GC) 6890N, Network GC System with mass spectrometry (MS) 5970 MSD (Agilent Hewlett–Packard, Geneva, Switzerland). The column used was J & W CP-Wax 52 CB (50 m × 320 μm × 1.2 μm). The initial temperature was 40 °C for 1 min and then was increased by 4.5 °C/min up to 65 °C. After that, the temperature increased by 10 °C/min up to 230 °C and remained at this temperature for 17 min. Compounds were identified by comparison based on a NIST 11 (National Institute of Standards and Technology) database. Gas carrier was helium at 1.0 mL/min flow.
2.8. Carotenoid Content
2.8.1. Extraction of Carotenoids from Carrot Juice
Carotenoids were extracted from carrot juice samples according to Purkiewicz et al. [
33], with some modifications. Briefly, a volume of 0.5 mL of juice was transferred to a 10-mL Teflon screw cap glass tube with 1.5 mL of n-hexane, 1.5 mL of acetone, and 5 mL of a 10% (
w/
v) sodium chloride solution used to avoid the formation of an emulsion. The mixture was then stirred on a vortex stirrer for 10 s and centrifuged at 662×
g for 2 min. The organic supernatant fraction was transferred to a second tube, and the extraction procedure was repeated four times more on the residual phase with 1.5 mL of n-hexane each time. The pooled organic extracts were washed with 2 mL of water, stirred for 10 s, and then centrifuged at 662×
g for 2 min. The separated hexane phase was moved to a 100-mL flat bottom flask, dried under reduce pressure in a rotary evaporator (bath temperature: 25 °C), kept under a nitrogen flow for 30 s, dissolved in 3 mL of acetone, transferred after a brief stirring in two 1.5-mL PP centrifuge tube, and kept at −18 °C until HPLC analyses (up to three days). Solvents were of analytical grade and purchased from Merck (Darmstadt, Germany).
2.8.2. Determination of Carotenoids by High-Performance Liquid Chromatography (HPLC)
Analyses were carried out on a HPLC apparatus from Jasco (Tokyo, Japan), equipped with two binary pumps (mod. PU-1580), a diode array UV-VIS detector (mod. MD-1510, quartz flow cell, optical path: 10 mm), and an autosampler (mod. AS-2055 Plus). Data were processed by the software ChromNAV (ver. 1.16.02) from Jasco. The solvent system consisted of two mobile phases: (A) water, (B) acetone; both solvents purchased from Merck were of chromatographic grade, filtered (0.45 μm), and degassed prior their use. The gradient program was the following: 0–5 min, 35% A; 5–9 min, 35 to 10% A; 9–12 min, 10% A; 12–14 min, 10 to 0% A; 14–17 min, 0% A; 17–19 min, 0 to 35% A; 19–30 min, 35% A as post run (total method time: 30 min). The flow rate and the injection volume were 0.8 mL/min and 5 μL, respectively. Chromatograms were acquired at 450 nm, whereas absorption spectra were recorded from 400 to 650 nm. Compound separation was performed by a Kinetex 2.6 μ C18 100A column (75 × 4.6 mm i.d., particle size: 2.6 μm) equipped with a guard cartridge Gemini-NX (4.0 × 3 mm i.d.), both from Phenomenex (Torrance, CA, USA). Colum temperature was maintained at 30 °C throughout analyses. Before HPLC determination, extracts were centrifuged at 15,000×
g for 3 min at 10 °C and then filtered in HPLC amber glass vials through RC syringe filters (diameter: 13 mm; pore dimension: 0.45 μm) from GVS Filter Technology (Indianapolis, IN, USA). Compound identification was assessed comparing peak retention times with those of a standard compound (β-carotene) and considering the results illustrated by Purkiewicz et al. [
33]. External standard mode was applied as a quantification method, constructing a β-carotene calibration curve using a range between 0.00025–0.04909 mg/mL (eight calibration points,
r > 0.99). Lutein was quantified using β-carotene as a reference compound at the following concentration levels: 0.00025–0.00491 mg/mL (five calibration points,
r > 0.99). The limit of detection (LOD) and the limit of quantification (LOQ) of the method for β-carotene were 0.00010 and 0.00023 mg/mL of juice, respectively.
2.9. Statistical Analysis
Microbial cell loads, color, and volatile profiles were analyzed using the one-way ANOVA option of Statistica software (v. 8.0; StatSoft, Tulsa, OK, USA). The significance of data obtained was evaluated using ANOVA followed by LSD test at p < 0.05. The volatile molecule profiles were analyzed using a principal component analysis (PCA) performed by Statistica software (v 8.0; StatSoft, Tulsa, OK, USA).
