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Food and Bioprocess Technology (2020) 13:1833-1847

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Food and Bioprocess Technology (2020) 13:1833–1847

https://doi.org/10.1007/s11947-020-02520-y

ORIGINAL RESEARCH

Non-thermal Processing of Pineapple (Ananas comosus [L.] Merr.)


Juice Using Continuous Pressure Change Technology (PCT): Effects
on Physical Traits, Microbial Loads, Enzyme Activities,
and Phytochemical Composition
Kathrin Vollmer 1,2 & Sara Santarelli 1 & Ana Lucía Vásquez-Caicedo 3 & Salima Varona Iglesias 3 & Jan Frank 2 &
Reinhold Carle 1,4 & Christof Björn Steingass 1,5

Received: 14 February 2020 / Accepted: 21 August 2020 / Published online: 11 September 2020
# The Author(s) 2020

Abstract
A comprehensive study using continuous pressure change technology (PCT) for the non-thermal processing of fresh
pineapple juice on pilot scale was conducted (1 L/min, 50 MPa, argon, 3 min, <35 °C). The immediate effects of a
single and a twofold PCT treatment on the most important quality parameters were examined and compared with
those of fresh and thermally pasteurised (90 °C) juices. In comparison to the fresh juice, both PCT-treated samples
exhibited slightly brighter and less yellowish colour (CIE L*a*b*). A significant reduction in the mean particle size
resulted in diminished centrifugable pulp contents and enhanced cloud stability. Moreover, a slightly improved
microbial quality (−0.9 to −1.2 log10 CFU/mL) in terms of total aerobic and yeast and mould counts was attained.
Noteworthy, PCT retained a high bromelain activity (−3 to −15% reduction) and efficiently inactivated polyphenol
oxidase. Water-soluble vitamins, phenolic compounds, and all further constituents assessed were mostly preserved.
However, the high residual peroxidase activity (−10 to −23%) and microbial loads are likely to affect juice quality
during storage. In contrast, thermal pasteurisation ensured a complete reduction in both microbial counts (−4.4 to
−4.5 log10 CFU/mL) and effective inactivation of peroxidase. However, bromelain activity was strongly affected
(−83%) by heat treatment, and colour was darkened and even less yellowish. Overall, this study highlighted the
potential of PCT for the production of fresh-like pineapple juices; however, its current limitations were revealed as
well.

Keywords Juice quality . Non-thermal processing . Pasteurisation . Pineapple . Pressure change technology

Abbreviations
* Christof Björn Steingass a* Green-red
christof.steingass@hs-gm.de AA Ascorbic acid
1 ANOVA Analysis of variance
Institute of Food Science and Biotechnology, Chair Plant Foodstuff
Technology and Analysis, University of Hohenheim, Garbenstrasse TPC Total phenolic content
25, 70599 Stuttgart, Germany b* Blue-yellow
2
Institute of Nutritional Sciences, Chair Food Biofunctionality, BRM Bromelain
University of Hohenheim, Garbenstrasse 28, C* Chromaticity
70599 Stuttgart, Germany CDU Casein digestive unit
3
Fraunhofer Institute for Interfacial Engineering and Biotechnology DHAA Dehydroascorbic acid
IGB, Nobelstrasse 12, 70569 Stuttgart, Germany ΔE* Total colour difference
4
Biological Science Department, Faculty of Science, King Abdulaziz ESI Electrospray ionisation
University, P.O. Box 80257, Jeddah 21589, Saudi Arabia GAE Gallic acid equivalent
5
Department of Beverage Research, Chair Analysis & Technology of h° Hue angle
Plant-based Foods, Geisenheim University, Von-Lade-Strasse 1, HCA Hierarchical cluster analysis
65366 Geisenheim, Germany
1834 Food Bioprocess Technol (2020) 13:1833–1847

HDMF 4-Hydroxy-2,5-dimethyl-3 studies. First promising results about batch, semi-continuous,


(2H)-furanone and continuous PCT applications have been reported by
HMF 5-(Hydroxymethyl)furfural Klingner et al. (2006), Bönsch et al. (2007), and Aschoff
HPLC-/UPLC-DAD High-/ultra-performance et al. (2016), respectively. PCT is based on a physical process.
liquid chromatography-diode Juices or other liquid food products are put under moderate
array detection pressure in the presence of an inert gas. At pressures of up to
L* Lightness 50 MPa for a short holding time, the gas dissolves in the liquid
MSn Multi-stage mass spectrometry food products and diffuses into plant and microbial cells.
PCA Principal component analysis Once the pressure is suddenly reduced to ambient
PCT Pressure change technology conditions, the inert gas expands and compartmentalised
PME Pectin methylesterase structures, e.g. plant or microbial cells, are disrupted.
POD Peroxidase Aschoff et al. (2016) stated that the mode of action is based
PPO Polyphenol oxidase on this dynamic decompression phase. In contrast, static tech-
TA Titratable acidity nologies, such as high-pressure processing, are premised on
TSS Total soluble solids much higher working pressures applied during a certain hold-
TAPC Total aerobic plate count ing time, in addition to chemical effects (e.g. hydrolysis and
YMC Yeast and mould count acidification) when using carbon dioxide as the process gas
LOD Limit of detection (Balasubramaniam et al. 2015; Kincal et al. 2005).
LOQ Limit of quantitation The most obvious advantages of PCT are the low working
temperatures (<40 °C) and the protective atmosphere due to
the inert process gas. The exclusion of oxygen inhibits the
Introduction destruction and oxidation of sensitive juice constituents. The
successful reduction of diverse food-related microorganisms
Pineapples (Ananas comosus [L.] Merr.) are one of the most in cell suspensions has been demonstrated by Klingner et al.
popular tropical fruits consumed worldwide. In 2017, (2006) and Bönsch et al. (2007). This is particularly essential
the global production of pineapples exceeded 27.4 million for the production of microbiologically safe juices (Miller and
tons. According to the global import statistics, the major por- Silva 2016). Applying PCT operated with nitrogen at 50 MPa
tion was consumed fresh and merely 7% were processed into (40 °C, 5 min) to a Pseudomonas fluorescence suspension
juices and concentrates, respectively, among others (FAO resulted in a significant reduction (−log N/N 0 of 6.4)
2019). (Bönsch et al. 2007). However, the authors have also pointed
Pineapple juices are commonly thermally pasteurised out that the type of microorganism (gram− > yeast >> gram+),
aiming at the extension of their shelf life by inactivating path- the applied pressure (5–25 MPa less effective than 50 MPa),
ogenic and spoilage microorganisms as well as enzymes. the temperature (10, 25, or 40 °C), and the process gas (nitro-
However, the fragile aroma, colour, and heat-sensitive gen, helium, argon, carbon dioxide, or nitrous oxide) deter-
micronutrients may be adversely affected (Lobo and Paull mine the efficiency of this technology. Furthermore, continu-
2017; Miller and Silva 2016), impairing the ‘fresh’ and ‘nat- ous PCT studies (25/50 MPa, nitrogen, <40 °C) conducted by
ural’ character of the juice. However, the latter is exactly what Aschoff et al. (2016) with freshly squeezed orange juice per-
consumers increasingly demand. In the European market, the mitted a total plate count reduction of at least 3.4 log10 CFU/
share of cooled premium juices with superior sensory quality mL. Partial and even total inactivation of pectin
steadily grew during the past years whereas that of pasteurised methylesterase (PME) and peroxidase (POD), respectively, a
juices storable at ambient temperatures gradually declined significant reduction of the mean particle size, thus improving
(AIJN 2019). cloud stability, a negligible brightening and reinforcement of
Innovative non-thermal technologies rendering both shelf the yellow colour were additionally achieved. However, a
life extension and retention of fresh-like quality traits possible slight reduction in micronutrients (carotenoids, vitamin C,
are therefore increasingly in demand, and thus, of utmost im- and hesperidin) was observed. In addition, PCT offers oppor-
portance for the juice industry. Pulsed light, ultraviolet light, tunities for continuous processing and the optional recovery of
pulsed electric fields, high-pressure, and supercritical carbon the process gas.
dioxide treatments have already been applied for non-thermal The aim of the present study was to process fresh pineapple
processing of pineapple juice or other liquid fruit products juice at pilot plant scale with a continuous PCT device apply-
(Vollmer et al. 2020; Paniagua-Martínez et al. 2018; ing one (PCT1) or two passages (PCT2), respectively, and to
Koutchma 2009; Kincal et al. 2005; Norton and Sun 2008). comprehensively investigate its immediate effects on relevant
Pressure change technology (PCT) is an emerging non- quality parameters. Physical traits, such as colour, particle size
thermal process, so far not sufficiently considered in previous and distribution, and cloud stability as well as microbial loads
Food Bioprocess Technol (2020) 13:1833–1847 1835

