Food Control 124 (2021) 107874

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Production of liquid smoke using fluidised-bed fast pyrolysis and its

application to green lipped mussel meat
Xing Xin a, Amy Bissett a, Joyce Wang a, Andrew Gan a, Kiri Dell b, Saeid Baroutian a, *
a
Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand
b
Faculty of Business and Economics, The University of Auckland, Auckland, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: Liquid smoke flavouring as an all-natural food additive is usually prepared by smouldering or a carbonisation
Bioactive compounds method. In this study, liquid smoke was prepared by fast pyrolysis technology using a fluidised bed reactor with a
Fast pyrolysis yield of 30 wt%. GC-MS analysis showed that main functional compounds were furans, carbonyls and phenols.
Liquid smoke
The fast pyrolysis liquid smoke presented a high antioxidant capacity with a total phenolic content of 21.9 mg
Smoked mussel
Preservation
GAE/mL. Cytotoxicity analysis indicated that it was as safe as commercial liquid smoke products. This fast
Toxicity pyrolysis liquid smoke was diluted and applied to treat green lipped mussel meat, and the preserving effects were
studied over 30-day refrigerated storage. The microbiological analysis results showed bacteria growth was
inhibited during the first 14 days of the storage period. The image and texture analysis results showed insig­
nificant changes to liquid smoked mussel meat during storage except for colour darkening and decreased
hardness in meat texture.

1. Introduction methods to prepare liquid smoke. The application includes to preserve

Food smoking process is popular due to the enhancement of flavours,
colours, and prolonged preserving quality of treated food (Ledesma,
Rendueles, & Díaz, 2017, pp. 201–243). Liquid smoke flavouring is an
all-natural food additive, and it is a safer alternative to the traditional
smoking process (Maga, 2009). It as a food ingredient is ‘generally
regarded as safe’ (GRAS) (Holley & Patel, 2005) with United States (75%
of the market) and Europe (30% of the market) making up the largest
consumers of liquid smoke (Alçiçek & Balaban, 2015b). Alternative
names for liquid smoke include smoke flavouring, wood condensate,
wood vinegar and pyroligneous acid (Maga, 2018).
Three liquid smoke production methods are: 1) smouldering - a slow,
low-temperature, flameless form of combustion under a limited oxygen
supply; 2) carbonisation - a slow pyrolysis using an external heat source
in the absence of oxygen; and 3) fast pyrolysis - similar to carbonisation
except higher heat temperatures are applied. In comparison with
smouldering and carbonisation, fast pyrolysis has advantages of reduced
environmental impacts, higher liquid yields, easier control of the pro­
cess (Bridgwater & Cottam, 1992), and more bioactive compounds
(Underwood & Graham, 1989).
Most of the previous studies used smouldering or carbonisation
and improve the quality of protein-based food such as meat, seafood and
cheese (Martinez, Salmerón, Guillén, & Casas, 2011). Previous studies
also investigated the effect of liquid smoke on mussels (Alçiçek &
Balaban, 2015b), salmon (Ayvaz, Balaban, & Kong, 2017), frankfurters
(Martin et al., 2010), beef (Dimakopoulou-Papazoglou & Katsanidis,
2017), pork (Martinez, Salmeron, Guillen, & Casas, 2004), etc. On the
other hand, limited literature of fast pyrolysis method exists in the
public domain due to the tendency for this method to be patented and
applied at a commercial scale (Emmerson, 2011).
In this study, liquid smoke was produced by fast pyrolysis of hickory
woodchips in a lab-scale fluidised bed reactor. Fractional condensation
was also used to selectively condense liquid smoke and other valuable
products. The obtained liquid smoke was analysed to understand its
chemical and biological characteristics. This fast pyrolysis liquid smoke
was used to preserve New Zealand green lipped mussel (Perna canalic­
ulus) meat. It is an iconic seafood product of New Zealand where
traditional smoking is still used. A comprehensive study on the chemical
characteristics, health benefit, safety, and application aspects of fast
pyrolysis liquid smoke is of significant interest in research and industry.

* Corresponding author.
E-mail address: s.baroutian@auckland.ac.nz (S. Baroutian).