4. Discussion
Over the past years, application of the non-thermal treatment HPH has been studied to improve shelf life and organoleptic and functional properties of vegetable and fruit juices [
8,
19,
22,
34,
35]. Another approach that has drawn the attention of researchers is the use of natural antimicrobials (such as essential oils or bacteriocins) [
22,
23] or biocontrol cultures [
9,
24]. Nevertheless, no data on their possible combined effect on fruit juice shelf life and functionality have been published yet. In this work, the microbial stability of extremely perishable carrot juice and its functionality were monitored for 12 and 7 days (stored at 4 and 10 °C, respectively) upon HPH treatment alone or in combination with a fermentation step with the biocontrol agent
L. lactis LBG2.
As already demonstrated in many publications [
17,
34,
35,
36], HPH treatment at 150 MPa reduces the naturally occurring microflora present in vegetable juice. In this study, a reduction ranging between 1.0 and 2.4 log cycles was observed for TMC depending on the number of HPH passes and inlet temperature of the treated juice. For instance, Patrignani et al. [
16] showed a decrease of yeast of about 2.0 log CFU/mL in kiwi juice following an HPH treatment at 200 MPa for two passes. Moreover, Patrignani et al. [
22] showed a reduction of three log cycles following an HPH treatment at 200 MPa × two passes on apple juice deliberately inoculated with
Saccharomyces cerevisiae at a level of 4.8 log CFU/mL. The HPH potential to inactivate microorganisms depends on both internal (chemical-physical characteristics of the matrix and microbial sensitivity) and external factors (HPH operational procedure) [
17,
37,
38]. Among the external factors, pressure degree and number of passes play an important role as much as the temperature generated during the dynamic pressure applied. In fact, it is estimated that the sample is subjected to an increase of around 2 °C/10 MPa during homogenization. Although for short treatment periods temperature increases were not observed [
39,
40], in the present work a thermal exchanger was applied to maintain temperature at 25 °C. For what concerns the number of passes through HPH at 150 MPa, the data obtained showed an additive antimicrobial effect when increasing the number of HPH passes applied but without linearity in terms of reduction of microbial load. Literature data concerning the additive effect of the number of HPH passes on microbial deactivation are contradictory. Some authors report a limited microbial deactivation following multiple HPH passes and have attributed this trend to the physiological diversity of microbial populations and the presence of resistant cells from the original microbiota of the matrix able to survive at high pressures [
41].
The HPH treatment with the selected parameters (150 MPa × three passes) determined an average reduction of about 1.6, 1.3, and 2.0 log CFU/mL for TMC, yeasts, and total coliforms, respectively. This initial reduction extended the shelf life of carrot juice. In fact, the spoilage threshold limit of TMC and yeasts in vegetable juices is usually considered to be 6.0 log CFU/mL [
42,
43]. These limits were exceeded in HPH-treated samples only for TMC after nine and five days when stored at 4 and 10 °C, respectively, while controls exceeded the limit after six and two days at 4 and 10 °C, respectively.
For what concerns color parameters, HPH increased a* and b* values while it decreased the L* value. The overall ΔE was 6.8 CIELAB units, in line with what was reported by Szczepańska et al. [
34], considering the high variability observed in our study. The effect of HPH on juice color seems strongly dependent on food matrices and treatments. In fact, Calligarsi et al. [
44] reported an increase in L* and b* values as well as a decrease in a* values upon HPH treatment on banana juice. Zhou et al. [
19] observed a luminosity loss and an increase of red color intensity in mango juice after HPH, while Tribst et al. [
45] reported a decrease of L* and a* values of mango nectar after HPH. Despite the initial variation due to HPH treatment which may be determined by oxidative process, color variations were less significant than those observed for the control over time. In fact, at the end of the storage period, HPH samples had higher L*, a*, and b* values than controls. According to literature data, HPH treatment does not impact the level of total carotenoids, especially at pressures ranging from 20 to 150 MPa [
46]. Even Szczepańska et al. [
36] reported that the level of total carotenoids can only be increased by applying 150 MPa, or higher pressures, for longer time (four passes or more). However, they also observed a different behavior depending on the type of carotenoid. For instance, 150 MPa with four passes increased β-carotene concentration but reduced lutein. In our work, 150 MPa for three passes reduced the concentration of β-carotene and lutein with respect to the untreated samples. Although starting from a lower concentration, storage at 4 °C did not further reduce the total carotenoids measured during the 12 days considered. On the other hand, storage at 10 °C promoted a degradation process from day two until day seven, following a first-order reaction [
46]. However, the sum of β-carotene and lutein, at the beginning and at the end of the storage, was still within the range of 30 to 300 mg/L given by the AlJN Code of Practice as a reference value for carrot juice and purees [
47].