(total aerobic plate count (TAPC), yeast and mould count Consulting, Neustadt an der Weinstraße, Germany). After di-
(YMC)), presence of desired and detrimental enzymes (bro- rect hot filling into 500-mL clear glass bottles and flushing the
melain (BRM), PME, POD, and polyphenol oxidase (PPO)), headspace of approx. 5% of the total volume with vapour
and content of the most important micronutrients, inter alia using a filling and sealing machine (Schmalbach-Lubeca,
vitamin C, B vitamins, and phenolic compounds, were Braunschweig, Germany), the juice was immediately cooled
assessed. In addition, both PCT-treated samples were com- to ambient temperature in a water bath.
pared with a fresh and thermally pasteurised (90 °C) pineapple
juice. Hereby, the thermal pasteurisation represents the current Pressure Change Technology-Treated Juices
state of the art applied in the industry, whereas the untreated
juice serves as the reference nearest to natural. These reference The fresh juice was transferred to the feed tank of a continuous
points were used as relevant boundaries to range in our results. PCT pilot plant located in a cleanroom with a controlled am-
bient temperature of 20 ± 1 °C (Fig. 1). At a continuous flow
rate of 1.0 L/min, the juice was pressurised to 50 MPa within a
Materials and Methods few seconds (<10 s) by a high-pressure pump. Subsequently,
the juice was homogeneously mixed with individually com-
Chemicals pressed argon applying a slight excess pressure of the gas (ca.
52 MPa) by an inline static mixer within the tubular PCT
All chemicals were purchased from Sigma-Aldrich reactor. The gas flow rate was 20 g/min resulting in
(Steinheim, Germany, and Buchs, Switzerland), Merck an argon to juice ratio of 20 g/L. The juice-argon mixture
(Darmstadt, Germany), VWR International (Leuven, was kept constant at 50 MPa and released after 3 min through
Belgium, and Fontenay-sous-Bois, France), Th. Geyer an exhaust valve into a filling tank previously flushed with
(Renningen, Germany), Roth (Karlsruhe, Germany), nitrogen. The pineapple juice was bottled manually (PCT1).
Extrasynthèse (Genay, France), Fluka Chemie (Buchs, Once the first passage was entirely completed, the remaining
Switzerland), or Herbstreith & Fox (Neuenbürg, Germany).
Ultrapure water was provided by an arium 611 UV
(Sartorius, Göttingen, Germany) ultrapure water system and
used throughout all experiments, if not stated otherwise.

Pineapple Juice Production

Approx. 210 kg of pineapples (Ananas comosus (L.) Merr. cv.


‘MD2’ (syn. ‘Extra Sweet’)), air-freighted from Ghana, was
purchased from a local fruit distributor (Schumacher,
Filderstadt-Bernhausen, Germany).

Fresh Juice

After one day of storage at ambient temperature, the entire


batch of pineapples was sorted, decrowned, washed with tap
water, peeled, and manually cut into quarters. The flesh was
shredded using a grating mill (Bucher-Guyer,
Niederweningen, Switzerland), and the juice extracted by
finishing through a 1.6-mm sieve (Alberto Bertuzzi SpA,
Brugherio, Italy). Large flesh particles were removed using a
decanter (GEA Westfalia Separator, Oelde, Germany). Juice
yield was 46% (w/w).

Thermally Pasteurised Juice

For thermal pasteurisation, the fresh juice was heated to a


targeted filling temperature of 90 °C (Lobo and Paull 2017)
at a continuous flow rate of 1.3 L/min applying a pilot plant Fig. 1 Schematic of the continuous pressure change technology (PCT)
scale tubular heat exchanger (Ruland Engineering & system applied
1836 Food Bioprocess Technol (2020) 13:1833–1847

juice was re-circulated from the filling to the feed tank follow- 5-(Hydroxymethyl)furfural (HMF) was analysed by
ing a further passage (PCT2) applying the above settings. UPLC-DAD following a modified procedure according to
The initial temperature of the juice prior to both PCT treat- IFU (1996b). The pineapple juices were initially filtered
ments was 21.0 ± 2.8 °C. Throughout the following processes, through regenerated cellulose membranes (0.45 μm,
the average juice temperature gradually increased. Chromafil RC-45/15 MS, Macherey-Nagel, Düren,
Pressurisation and mixing within the PCT reactor resulted in Germany) into amber glass vials. An Acquity H-class UPLC
a slight increase to 22.0 ± 1.4 °C, while the main rise to 33.5 ± system equipped with an eλ photodiode array detector (all
0.7 °C was caused during the dynamic decompression phase. from Waters, Milford, MA, USA) was applied for quantita-
The latter rise in temperature may result from two individual tion. UPLC separation at 25 °C was achieved with a Kinetex
effects that reversely occur during decompression of the juice- reversed-phase C18 core-shell column (100 × 2.1 mm, 1.7-μm
argon mixture at the exhaust valve. These include the cooling particle size, 100-Å pore size) and a guard column of the same
due to the sudden outgassing and rapidly expanding gas based material (both from Phenomenex, Torrance, CA, USA). The
on the Joule-Thomson effect and the predominating heating mobile phase consisted of ultrapure water/methanol (90:10,
caused by shear forces. v/v). Applying an isocratic elution, total run time was 5 min
Immediately after juice production, two representative at a flow rate of 0.35 mL/min. Injection volume was 3 μL.
samples of the four juice types each were taken, filled into HMF was detected at 284 nm. Linear external calibration
containers headspace-flushed with nitrogen, and stored at curves were prepared with ultrapure water and HMF-free
−80 °C. Immediately prior to analyses, juices were thawed pineapple juice, respectively. Limit of detection (LOD) and
in a water bath (20 °C). quantitation (LOQ) were ascertained based on the signal-to-
noise (S/N) ratio of the calibration curves. LOD (S/N of 3:1)
Analysis of Physicochemical Juice Properties and LOQ (10:1) were determined at 0.001 and 0.016 mg/L in
ultrapure water and 0.3 and 1.8 mg/L in pineapple juice,
The pH value, the density, and the total soluble solids (TSS) respectively.
were determined using a pH meter (inoLab pH 720, WTW,
Weilheim, Germany), a density meter (DMA 48, Anton Paar,
Graz, Austria), and a digital refractometer (RX-5000, Atago, Colour Measurement
Tokyo, Japan), respectively.
The titratable acidity (TA, expressed as citric acid in g/100 CIE L*a*b* colour values were determined in triplicate using
mL of juice) and the formol number (in mL 0.1 mol NaOH/ an UltraScan Vis spectrophotometer (HunterLab, Reston, VA,
100 mL juice) were analysed according to the methods of the USA) in reflection mode and with standard illumination (D65/
International Federation of Fruit Juice Producers (IFU 1984, 10°). L* (lightness), a* (green-red), and b* (blue-yellow)
1996a) using an automatic titration system (Titrino 718 values were used to additionally calculate chromaticity (C*),
STAT, Metrohm, Herisau, Switzerland). The titration solution hue angle (h°), and the total colour difference (ΔE*).
was normalised using potassium hydrogen phthalate.
The total phenolic content (TPC) was assessed with the
colorimetric Folin-Ciocalteu assay according to Singleton Particle Size Analysis
et al. (1999) with modifications. Briefly, 20 μL of centrifuged
(14,100×g, 10 min, MiniSpin plus, Eppendorf, Hamburg, Particle size distributions were analysed using a static light
Germany) and filtered (polytetrafluoroethylene, 0.45 μm, scattering particle analyser (Horiba LA-950, Retsch
Acrodisc CR 13, Pall, Ann Arbor, MI, USA) pineapple juice Technology, Haan, Germany). Mean particle diameters
were mixed with 980 μL of ultrapure water and 100 μL of (d4,3); the particle diameters of the respective 10, 50, and
Folin-Ciocalteu reagent. After 3 min and the addition of 800 90% quantiles (d10, d50, and d90); and the particle size distri-
μL aqueous sodium carbonate solution (75 g/L), the mixture butions are given as volume-weighted diameters.
was incubated for 60 min at ambient temperature.
Subsequently, the absorbance was measured at 760 nm using
an UV/Vis spectrometer (Lambda 35, PerkinElmer, Cloud Stability Monitoring
Singapore). Gallic acid was used for external linear calibration
and TPC expressed as gallic acid equivalents (GAE) in mg/ As described by Aschoff et al. (2016), cloud stability was
100 mL juice. monitored by filling 20 mL of pineapple juice into 25-mL
The centrifugable pulp content of the juices was deter- graduated cylinders stored for two weeks at 4 °C.
mined according to IFU (1991) using a Heraeus Labofuge Sedimentation of cloud was visually inspected after 1, 2, 7,
400 R centrifuge (Thermo Fisher Scientific, Osterode, and 14 days.
Germany).
Food Bioprocess Technol (2020) 13:1833–1847 1837