https://doi.org/10.1016/j.foodcont.2021.107874
Received 5 May 2020; Received in revised form 25 November 2020; Accepted 10 January 2021
Available online 14 January 2021
0956-7135/© 2021 Elsevier Ltd. All rights reserved.
X. Xin et al.
2. Materials and methods
2.1. Liquid smoke production
Hickory woodchips were purchased from Smokai Limited (New
Zealand). The particle size was in the range of 2–4 mm to meet the re­
quirements of a fluidised bed reactor, and the moisture content was 9.2
wt% as received.
A bubbling fluidised bed reactor was employed to prepare liquid
smoke by fast pyrolysis process. This reactor as illustrated in Fig. 1
consisted mainly of a biomass hopper, a feeding auger, a fluid bed, an
electric nitrogen gas heater, two cyclones, three condensers (shell and
tube type) and a cotton filter for gas filtration.
Woodchips were loaded in biomass hopper and silica sand loaded
into the fluid bed. The fluidised bed reactor was heated to the target
temperature of 420 ◦ C. Nitrogen gas at a flowrate of 60 SLPM (standard
litre per minute) was preheated to 420 ◦ C by the gas heater and was
purged into the bed from the bottom. Before fast pyrolysis, woodchips
were fed into the bed bottom via the auger at a feeding rate of 1.2 kg/h.
The woodchips were pyrolysed in the bed, and converted to smoke,
pyrolytic gas and biochar. All products were entrained with nitrogen gas
flow to cyclones 1 and 2 in series. The two cyclones collected only
biochar by maintaining at a temperature of approximately 350 ◦ C to
avoid any condensation. The smoke and pyrolytic gas were entrained to
condensers 1, 2 and 3 in series. The temperature in condenser 1 was set
to 120–130 ◦ C by maintaining a balance of heat exchange between hot
smoke and air. Condenser 1 collected only thick tar containing high
boiling-point compounds. Condenser 2 was kept at 20–25 ◦ C by
exchanging heat with tap water flowing through the condenser’s tubes,
and liquid smoke was collected at this stage. The smoke was further
cooled to 5 ◦ C in condenser 3 to collect remained smoke condensate.
Condenser 3 exchanged heat with chilled water at the same tempera­
ture. A gas filter was connected to condenser 3 to capture smoke aero­
sols. Wood smoke usually contained aerosols, which were hard to be
captured by shell and tube condensers. Lastly, pyrolytic gas and nitrogen
gas were vented to a fume cabinet.
After fast pyrolysis, the raw liquid smoke from condensers 2 and 3
was mixed and centrifuged at 1000 rpm for 10 min. An oil phase product
was separated from the raw liquid smoke. A full-strength liquid smoke
was obtained and stored at 5 ◦ C.
2.2. Liquid smoke characterisation
2.2.1. Chemical characterisation
Gas Chromatography-Mass Spectrometry (GC-MS) analysis was
conducted twice to identify chemical compounds contained in the fast
pyrolysis liquid smoke. The liquid smoke was mixed with dichloro­
methane in a volume ratio of 1/2 (liquid smoke/dichloromethane)
before GC-MS analysis. Then the mixture was agitated at 200 rpm for 6
h. Dichloromethane was purchased from Sigma-Aldrich (New Zealand).
The dichloromethane extract samples were injected to a Shimadzu
Fig. 1. Schematic of bubbling fluidised bed reactor for fast pyrolysis.
2
Food Control 124 (2021) 107874
GCMS QP5000 instrument (Kyoto, Japan) equipped with a DB-5HT
column (30 m × 0.25 mm × 0.1 μm). An injection volume of 1 μL
with a split ratio of 100:1 was used, and the injection temperature was
set at 280 ◦ C. The oven temperature was set initially at 50 ◦ C and
increased to 250 ◦ C by a rate of 20 ◦ C/min, then was held for 5 min.