To boost HPH effects and extend carrot juice shelf life, a fermentation step was performed upon HPH treatment using the biocontrol agent
L. lactis LBG2. The selection of the biocontrol agent applied in this work was based on the results reported by Siroli et al. [
9] that showed the good acidifying and nisin-producing capacity of
L. lactis LBG2 on the same substrate. However, in this work the fermentation process by LBG2 strain was optimized by reducing fermentation times from 24 to 7 h. In fact, samples treated with HPH and fermented by LBG2 showed a drop in pH to 4.6 and an increase of the biocontrol agent up to 9.0 log CFU/mL after 7 h of fermentation. The rapid fermentation kinetic represents an important feature applicable at an industrial level to reduce energy and working costs [
48]. Moreover, lowering the pH, production of organic acids, and bacteriocins can prevent possible contamination by undesirable microorganisms, such as spoilage and pathogens [
49]. In this regard, the implementation of the fermentation step with LBG2 did not change the microbial profile already observed immediately after HPH treatment but it exerted an important effect on the microbial stability of the juices during their storage. In fact, using the combined treatments, microbiological shelf life of carrot juice was extended to more than 12 and 7 days when stored at 4 and 10 °C, respectively. In fact, the acceptance threshold for TMC and yeasts, reported as 6.0 log CFU/mL for vegetable juices [
42,
43], have never been overcome during storage, either at 4 or 10 °C. The observed antimicrobial effect is also correlated to nisin production by LBG2. In fact, at the end of the fermentation process, the presence of 13 mg/L of nisin in fermented carrot juice was determined. The presence of nisin was also detected during the storage at 4 and 10 °C, however a decrease of its concentration was observed over time. According to literature data, nisin production occurs mainly in the late exponential growth phase and the beginning of the stationary phase [
9]. Then the physical and compositional characteristics of the substrate may induce modifications of the activity and stability of nisin that can also be degraded by proteases [
50].
Initial color modifications were more dependent on the HPH treatment; in fact, even the total color difference (5.3 CIELAB units) was in line with what was reported by Szczepańska et al. [
36] for carrot juices treated with 150 MPa for three passes. However, the fermentation step and subsequent acidification of the product did not significantly impact the L* parameter while it maintained the three values (L*, a* and b*) during the storage time, particularly at 4 °C. In fact, samples that underwent the combined treatment had the highest L*, a*, and b* values among the samples considered. The fermentation step had a positive effect also on β-carotene and lutein. In fact, the initial concentration of β-carotene was higher in fermented samples than in those treated with only HPH. Although a reduction (6–7 mg/L) was observed at the end of the shelf life, its concentration remained the highest. A similar profile was also observed for lutein. Demir et al. [
51,
52] reported that acidified carrot juice, especially with lactic acid, significantly increased the β-carotene content and preserved its stability over time. In fact, acidification can help to release bound carotenoids by making them easily extractable during juice preparation. Moreover, low pH may inhibit the oxidative process by protecting compounds such as β-carotene.
Analyses of the volatile compounds detected in all the samples showed that treatments with HPH or HPH and LBG2 had an impact on the final profiles. HPH itself determined a profile where the relative abundance of terpenes and terpenoids was higher than in control and fermented samples. Modifications in the relative abundance of compounds upon HPH treatment were also described by Patrignani et al. [
23], who reported a reduction of the percentage of aliphatic aldehydes and an increase in benzaldehyde and terpineol in apricot juice. As already reported by Siroli et al. [
9], samples fermented by the biocontrol agent LBG2 are characterized by volatile molecules deriving from
L. lactis fermentation. In fact, the latter samples were characterized by a higher abundance of ketones (diacetyl, 3,4-dimethyl-2-pentanone, 2,6-dimethyl-4-heptanone), alcohols (1-octanol, 3-methyl-1-butanol, Terpinen-4-ol), and acids (acetic acid) that last during all the storage periods. These volatile molecules have been previously associated with a positive sensory impact in different fermented juices [
53,
54,
55]. Moreover, as observed by Fukuda et al. [
56] and Siroli et al. [
9], the microbial detoxification of initially present terpene molecules determined a reduction of their abundance. Similarly, myristicin represents an anti-nutritional compound naturally present in carrots [
57]. A significant reduction of its abundance in samples fermented with LBG2 represents an interesting tool that can be used to enhance the nutritional properties of the fermented carrot juice. In fact, it has been already reported that lactic acid fermentation can act as a food detoxification process against anti-nutritional factors such as phytates, saponins, tannins, cyanogens, or trypsin inhibitors [
58]. PCA analyses of the volatile compounds showed that samples treated with HPH and the biocontrol agent were different with respect to control and HPH-treated samples, however, they did not change significantly over time, showing the stability of the volatilome during the storage period.