Microbial Counts PPO activity was analysed by spectrophotometry accord-


ing to Baur et al. (2004) with slight modifications. Briefly, 1.5
Total aerobic plate count (TAPC) as well as yeast and mould mL of the reaction buffer (2 mM sodium dodecyl sulphate
count (YMC) were determined according to IFU (1996c,d) dissolved in McIlvaine buffer, pH 6.5), 0.2 mL of an L-
with modifications. In brief, serial dilutions were prepared proline solution (0.5 M in reaction buffer), and 0.1 mL of
using sterile saline solution containing 0.1% (w/v) peptone the enzyme extract were mixed. After adding 0.2 mL substrate
broth (VWR International). One hundred microlitres of juice solution (25 mM 4-methylcatechol in reaction buffer), the
and appropriate dilutions was spread out in triplicate on or- absorbance was immediately monitored every 2 s for 5 min
ange serum agar plates. For the cultivation of yeasts and using the above-mentioned UV/Vis spectrometer. PPO activ-
moulds, the orange serum agar additionally contained 34 μg/ ity was expressed in nkat/g pineapple juice.
mL of chloramphenicol (≥98%) to inhibit bacterial growth.
All plates were incubated at 30 °C for 48 h. Both counts were Analysis of Micronutrients and Sugars
expressed in colony-forming units (CFU)/millilitre of juice.
Plates with <10 CFUs (<2 log10 CFU/mL) were not evaluated. Vitamin C

For the quantitation of vitamin C, i.e. ascorbic and


Enzyme Assays dehydroascorbic acid (AA and DHAA), the sample prepara-
tion described by Aschoff et al. (2015) was applied using the
BRM activity was assessed spectrophotometrically following buffers previously reported (Vollmer et al. 2020). For HPLC
Jutamongkon and Charoenrein (2010) and expressed as casein analysis, an Agilent 1100 Series system equipped with a
digestive units (CDU). G1315B diode array detector (DAD) was used (all from
PME, POD, and PPO were initially extracted from the Agilent Technologies, Waldbronn, Germany). HPLC param-
pineapple juices according to Hirsch et al. (2008) with slight eters, including column and mobile phase, were set as reported
modifications. In brief, 20 g of pineapple juice was blended previously (Difonzo et al. 2019).
with 20 mg saponin, 600 mg polyvinylpolypyrrolidone, and
175.5 mg NaCl. After adjusting the pH value to 6.0 (10 and B Vitamins
1 M NaOH), the mixture was stirred (2 h, 4 °C) and subse-
quently centrifuged (25,000×g, 4 °C, 30 min, Suprafuge 22, Vitamins B 1 (thiamin), B2 (riboflavin), B3 (niacin), B5
Heraeus Instruments, Osterode, Germany). The supernatant (pantothenic acid), and B6 (pyridoxine) were quantitated
was decanted, and the solid remainder washed twice with utilising microbiological microtiter plate tests (VitaFast, R-
ultrapure water. The combined supernatant and washing water Biopharm, Darmstadt, Germany). Sample preparation and
was made up to 40 mL with water. Boiled enzyme extracts quantitation of B vitamins were conducted according to the
were used as blanks. Extracts were stored at −80 °C until supplier’s instructions. An aliquot of 1 mL pineapple juice
further analyses. was diluted with 20 mL citrate buffer (pH 4.5).
PME activity was determined by titration according to Subsequently, 300 mg α-amylase from Aspergillus oryzae
Hirsch et al. (2008) applying an automatic titration system (~1.5 U/mg) and additional 10 mg acid potato phosphatase
(Titrino 907 STAT, Metrohm). Two millilitres enzyme extract (0.5–3.0 U/mg) for the analysis of B2 and B6 were admixed
and 58 mL substrate solution were employed for titrimetric following incubation for 1 h in the dark at 37 °C (incubator U
analysis. The substrate solution (0.5%, w/v, pectin Classic CU 30ü, Memmert, Schwabach, Germany) with occasional shak-
201, degree of esterification 70–74%, in 0.15 M NaCl) was ing. After adding 19 mL of ultrapure water, the mixture was
prepared as detailed by Aschoff et al. (2016). PME activity, centrifuged at 4500×g and 20 °C for 10 min (Heraeus
expressed in nkat/g of pineapple juice, was calculated as re- Labofuge 400 R). The supernatant was sterile-filtered (0.8/
ported by Hirsch et al. (2008). 0.2 μm, polyethersulfone, Acrodisc Supor, Pall, Ann Arbor,
POD activity was assessed spectrophotometrically accord- MI, USA), and the filtrate immediately used for quantitation
ing to Hirsch et al. (2008) with slight modifications. Briefly, of B vitamins.
0.4 mL of the enzyme extract diluted 1:2 with McIlvaine buff-
er (pH 6.5) if required was mixed with 1.1 mL of the substrate Phenolic Compounds, Furanones, and Other Metabolites
solution (12 mM tropolone and 3.3 mM hydrogen peroxide
dissolved in McIlvaine buffer, pH 6.5). The absorbance was Prior to HPLC analysis, the pineapple juices were centrifuged
immediately recorded at 418 nm every 2 s for 10 min using a (14,100×g, 10 min, MiniSpin plus, Eppendorf, Hamburg,
UV/Vis spectrophotometer (Lambda 35, PerkinElmer, Germany) and filtered through polytetrafluoroethylene mem-
Singapore). POD activity was expressed in nkat/g of pineap- branes (0.45 μm, Acrodisc CR 13, Pall, Ann Arbor, MI, USA)
ple juice. into amber glass vials. Individual compounds were identified
1838 Food Bioprocess Technol (2020) 13:1833–1847