Helium gas was used as the carrier with a flow rate of 3 mL/min. Mass
spectra were operated in electron ionization mode at 70 eV, and the
mass range was 50–300 amu for acquisition. Chemical components were
identified by comparing the mass spectra with those in the library NIST
and by comparing the data results with previous studies (Bridgwater,
2012; Montazeri, Oliveira, Himelbloom, Leigh, & Crapo, 2013). Their
relative proportions were calculated based on the peak area of total
ionization chromatogram (TIC).
The concentration (mg/mL) of residual matters in the fast pyrolysis
liquid smoke was measured gravimetrically following a previous study
(Vidal, Goicoechea, Manzanos, & Guillén, 2017). Three 5 mL of aliquots
were transferred into three pre-dried aluminium pans, respectively.
These samples were dried in a 40 ◦ C oven until they reached constant
weights. The results indicated the content of residual matters in the
liquid smoke, as some light molecules were volatilised during
oven-drying.
The pH value of the fast pyrolysis liquid smoke was measured using a
pH meter (Mettler FE28).
2.2.2. Determination of total phenolic content
The total phenolic content (TPC) was determined by a spectropho­
tometric assay method, Folin–Ciocalteu assay described in a previous
study (Putnam, Bombick, Avalos, & Doolittle, 1999). TPC value in­
dicates the antioxidant capacity of liquid smoke, as phenols are the
essential antioxidants in liquid smoke (Singleton, Orthofer, &
Lamuela-Raventós, 1999). Particularly, methoxyphenols are efficient
scavengers of free radicals due to their hydroxyl groups (Soldera,
Sebastianutto, & Bortolomeazzi, 2008). Chemicals for analysis included
gallic acid, sodium carbonate and Folin-Ciocalteu’s phenol reagent from
Sigma-Aldrich (New Zealand).
Gallic acid was used to prepare standard solutions with concentra­
tions of 1.000, 0.500, 0.250, 0.100, 0.050 and 0.025 mg/mL. After
preparation, 200 μL of the liquid smoke sample, standard solution and
distilled water (blank) aliquots were pipetted into a 96-well plate. Then
100 μL of 10-fold diluted Folin-Ciocalteu reagent and 800 μL of 750 mM
sodium carbonate aliquots were pipetted into each well. After incu­
bating for 30 min in the dark, the absorbance of each well was measured
using a PerkinElmer ultraviolet–visible spectrophotometer at 765 nm.
Absorbance values of measured samples were then compared against the
calibration curves to determine the equivalent values. The TPC results
were reported as mg gallic acid equivalent/mL (mg GAE/mL).
2.2.3. Cytotoxicity assay
A cell line derived from murine fibroblast (L cells, ATCC® CRL-
2648™) was purchased from In Vitro Technology (New Zealand) in May
2019. Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC®
30–2002™) and fetal bovine serum for culturing the cell line were
purchased from the same place. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide), PBS (phosphate buffered saline) and
DMSO (dimethyl sulfoxide) were from Sigma-Aldrich (New Zealand).
The effect of fast pyrolysis liquid smoke on L cells viability was
determined using the MTT colourimetric assay (Kjällstrand & Petersson,
2001; Mosmann, 1983). A completed culture medium, DMEM supple­
mented with 10% fetal bovine serum, was prepared for cell culturing.
For cytotoxicity analysis, L cells were seeded in a 96-well plate each at a
density of 10 × 103 cells per well. Then they were grown at 37 ◦ C in a
humidified atmosphere for 24 h. Distilled water as control and liquid
smoke samples diluted with the completed medium (DMEM supple­
mented with 10% fetal bovine serum) to concentrations in the range of
7–1919 μg/mL were added to the wells and incubated for 24 h at 37 ◦ C
and 5% CO2.
X. Xin et al.
After the treatment, supernatants were removed, and 10 μL of MTT
solution (5 mg/mL of MTT in PBS) was added to each well and incubated
for 4 h. Then, 100 μL of DMSO was added onto the supernatant-drained
cell layer to dissolve the precipitate (formazan product). The absorbance
of the supernatant was immediately measured at 540 nm using a
microplate reader (Thermo Scientific Multiskan GO). The anti­
proliferation rate (or inhibition rate or percentage death) was calculated
according to Equation (1):
Ac − Ak
Antiproliferation rate (%) = × 100 (1)
Ac
where Ak is the absorbance value of the sample group; Ac is the absor­
bance value of the control group.