by HPLC-DAD-ESI-MSn analysis as detailed elsewhere Paull 2017). The consistent values determined among all
(Vollmer et al. 2020; Difonzo et al. 2019; Steingass et al. juices indicated that neither concentrations nor dilutions oc-
2015). For quantitation, the above-mentioned Acquity H- curred during processing.
class UPLC system with an eλ photodiode array detector A maximum of 2 mg HMF/100 mL of pineapple juice is
was used as reported previously (Difonzo et al. 2019). specified as an absolute quality requirement (AIJN 2016).
Detection wavelengths were 280 and 320 nm. Quantitation HMF may be formed during intense thermal processing or
was achieved by external calibrations of adequate reference during storage at elevated temperatures by acid-catalysed de-
standards (Vollmer et al. 2020; Difonzo et al. 2019). hydration of reducing sugars or as an intermediate of the
Maillard reaction of reducing sugars and amino compounds.
Sugars HMF being absent in fresh, untreated juices represents a well-
documented indicator of excessive heat treatment (Lozano
Juices were pre-filtered (4–12 μm, cellulose, MN 615 ¼, 2006). During this study, HMF was not detected among any
Macherey-Nagel, Düren, Germany), diluted with ultrapure of the pineapple juices assessed (LOD: 0.03 mg/100 mL). The
water (1:6250), and membrane filtered (0.45 μm, polyamide, generation of HMF, which has been reported to depend on
Chromafil AO-45/15 MS, Macherey-Nagel, Düren, both the juice composition and the process parameters
Germany) into amber glass vials. D-Glucose, D-fructose, and (Lozano 2006), may have been prevented due to good
sucrose were quantitated by high-performance anion-ex- manufacturing practice and the omission of juice
change chromatography with pulsed amperometric detection concentration.
(ICS-3000 ion chromatography system, Dionex, Sunnyvale,
CA, USA) as described by Pöhnl et al. (2017).
Colour Profile
Statistics
Lightness (L*) and yellowness (+b*) are the two main chro-
Two representative samples were drawn for each juice type matic parameters characterising the colour of pineapple juice.
and analysed in duplicate, if not stated otherwise. Results are As illustrated in Table 1, the fresh juice displayed average
displayed as means ± standard deviations. Statistically signif- values of L* = 39.1 and b* = 10.5. The PCT-treated juices
icant differences (p ≤ 0.05) among means were identified by were slightly brighter and less yellowish as indicated by ele-
one-way analysis of variance (ANOVA) and Tukey’s post- vated L* and smaller b* values, respectively. Both effects were
hoc multiple comparison test using SAS University Edition slightly more pronounced after the second PCT treatment.
software (SAS Institute, Cary, NC, USA), assuming normal Such a brightening concomitantly with a slightly more intense
distribution and homogeneity of variances. yellow colour has also been observed by Aschoff et al. (2016)
Unsupervised principal component analysis (PCA) and hi- after treating fresh orange juice with PCT. The authors as-
erarchical cluster analysis (HCA) were calculated with Solo sumed that these alterations probably resulted from changes
software release 8.2.1 (Eigenvector Research, Wenatchee, in light scattering owing to smaller particle sizes. The latter
WA, USA) applying venetian blinds cross-validation method was also observed in our PCT-treated juices, as discussed in
and Ward’s method of agglomeration, respectively. Data was the ‘Particle Size, Centrifugable Pulp Content, and Cloud
pre-processed using the ‘autoscale’ function of the software. Stability’ section. In contrast, the thermally pasteurised juice
turned out to be the darkest (L* = 37.9) and less yellowish (b*
= 8.2) variant. The darkening may be attributed to non-
Results and Discussion enzymatic browning (Lozano 2006), possibly due to heating
to 90 °C.
General Quality Traits CIE L*a*b* colour values were in accordance with our
visual impression. Overall, the calculated total colour differ-
All pineapple juices displayed a constant pH value of 3.7 ences (ΔE*) to the fresh juice amounted to 2.8 for the thermal-
(Table 1). The relative density 20/20 (density), the TSS, the ly pasteurised and to 4.9 and 6.6 for the two PCT-treated
TA, and the formol number amounted to 1.045–1.048 (1.044– juices, respectively. This clearly illustrated that the impact of
1.047 g/cm3), 11.7–11.9 °Brix, 0.5 g/100 mL, and 7.7–9.2 mL the PCT treatments on the colour profile was significantly (p ≤
0.1 mol NaOH/100 mL, respectively, thus complying with the 0.05) higher compared with that of the thermal pasteurisation.
AIJN (2016) reference guidelines for pineapple juice (relative However, consumers will decide about the final acceptance of
density 20/20: ≥1.045; TSS: ≥11.2 °Brix; TA: 0.32–1.15 g/ the colour changes observed. The sensory analysis of PCT-
100 mL; formol number: 8–20 mL 0.1 mol NaOH/100 mL). treated fruit juices will be the subject of ongoing research.
The TSS/TA ratios of 24.5–24.7 g/g were within the range of
20–40 g/g reported for high-quality pineapple fruit (Lobo and
Food Bioprocess Technol (2020) 13:1833–1847 1839

Table 1 General juice quality traits, colour values, and particle diameters of fresh, thermally pasteurised, and PCT-treated pineapple juices

Parameters Pineapple juice

Fresh Thermally pasteurised PCT1 PCT2

General juice quality traits


pH - 3.7 ± 0.0a 3.7 ± 0.0a 3.7 ± 0.0a 3.7 ± 0.0a
Density g/cm3 1.047 ± 0.001 a 1.045 ± 0.001 a 1.044 ± 0.002 a 1.045 ± 0.000 a
Total soluble solids (TSS) °Brix 11.8 ± 0.1b 11.9 ± 0.0a 11.7 ± 0.0b 11.8 ± 0.0ab
Titratable acidity (TA) g/100 mL 0.5 ± 0.0a 0.5 ± 0.0a 0.5 ± 0.0a 0.5 ± 0.0a
TSS/TA mL/g 23.5 ± 0.1a 23.5 ± 0.1a 23.6 ± 0.1a 23.6 ± 0.0a
Formol number (FN) mL 0.1 mol NaOH/100 mL 9.0 ± 0.1a 7.7 ± 0.1b 8.9 ± 0.1a 9.2 ± 0.1a
Total phenolic content (TPC) mg GAE/100 mL 73.4 ± 1.6a 74.7 ± 0.1a 74.9 ± 1.5a 76.9 ± 0.9a
Centrifugable pulp content % v/v 4.5 ± 0.7a 2.5 ± 0.7a tr. n. d.
5-(Hydroxymethyl)furfural (HMF) mg/100 mL n. d. n. d. n. d. n. d.
CIE L*a*b* colour
L* - 39.1 ± 0.1c 37.9 ± 0.1d 43.7 ± 0.1b 45.4 ± 0.1a
a* - −0.6 ± 0.1 a −1.3 ± 0.0c −0.7 ± 0.0ab −0.9 ± 0.1b
b* - 10.5 ± 0.0a 8.2 ± 0.1c 9.2 ± 0.2b 8.8 ± 0.4bc
h° - 93.1 ± 0.2d 99.2 ± 0.3a 94.4 ± 0.1c 95.5 ± 0.1b
C* - 10.5 ± 0.0a 8.3 ± 0.1c 9.2 ± 0.3b 8.9 ± 0.4bc
ΔE* - - 2.8 ± 0.1c 4.9 ± 0.1b 6.6 ± 0.3a
Particle diameters
d4,3 μm 5.5 ± 0.2a 3.4 ± 0.0b 2.4 ± 0.0c 2.2 ± 0.0c
d10 μm 4.3 ± 0.0a 1.6 ± 0.0b 1.3 ± 0.1c 1.2 ± 0.1c
d50 μm 5.4 ± 0.1a 2.7 ± 0.1b 2.2 ± 0.0c 2.0 ± 0.1c
d90 μm 6.7 ± 0.4a 6.1 ± 0.1a 3.8 ± 0.1b 3.5 ± 0.1b

a* , red-green; b* , yellow-blue; C* , chromaticity; d4,3, volume-weighted mean particle diameter; d10/d50/d90, volume-weighted particle diameters of the
respective 10%, 50%, and 90% quantiles; ΔE* ; total colour difference; h°, hue angle; L* , lightness; n. d., not detected (<LOD); tr., trace (<LOQ)
Different superscript letters within one row indicate significant (p ≤ 0.05) differences among means determined by ANOVA and Tukey’s test

Particle Size, Centrifugable Pulp Content, and Cloud distributed. In contrast, both PCT-treated juices displayed a
Stability right-skewed distribution and a slightly narrower range, con-
siderably shifted towards smaller particle sizes (PCT1: 1.2–
Pineapple juices are inherently prone to poor cloud stability. 8.8 μm; PCT2: 1.0–7.7 μm). These effects were also visible
Will (1995) and Will et al. (1999) have reported that this is with regard to the volume-weighted particle diameters of the
mainly attributed to the presence of prevailing coarse (~100 respective 10, 50, and 90% quantiles (significantly smaller
μm) and the lack of fine cloud particles (~0.5 μm), in addition d10, d50, and d90 values of the PCT-treated samples compared
to the differing serum composition of pineapple juice (being with the fresh juice, see Table 1). This shift towards smaller
rich in glucomannans and low in pectin) in comparison with particle sizes may be attributed to mechanical stress during the
those from other fruits. As the sedimentation velocity accord- decompression phase. The dissolved inert gas instantly ex-
ing to Stokes’ law increases with the square of the particle pands through the exhaust valve, causing shear stress and
size, the latter is a crucial parameter determining cloud stabil- resultantly, the disruption of juice particles. Interestingly, ther-
ity. Heavy and coarse cloud particles settle more quickly, mal pasteurisation also resulted in smaller, and concurrently,
resulting in the rapid separation of a clear upper serum and slightly larger particles than those of the fresh juice.
sediment at the bottom, which does not comply with con- Consequently, this right-skewed distribution was the broadest
sumers’ expectations (Will et al. 1999; Will 1995). (1.5–22.8 μm) observed among all four juices. An increase in
In our study, the particle sizes of all pineapple juices ranged particle size after thermal treatments has also been observed
between approx. 1 and 23 μm, displaying monomodal distri- by Baron et al. (2006) and Katiyo et al. (2017), as a result of
butions (Fig. 2). Interestingly, the particle sizes of the fresh enhanced Brownian motion and inter-particle forces
juice ranging between 2.6 and 13.2 μm were largely normally (Genovese et al. 2007).
1840 Food Bioprocess Technol (2020) 13:1833–1847

Fig. 2 Representative volume-weighted particle size distribution of fresh,


thermally pasteurised, and PCT-treated pineapple juices

Overall, the fresh juice was characterised by a volume-


weighted mean particle diameter (d4,3) of 5.5 μm, followed
by 3.4 for the thermally pasteurised and 2.4 and 2.2 μm for the
two PCT-treated juices, respectively (see Table 1). These
smaller particle sizes may result in the clearly reduced
centrifugable pulp content of both PCT-treated juices (see
Table 1). After the first PCT treatment, traces of centrifugable
pulp were still visible (<1.0%), whereas a second passage
resulted in a complete depletion. In comparison, the
centrifugable pulp contents of the fresh and the thermally
pasteurised juice amounted to 4.5 and 2.5% of the total juice
volume, respectively.
The cloud stability tests at 4 °C are illustrated in Fig. 3.
Initially, the cloud particles were homogeneously suspended
in all juices (day 0). In the fresh and thermally pasteurised
juices, the onset of sedimentation was already visible after
6 h (not shown). The sedimentation in these two samples
proceeded rapidly, resulting in a turbid serum due to
suspended fine particles and a considerable sediment forma-
tion. By contrast, the PCT-treated juices still displayed a ho-
mogeneous, turbid phase. During the subsequent storage, the
serum of the fresh and thermally pasteurised juices gradually
clarified and cloud became more compact (days 7 and 14).
This was more pronounced in the thermally pasteurised than
in the fresh juice. Quick sedimentation of cloud in thermally
pasteurised pineapple juices has also been pointed out by Will
et al. (1999). In contrast, both PCT-treated juices displayed
improved cloud stability, possibly resulting from the above- Fig. 3 Cloud stability monitoring of fresh, thermally pasteurised, and
PCT-treated pineapple juices during 14 days of storage at 4 °C. Arrows
mentioned shifts towards reduced particle sizes and their
mark the upper margin of the cloud sediment.
narrowed distribution (Fig. 2). However, initial sedimentation
was also observed in the PCT-treated samples as early as day 1
that further manifested with progressing storage. Will et al.
Food Bioprocess Technol (2020) 13:1833–1847 1841

(1999) have emphasised that particle sizes <1 μm are a pre- several process parameters, including working pressure, tem-
requisite for satisfactory cloud stabilities. Thus, the particles in perature, and the process gas. The combination of the latter
the PCT-treated samples were possibly still too large. three parameters determines the amount of gas dissolved in
However, their less compact sediment may be easily re- the liquid (Klingner et al. 2006). The juice matrix itself may
suspended prior to consumption, e.g. by shaking. As the vis- have a significant effect as, e.g. salt (Sun et al. 2001) and sugar
cosity is inversely proportional to the sedimentation velocity contents (Illera et al. 2019) influence the solubility of gases in
in Stokes’ law, the cloud stability may be further improved by liquids. In addition, the tissue and cell wall structure may
thickening agents. The European regulation (EC) No 1333/ affect the efficiency of the PCT treatment. Whereas orange
2008 (European Union 2008) allows the admixture of up to endocarp consists of comparatively soft juice vesicles, the
3 g pectin (E 440) per kg pineapple juice. Will (1995) and Will firm pineapple flesh is composed of cells rich in hemicellulose
et al. (1999) have shown that this improved cloud stability, and cellulose (Vidal-Valverde et al. 1982). Consequently, the
however, adversely affected the organoleptic properties of inactivation of microorganisms in pineapple juice by PCT
pineapple juice. The observed ‘homogenisation’ effect of the requires further optimisation. The impact of the residual mi-
PCT treatment may permit to significantly reduce the allowed crobial loads on the shelf life and safety of the PCT-treated
pectin dosage, and thus, not impairing the sensory appearance juices may be subject of continuative studies. Another possi-
of PCT-treated pineapple juices. ble reason for the comparatively inefficient reduction of plate
counts observed herein may be the disintegration of multicel-
lular microbial aggregates during PCT treatment. Similarly,
Microbial Quality Joyce et al. (2011) have observed such an effect after sonicat-
ing bacterial suspensions. This hypothesised disintegration of
In the fresh pineapple juice, TAPC and YMC amounted to 4.5 microbial cell aggregates, which possibly counteracts the re-
and 4.4 log10 CFU/mL, respectively (Table 2). Thermal duction of the plate counts, merits further investigation.
pasteurisation reduced both counts below the detection limit
(<2 log10 CFU/mL). In comparison, PCT resulted in average
reductions of both counts by 0.9 and 1.1–1.2 log10 CFU/mL Enzyme Activities
after the first and the second passage, respectively. The
microbial quality was thus merely slightly improved by the The activities of quality-relevant enzymes are listed in
PCT treatment, while Aschoff et al. (2016) reported a total Table 2. Pineapples are well-known sources of the protease
reduction of the TAPC of initially 3.4 log10 CFU/mL after BRM (Lobo and Paull 2017; Mehrlich and Felton 1980). Its
applying similar PCT settings (50 MPa, nitrogen, <40 °C, 2 activity amounted to 106.3 ± 1.1 CDU in the fresh juice.
L/min, 1.3 min) to orange juice. Bönsch et al. (2007) have Thermal pasteurisation caused a drastic drop to 17% of the
demonstrated the efficient reduction (−log N/N0) of diverse initial activity, being in line with our previous study (Vollmer
food-related microorganisms in cell suspensions by PCT et al. 2020). In contrast, BRM activity was largely retained
batch treatments (50 MPa) of up to 7.5. The latter authors applying PCT (<35 °C) and merely reduced to 97 and 85%
stated that the inactivation efficiency is dependent on, inter residual activity after one and two passages, respectively.
alia, the type of microorganism (gram− > yeast >> gram+) and Owing to the putative health-promoting effects of BRM