The IC50, which is the concentration of liquid smoke that inhibited
cell survival by 50%, was obtained using the non-linear regression
fitting model based on the Four-Parameter Logistic dose-response curve
(Sebaugh, 2011).
2.3. Liquid smoke application
2.3.1. Liquid smoking of mussel meat
Fresh New Zealand green lipped mussels were obtained from a local
supermarket in Auckland, New Zealand. The shells were removed, and
the mussel meat was washed with distilled water. The samples were then
air-dried at room temperature for 10 min.
The fast pyrolysis liquid smoke was diluted with distilled water to
concentrations of 5, 10 and 15 vol%. Dilution of liquid smoke is usually
required before food treatment to soften the flavours. After removing
mussels’ shells, the meat pieces were dipped in each of the three diluted
liquid smoke samples for 30 s. The liquid smoked mussel meat and non-
smoked as control were vacuum-packed in plastic bags, and there were
three pieces of mussel meat in each bag. Then they were stored in a
refrigerated incubator at 4 ◦ C. The microbiological, colour, texture and
water activity analysis was conducted on day 0, day 14 and day 30,
repeatedly.
2.3.2. Microbiological analysis
Chemicals and media for analysis were peptone water, plate count
agar (PCA) and violet red bile glucose agar (VRBGA) from Sigma-Aldrich
(New Zealand). The total viable bacterial counts (TBC) and the
Enterobacteriaceae bacterial counts of treated and non-treated mussel
meat were determined following a previous study (Siskos, Zotos, Meli­
dou, & Tsikritzi, 2007). Before analysis, mussel meat samples (25 g)
were homogenized with 0.1% peptone water (130 g) at room tempera­
ture using a laboratory paddle blender (Stomacher 400). Then 1 mL of
each homogenate was transferred to a sterilized Petri dish.
Plate count agar (PCA) was used as the microbiological growth
medium for TBC analysis. Homogenate samples were mixed with 10 mL
of PCA in the Petri dishes and then were incubated at 30 ◦ C for 72 h.
Violet red bile glucose agar (VRBGA) was used as the medium for
Enterobacteriaceae analysis. Homogenate samples were mixed with 10
mL of VRGBA in Petri dishes and then were incubated at 30 ◦ C for 24 h.
Colonies on the Petri dishes were counted after the incubation. The
microbial counts were reported as colony-forming unit per gram (CFU/
g).
2.3.3. Image analysis
The method of image analysis followed a previous study (Alçiçek &
Balaban, 2015b). The illumination system used two fluorescent light
bulbs (Lumichrome F15W1XX, colour temperature = 6500 K, colour
retention index = 98, Lumiram, Larchmont, NY, USA). One fluorescent
bulb was located at the top for front lighting, and the other at the bottom
for bottom lighting. Images of the mussel meat samples were taken using
a Nikon D300 digital camera with an 18–200 mm zoom Nikkor lens
mounted at the top of a lightbox. The LensEye software (Version 10.3.4,
3
Food Control 124 (2021) 107874
ECS, Gainesville, FL, USA) was used to analyse the visual attributes of
the images.
The colour of each mussel sample was determined by L*a*b* co­
ordinates (Caglak, Cakli, & Kilinc, 2008). The L* coordinate represents
the lightness, where it varies from 100 (white) to 0 (black). The a* co­
ordinate represents the colour from red (+a*) to green (-a*), while the
b* coordinate represents the colour from yellow (+b*) to blue (-b*).
2.3.4. Texture profile and water activity analysis
The texture profile analysis of mussel meat was conducted using a
Brookfield CT3 Texture Analyzer (USA). A cylindrical flat acrylic probe
(TA5) with a diameter of 12.7 mm was used for compression. The trigger
speed of the probe was set at 1.5 mm/s. Samples were compressed on the
thickest location twice to 60% of the original thickness. The software,
TextureProCT, automatically acquired the force-deformation data of the
compression and calculated TPA (texture profile analysis) parameters
including hardness (g), cohesiveness, springiness (mm) and chewiness
(mJ).