Table 2 Microbial loads and enzyme activities of fresh, thermally pasteurised, and PCT-treated pineapple juices

Parameters Pineapple juice

Fresh Thermally pasteurised PCT1 PCT2

Microbial loads
Total aerobic plate count (TAPC) log10 CFU/mL 4.5 ± 0.1a n. d. 3.6 ± 0.1b 3.3 ± 0.1b
Yeast and mould count (YMC) log10 CFU/mL 4.4 ± 0.0a n. d. 3.5 ± 0.1b 3.3 ± 0.1b
Enzyme activities
Bromelain (BRM) CDU 106.3 ± 1.1a 17.8 ± 1.3d 102.6 ± 0.0b 90.1 ± 0.6c
Pectin methylesterase (PME) nkat/g n. d. n. d. n. d. n. d.
Peroxidase (POD) nkat/g 7.0 ± 0.2a n. d. 5.4 ± 0.4a 6.3 ± 1.2a
Polyphenol oxidase (PPO) nkat/g 3.2 ± 0.6 n. d. n. d. n. d.

n. d., not detected (<LOD)


Different superscript letters within one row indicate significant (p ≤ 0.05) differences among means determined by ANOVA and Tukey’s test
1842 Food Bioprocess Technol (2020) 13:1833–1847

(Lobo and Paull 2017; Maurer 2001), the maximum retention treatment than POD and most likely inactivated by shear
of this mixture of proteolytic enzymes is highly desirable with forces occurring at the exhaust valve during decompres-
regard to the biofunctionality of the juice. sion. Future research may elucidate the reasons for the
PME negatively affects cloud stability of fruit juices as the observed differences and underlying inactivation mecha-
enzyme catalyses the demethylation of pectin. Free carboxyl nisms. The inactivation of POD and PPO in pineapple
groups arise, promoting the generation of coarse aggregates, puree by high-pressure and thermal treatments has already
cross-linked via bivalent cations resulting in undesired sedi- been studied by Chakraborty et al. (2015).
mentations and an overall quality loss (Terefe et al. 2014). In
our study, PME activity was not detected among all pineapple Micronutrients and Sugars
juices. Low PME activities have previously been reported by
Cautela et al. (2018) in fresh ‘Smooth Cayenne’ pineapple Vitamin C
juice. The activity may thus depend on the cultivar.
Pineapple juices contain low pectin levels (Will et al. 1999). All pineapple juices contained vitamin C at concentra-
The absence of the PME substrate renders high activities un- tions between 46.9 and 48.7 mg/100 mL, with a high
likely. PME is efficiently inactivated by thermal (Cautela et al. proportion of ascorbic acid (84–90%) compared with
2018; Terefe et al. 2014), but only partly by PCT treatment dehydroascorbic acid (10–16%; Table 3). These vitamin
(<40 °C), as demonstrated for orange juice (Aschoff et al. C concentrations were almost as high as those reported
2016). Cultivars exerting none or only low PME activities, for orange cv. ‘Navel Late’ juice (Aschoff et al. 2016).
such as ‘MD2’, may be recommended for the production of However, vitamin C contents may substantially vary
fresh-like pineapple juices. among individual pineapple varieties (Lobo and Paull
POD catalyses the oxidation of inter alia phenolic com- 2017). Thermally pasteurised juices from ‘Queen’ and
pounds in the presence of hydrogen peroxide. Undesirable ‘MD2’ pineapples have previously been reported to con-
discolouration and off-flavour formation may be the conse- tain vitamin C levels of 7.7 and 50.6 mg/100 mL, re-
quences, adversely affecting product quality (Terefe et al. spectively (Vollmer et al. 2020; Difonzo et al. 2019).
2014; Vámos-Vigyázó 1981). In pineapples, POD activity Surprisingly, thermal pasteurisation did not exert any ad-
gradually declines with progressing ripening; however, it is verse effects on vitamin C in our study, although it is known
still moderate in ripe fruit (Mehrlich and Felton 1980). In our for being susceptible to heat (Miller and Silva 2016). In com-
fresh pineapple juice, POD activity amounted to ~7.0 nkat/g. parison, vitamin C contents declined by almost 60% during
An effective inactivation was achieved by thermal thermal pasteurisation (90 °C/5 min) at laboratory scale in our
pasteurisation, being in agreement with literature (Vervoort previous study (Vollmer et al. 2020). Noteworthy, vitamin C
et al. 2011). Due to its high thermal stability, POD often serves contents of 57.6–61.5 and 32.6–38.4 mg/100 mL have previ-
as an indicator of sufficient heat treatments during food pro- ously been reported for thermally pasteurised pineapple juice
cessing, assuming that less thermally stable enzymes have processed at pilot plant and laboratory scale, respectively
already been inactivated (Miller and Silva 2016; Terefe et al. (Difonzo et al. 2019).
2014). Non-thermal PCT application also reduced POD; how- Interestingly, the use of PCT completely retained vi-
ever, the residual activities were still in the range of 77–90%, tamin C regardless of whether the pineapple juice was
possibly limiting the shelf life of the respective juices. treated once or twice. Hereby, dehydroascorbic acid
Conversely, Aschoff et al. (2016) have reported a complete levels slightly increased at the expense of ascorbic acid
inactivation of POD in orange juice after applying PCT with (statistically not significant at p ≤ 0.05). Conversely,
25 MPa and 50 MPa, respectively. They suggested that shear Aschoff et al. (2016) have observed a loss of 5–7% in
forces occurring during the decompression phase are respon- vitamin C due to declining dehydroascorbic acid levels
sible for this effect. However, the initial POD activity of the during PCT treatment of orange juice. Most likely, the
fresh orange juice in the afore-mentioned study of merely 1.0 inert working gas argon and, more importantly, flushing
± 0.1 nkat/g was considerably low compared with the 7.0 of the expansion tank with nitrogen that has not been
nkat/g determined herein. implemented by Aschoff et al. (2016) may have
PPO catalyses the hydroxylation and oxidation of phe- prevented the degradation of vitamin C in our study.
nolic compounds, resulting in yellow-coloured and reac-
tive o-quinones. The latter polymerise in a non-enzymatic B Vitamins
reaction, generating brown melanins impairing product
colour (Terefe et al. 2014). In our study, PPO activity Pineapples contain B vitamins, which are involved in
amounted to 3.2 ± 0.6 nkat/g in the fresh juice and was many important functions in the human body (Lobo
completely inactivated by both thermal pasteurisation and and Paull 2017). Table 3 summarises the concentrations
PCT. Concluding, PPO was more sensitive to the PCT of vitamin B1 (thiamin), B2 (riboflavin), B3 (niacin), B5
Food Bioprocess Technol (2020) 13:1833–1847 1843

Table 3 Micronutrients and sugars of fresh, thermally pasteurised, and PCT-treated pineapple juices