For water activity, a piece of mussel meat sample was minced and
placed in a plastic dish. Water activity was measured using LabMaster-
aw 111–9971. The plastic dish was placed in the machine chamber,
and three readings were taken after equilibration was reached at 25 ◦ C.
2.3.5. Statistical analysis
Analyses in this study were conducted in triplicates, and the results
were presented as mean values ± standard deviation (n = 3). Analysis of
variance (ANOVA) was performed, and the difference between means
was analysed using Duncan’s test. Statistical significance was considered
at p < 0.05. All statistical analysis was performed using SPSS 9.05
(Chicago, USA).
3. Results and discussion
3.1. Fast pyrolysis
The product distribution of hickory wood fast pyrolysis is presented
in Fig. 2. The products from the fluidised bed reactor in series were
biochar, heavy tar, raw liquid smoke, aerosol oil and pyrolytic gas.
Biochar was firstly obtained from cyclones 1 and 2 with a yield of 20.5
wt%. The yield of heavy tar collected in condenser 1 was 5.4 wt%. The
raw liquid smoke obtained from condensers 2 and 3 gave the highest
yield of 29.9 wt%. The yields of aerosol oil from gas filter and pyrolytic
gas were 21.4 wt% and 22.8 wt% respectively. Fast pyrolysis was con­
ducted once to produce enough liquid smoke for this research. Previous
studies have proved that fluidised bed reactors had a good
Fig. 2. Distribution of products obtained by fast pyrolysis of hickory wood
at 420 ◦ C.
X. Xin et al. Food Control 124 (2021) 107874

reproducibility from the viewpoint of process engineering (±3% errors). Table 1

(Xin, Torr, Mercader, et al., 2019; Xin, Torr, Pang, et al., 2019). Compounds identified by GC-MS analysis with the relative percentages of peak
Raw liquid smoke from condensers 2 (20–25 ◦ C) and 3 (5 ◦ C) gave area.
yields of 27.0 wt% and 2.9 wt%, respectively. This raw liquid smoke was Compounds Retention time Peak area (%)
centrifuged to remove any remained oil, and the final product liquid (min)
1 2
smoke was composed of 28 wt% of hickory woody feedstock. In com­
Furans 34.99 36.01
parison, a previous study showed a lower yield (5–12 wt%) of liquid
Furfural 3.40 15.26 15.80
smoke in slow pyrolysis (Yuniningsih & Anggraini, 2013). 2-Furanmethanol 3.83 5.59 5.72
Any oil residue has to be removed from liquid smoke after smoke Tetrahydro-2,5-dimethoxy-furan 4.43 2.22 2.27
condensation (Simon, de la Calle, Palme, Meier, & Anklam, 2005). 2(5H)-Furanone 5.26 8.00 8.33
Fractional condensation showed a significant advantage over one stage 5-Methyl-5H-furan-2-one 6.06 1.30 1.30
3-Methyl-2(5H)-furanone 7.47 1.27 1.28
condensation method, as it efficiently separated liquid smoke and a 2-Furanone, 2,5-dihydro-3,5-dimethyl 8.43 0.68 0.66
bio-oil product, including heavy tar and aerosol oil. The oil product 4-Methyl-5H-furan-2-one 10.32 0.67 0.65
contained little water but substantial fragments from lignin and cellu­ Phenols 35.13 34.97
lose. Besides burning it for heat energy, the potential application in­ Phenol 7.69 1.08 1.08
m-Cresol 12.12 1.10 1.17
cludes biocides, wood preservatives, and resins (Bridgwater, 2012;
Guaiacol 12.63 4.25 4.36
Tiilikkala, Fagernäs, & Tiilikkala, 2010). Creosol 18.59 2.62 2.71
Catechol 19.36 1.47 1.36
3.2. Chemical characterisation 3-Methoxy catechol 22.61 1.05 0.95
3-Methyl catechol 23.04 0.64 0.58
4-Ethyl guaiacol 23.86 1.04 1.06
3.2.1. Chemical composition 4-Methyl catechol 24.89 0.48 0.47
Table 1 presents the identified compounds of the full-strength liquid 4-Vinyl guaiacol 25.97 0.53 0.53
smoke using GC-MS analysis. The identified compounds were mostly Syringol 28.41 5.91 5.98
furans, carbonyls and phenols. They were the main contributors to 2-Methoxy-3-allylphenol 28.71 1.08 1.00
Vanillin 31.05 0.45 0.44
liquid smoke’s functional properties including flavouring, colouring,
4-Methyl syringol 34.25 4.70 4.75
antimicrobial and antioxidant effects (Maga, 2018). Other identified 1,2,3-Trimethoxy-5-methyl-benzene 38.97 1.71 1.69
compounds were alcohols, acids and nitrogenated compounds. The 1-(3,5-Dimethoxyphenyl)ethanone 41.13 0.78 0.76
primary carcinogens in wood smoke (Zachara, Gałkowska, & Juszczak, trans-4-Propenylsyringol 43.37 0.81 0.81
2,6-Dimethoxy-4-prop-1-enylphenol 46.01 0.46 0.46
2017), poly-aromatic hydrocarbons (PAHs) were not found in this study.