Parameters Pineapple juice

Fresh Thermally pasteurised PCT1 PCT2

Water-soluble vitamins
Vitamin C* (mg/100 mL) 48.3 ± 0.8a 47.7 ± 0.1a 48.7 ± 1.5a 46.9 ± 2.6a
Ascorbic acid (AA) 42.6 ± 0.1a 43.0 ± 0.1a 41.3 ± 1.6a 39.6 ± 2.2a
Dehydroascorbic acid (DHAA) 5.7 ± 0.6b 4.7 ± 0.0b 7.4 ± 0.1a 7.3 ± 0.4a
B vitamins* (μg/100 mL) 431.1 ± 15.7a 401.7 ± 9.2a 404.7 ± 5.3a 441.6 ± 36.3a
Thiamin (B1) 36.8 ± 0.6a 43.9 ± 0.0a 39.2 ± 2.8a 40.0 ± 1.8a
Riboflavin (B2) tr. tr. tr. tr.
Niacin (B3) 173.2 ± 8.9a 142.4 ± 3.2a 153.5 ± 8.9a 177.8 ± 36.3a
Pantothenic acid (B5) 131.9 ± 2.6a 131.7 ± 8.1a 133.3 ± 7.6a 136.9 ± 3.7a
Pyridoxine (B6) 89.2 ± 3.5a 83.8 ± 2.1a 78.8 ± 6.7a 87.0 ± 1.9a
Phenolic compounds and other metabolites
Amino acids and amines* (mg/100 mL) 5.5 ± 0.2ab 4.9 ± 0.1b 6.0 ± 0.3ab 6.7 ± 0.5a
L-Tyrosine (no. 1) 4.1 ± 0.2bc 3.5 ± 0.1c 4.6 ± 0.3ab 5.3 ± 0.4a
Serotonin (no. 2) 1.4 ± 0.0a 1.4 ± 0.0a 1.4 ± 0.0a 1.4 ± 0.0a
Furanones* (mg/100 mL) 4.0 ± 0.0a 4.0 ± 0.0a 3.9 ± 0.0ab 3.9 ± 0.1b
4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) (no. 3) 0.4 ± 0.0a 0.4 ± 0.0a 0.4 ± 0.0a 0.4 ± 0.0a
HDMF hexoside (no. 4) 2.1 ± 0.0b 2.2 ± 0.0a 2.0 ± 0.0c 2.0 ± 0.0c
HDMF malonyl hexoside (1) (no. 5) 0.6 ± 0.0a 0.6 ± 0.0a 0.6 ± 0.0a 0.6 ± 0.0a
HDMF malonyl hexoside (2) (no. 6) 0.9 ± 0.0a 0.9 ± 0.0a 0.9 ± 0.0a 0.9 ± 0.0a
Phenolic compounds* (mg/100 mL) 37.8 ± 0.3b 38.8 ± 0.0a 37.6 ± 0.3b 37.5 ± 0.1b
Syringoyl hexoside (no. 7) 2.0 ± 0.2a 1.4 ± 0.1b 2.2 ± 0.0a 2.3 ± 0.0a
Sinapoyl hexoside (no. 8) 0.9 ± 0.0b 1.2 ± 0.0a 0.8 ± 0.0c 0.7 ± 0.0d
S-Coniferyl-L-cysteine (no. 9) 1.9 ± 0.0b 1.9 ± 0.0b 1.9 ± 0.0b 2.0 ± 0.0a
S-Sinapyl-L-cysteine (no. 10) 4.1 ± 0.1a 4.1 ± 0.0a 4.1 ± 0.0a 4.2 ± 0.0a
Caffeoylisocitrate (no. 11) 0.9 ± 0.0a 0.7 ± 0.0b 0.9 ± 0.0a 0.9 ± 0.0a
S-Coniferylglutathione (no. 12) 6.2 ± 0.0b 6.5 ± 0.1a 6.0 ± 0.0c 5.8 ± 0.0d
S-Sinapylglutathione (no. 13) 12.1 ± 0.1b 12.9 ± 0.0a 11.7 ± 0.1bc 11.4 ± 0.1c
p-Coumaroylisocitrate (2) (no. 14) 1.3 ± 0.0b 1.5 ± 0.0a 1.3 ± 0.1b 1.2 ± 0.0b
N-L-γ-Glutamyl-S-coniferyl-L-cysteine (no. 15) 2.9 ± 0.1a 2.8 ± 0.0a 2.8 ± 0.0a 2.8 ± 0.0a
N-L-γ-Glutamyl-S-sinapyl-L-cysteine (no. 16) 5.6 ± 0.0b 5.8 ± 0.0a 5.7 ± 0.1b 5.9 ± 0.0a
(di-E,E)-N,N´-Diferuloylspermidine (no. 17) 0.2 ± 0.0a 0.2 ± 0.0a 0.2 ± 0.0a 0.2 ± 0.0a
Sugars
Total sugars* (g/100 mL) 9.7 ± 0.a 9.9 ± 0.1a 9.6 ± 0.1a 10.1 ± 0.4a
D-Glucose (Glc) 1.7 ± 0.1a 1.8 ± 0.1a 1.7 ± 0.1a 1.8 ± 0.1a
D-Fructose (Frc) 1.4 ± 0.1a 1.5 ± 0.1a 1.4 ± 0.1a 1.5 ± 0.1a
Sucrose (Suc) 6.7 ± 0.1a 6.8 ± 0.0a 6.6 ± 0.0a 6.8 ± 0.1a

tr., trace (<LOQ)


*
Calculated as sum of individual compounds
Different superscript letters within one row indicate significant (p ≤ 0.05) differences among means determined by ANOVA and Tukey’s test

(pantothenic acid), and B 6 (pyridoxine), together vitamins were not significantly affected by thermal and
amounting to 401.7–441.6 μg/100 mL across all pine- PCT treatments. Some B vitamins have been reported as
apple juices. The individual concentrations were sub- being sensitive to thermal processing (Miller and Silva
stantially lower than those reported by the USDA 2016; Eitenmiller et al. 2008). However, with the exception
(2019) for raw ‘MD2’ pineapples, possibly resulting of vitamin B1 and B6 model solutions subjected to high-
from processing or seasonal fluctuations. The B pressure processing (99–100% retention) (Sancho et al.
1844 Food Bioprocess Technol (2020) 13:1833–1847

1999), studies on the effects of non-thermal processing on B cysteine (9 and 10) conjugates. Minor phenolic compounds
vitamins are still widely lacking (Zhang et al. 2011). included phenolic glycosides (7 and 8), esters (11 and 14), and
one amide (17).
Phenolic Compounds, Furanones, and Other Metabolites Total phenolic compounds in the fresh juice
amounted to ~37.8 mg/100 mL. Thermal pasteurisation
The concentrations of phenolic compounds, genuine pineap- caused a slight, but significant (p ≤ 0.05), rise to 38.8
ple furanones, L-tyrosine, and serotonin are displayed in mg/100 mL, being in agreement with our previous re-
Table 3. The most abundant constituents were S-coniferyl sults (Vollmer et al. 2020). Compared with this previous
and S-sinapyl glutathione (12 and 13), in addition to their study, the stabilisation of phenolic compounds was con-
corresponding N-L-γ-glutamyl-L-cysteine (15 and 16) and L- siderably less pronounced. This may be attributed to the

Fig. 4 Score plots of the principal component analysis (PCA) calculated corresponding loading plots are displayed in a' and b'. Ellipses in the score
on the basis of general juice quality traits, microbial loads, and enzyme plots illustrate clusters from hierarchical cluster analysis (HCA).
activities (a) and levels of genuine juice constituents (b). The Abbreviations are defined in Tables 1, 2, and 3.
Food Bioprocess Technol (2020) 13:1833–1847 1845

differences in production scales (laboratory vs. pilot of the PCT-treated juices was particularly influenced by the
plant scale) as already mentioned in the section about loadings of the lightness (L*) and the total colour difference
vitamin C. The content of phenolic compounds (ΔE*). The thermally pasteurised juice was separated by PC2
remained almost unaffected in both PCT-treated juices. due to the elevated hue angle (h°), but also due to the complete
In contrast, Aschoff et al. (2016) have reported signifi- inactivation of microorganisms and most enzymes. Their
cant losses of hesperidin during PCT treatment of or- slightly elevated total soluble solids (TSS) further promoted
ange juice. However, the high residual POD activities of this clustering, although the differences were rather small be-
our samples may promote the oxidation of phenolic tween the individual treatments.
compounds during prolonged storage. The second PCA illustrated in Fig. 4b,b' was calcu-
The pineapple juices contained 4-hydroxy-2,5-dimethyl- lated on the basis of the genuine juice constituents
3(2H)-furanone (HDMF, 3) and some glucosides thereof (4, (Table 3). The first two PCs explained 73% of the total
5, and 6), being consistent with a previous study (Vollmer variance of this data set (PC1: 49%, PC2: 24%). The
et al. 2020). Owing to its ‘sweet and pineapple- and cara- thermally pasteurised juices formed one clear-cut clus-
mel-like’ odour and low odour threshold in water of 0.001 ter. The location of this cluster in the upper right quad-
mg/100 mL (Tokitomo et al. 2005), HDMF may be of vital rant was correlated with the position of the loadings of
importance for the sensory quality of the pineapple juices HDMF hexoside (4) and several phenolic compounds
assessed. The latter contained 3.9–4.0 mg/100 mL of this (8, 12, 13, and 14) that were found in slightly elevated
key odorant of fresh pineapples, irrespective of the treatments concentrations in the thermally pasteurised juice
applied. These values were substantially lower than the 10.4– (Table 3). The fresh and the PCT-treated samples
14.0 and 18.0–20.6 mg/100 mL reported previously for juices formed one widespread cluster that was clearly separat-
from ‘MD2’ and ‘Queen’ pineapples, respectively (Vollmer ed from the thermally pasteurised juice. This differenti-
et al. 2020; Difonzo et al. 2019). ation was inter alia promoted by the elevated levels of
dehydroascorbic acid (DHAA).
Sugars Concluding, multivariate statistics classified both PCT-
treated juices to the fresh juice with regard to the phytochem-
The pineapple juices contained sucrose (6.6–6.8 g/100 mL), ical composition and to the thermally pasteurised juice with
followed by D-glucose (1.7–1.8 g/100 mL) and D-fructose respect to the remaining physical and biochemical quality
(1.4–1.5 g/100 mL), thus being largely within the typical parameters.
ranges of pineapple juice (AIJN 2016). The proportions of
sucrose (67–69%) and reducing sugars (32–33%) matched
with those reported by Mehrlich and Felton (1980). The dif- Conclusions
ferent treatments had no effect on the afore-mentioned three
sugars. These findings were consistent with those of Vervoort To the best of our knowledge, continuous PCT was
et al. (2011), who have reported that neither thermal used for the first time for the non-thermal processing
pasteurisation (72 °C/20 s) nor high-pressure processing of pineapple juice on pilot scale. Our experiments not
(600 MPa/60 s/5 °C initial temperature) altered the sugar pro- only emphasised the prospects of this innovative tech-
file of orange juice. nology, but also revealed current limitations and the
need for further process optimisation. In particular, the
Statistical Pattern Recognition high retention of BRM activity and the significant re-
duction of the mean particle diameter, which substan-
The first two PCs of the PCA, calculated on the basis of tially reduced centrifugable pulp contents and enhanced
general quality traits, colour values, particle sizes (Table 1) cloud stability, proved to be the most conspicuous ad-
in addition to microbial counts and enzyme activities vantages of PCT compared with thermal pasteurisation.
(Table 2), explained 90% of the total variance (PC1: 56%, However, the high residual POD activity and the unex-
PC2: 34%) as illustrated in Fig. 4a,a'. The fresh juice samples pectedly small reduction of the microbial loads may
formed one single cluster, located in the lower right quadrant. adversely affect quality and limit the shelf life of
Its position and separation along PC1 were mainly related to PCT-treated pineapple juices. A second passage through
the loadings of microbial counts (TAPC and YMC) and en- the PCT device was less effective than expected. Hence,
zyme activities (BRM, POD, and PPO) as well as those related ongoing research may particularly focus on further op-
to the particle size distribution (d values). Both PCT-treated timisation of process parameters and the design of the
juices were closely clustered in the upper left quadrant. They PCT apparatus to overcome the above obstacles.
formed one joint HCA cluster with the thermally pasteurised Complementing the results presented herein, future re-
juice but were separated from the latter along PC2. Positioning search may additionally assess the long-term preservation
1846 Food Bioprocess Technol (2020) 13:1833–1847

effects of PCT and the storage stability of the PCT-treated Baron, A., Dénes, J.-M., & Durier, C. (2006). High-pressure treatment of
cloudy apple juice. LWT - Food Science and Technology, 39(9),
juices ensuring adequate shelf life and food safety, as well as
1005–1013. https://doi.org/10.1016/j.lwt.2006.02.016 .
the aroma-determining volatiles, the descriptive sensory pro- Baur, S., Klaiber, R. G., Koblo, A., & Carle, R. (2004). Effect of different
file, and the consumer acceptance of PCT-treated juices. washing procedures on phenolic metabolism of shredded, packaged
iceberg lettuce during storage. Journal of Agricultural and Food
Acknowledgements We gratefully acknowledge Klaus Mix (University Chemistry, 52(23), 7017–7025. https://doi.org/10.1021/jf048961a .
of Hohenheim) for his excellent assistance during pineapple juice pro- Bönsch, K., Wecks, M., Ondruschka, J., & Staudt, R. (2007). The effect
duction, and we thank Tobias Pöhnl for his helpful support in sugar of a new pressure change technology (PCT) on microorganisms: An
analysis. We are also grateful to Domenico Albrich and Katharina Henn innovate concept for food safety. Chemical Engineering &
for their contributions to the analytical work as part of their master pro- Technology, 30(6), 755–757. https://doi.org/10.1002/ceat.
gramme. 200600402.
Cautela, D., Castaldo, D., & Laratta, B. (2018). Thermal inactivation of
Funding Open Access funding provided by Projekt DEAL. pectin methylesterase in pineapple juice. Journal of Food
Measurement and Characterization, 12(4), 2795–2800. https://doi.
org/10.1007/s11694-018-9894-1 .
Compliance with Ethical Standards Chakraborty, S., Rao, P. S., & Mishra, H. N. (2015). Kinetic modeling of
polyphenoloxidase and peroxidase inactivation in pineapple
Conflict of Interest The authors declare that they have no conflict of (Ananas comosus L.) puree during high-pressure and thermal treat-
interest. ments. Innovative Food Science and Emerging Technologies, 27,
57–68. https://doi.org/10.1016/j.ifset.2014.11.003.
Open Access This article is licensed under a Creative Commons Difonzo, G., Vollmer, K., Caponio, F., Pasqualone, A., Carle, R., &
Attribution 4.0 International License, which permits use, sharing, adap- Steingass, C. B. (2019). Characterisation and classification of pine-
tation, distribution and reproduction in any medium or format, as long as apple (Ananas comosus [L.] Merr.) juice from pulp and peel. Food
you give appropriate credit to the original author(s) and the source, pro- Control, 96, 260–270. https://doi.org/10.1016/j.foodcont.2018.09.
vide a link to the Creative Commons licence, and indicate if changes were 015.
made. The images or other third party material in this article are included
Eitenmiller, R. R., Ye, L., & Landen, W. O. (2008). Vitamin analysis for
in the article's Creative Commons licence, unless indicated otherwise in a
the health and food sciences (2nd ed.). Boca Raton: CRC Press.
credit line to the material. If material is not included in the article's
FAO (2019). FAOSTAT. Food and Agriculture Organization of the
Creative Commons licence and your intended use is not permitted by
United States. http://www.fao.org/faostat/en/#data. Accessed 14
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July 2019.
permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Genovese, D. B., Lozano, J. E., & Rao, M. A. (2007). The rheology of
colloidal and noncolloidal food dispersions. Journal of Food
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Hirsch, A. R., Förch, K., Neidhart, S., Wolf, G., & Carle, R. (2008).
Effects of thermal treatments and storage on pectin methylesterase
and peroxidase activity in freshly squeezed orange juice. Journal of
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