Syringe aldehyde 46.31 0.87 0.81
A relatively low (420 ◦ C) pyrolysis temperature was maintained in this (E)-2,6-Dimethoxy-4-1-enylphenol 48.63 1.89 1.89
study to minimise their formation. Because mixed oxygenates from 4-Acetylsyringol 50.25 0.66 0.62
fragments of woody biomass were the main products at pyrolysis tem­ Homosyringic acid 52.38 0.89 0.85
peratures in the range of 400–450 ◦ C, and formation of PAHs was Sinapic acid 62.45 0.66 0.64
Carbonyls 27.39 26.59
induced at higher temperatures (Diebold, 1997).
1-Acetoxy-2-propanone 4.13 4.13 4.29
Furfural and 2(5H)-furanone shown in Table 1 were two major 2-Methyl-2-cyclopenten-1-one 5.03 3.66 2.91
compounds in the furans group. Furans are pyrolysis products of 2-Hydroxy-2-cyclopenten-1-one 5.55 8.08 7.96
hemicellulose and cellulose (Montazeri et al., 2013), and they contribute Acetonylacetone 5.76 0.56 0.59
n-Propyl acetate 6.47 0.46 0.35
to overall odour and aroma of liquid smokes (Michailof et al., 2014).
Furo[3,4-b]furan-2,6(3H,4H)-dione 6.88 2.90 3.02
Syringol and its derivatives were important phenolic compounds shown 3,4-Dimethyl-2-cyclopenten-1-one 8.04 0.37 0.35
in Table 1, which could boost the flavour properties and make hickory 3-Methyl-1,2-cyclopentanedione 9.50 5.67 5.69
wood a popular smoke source for food flavouring (Maga, 2009). Cate­ 2,3-Dimethyl-2-cyclopenten-1-one 10.03 0.67 0.61
chol and its derivatives (Table 1) contributed to liquid smoke’s antiox­ 3-Ethyl-2-hydroxy-2-cyclopenten-1-one 14.26 0.89 0.83
Nitrogenated compounds 1.90 1.86
idant capacity (Yang et al., 2016). Phenols contributed to liquid smoke’s
2-Nitropentane 3.20 0.86 0.87
flavouring, colouring, antioxidant and antimicrobial properties (Silva N,N-Diamylmethylamine 8.22 1.04 1.00
et al., 2018). Alcohols 0.40 0.37
2-Hydroxy-2-cyclopenten-1-one and 3-methyl-1,2-cyclopentane­ 1-Cyclopropyl-4-methyl-1,3- 14.96 0.40 0.37
dione were found to be the main carbonyl compounds shown in cyclohexanediol
Acids 0.18 0.20
Table 1. They were formed from thermal degradation of cellulose and 3-Methyl-3-butenoic acid 3.66 0.18 0.20
hemicellulose and often detected in smoke flavourings (Guillén &
Ibargoitia, 1996). Carbonyls affected the colour and textural properties
of smoked food via Maillard reactions with proteins (Lund & Ray, 2017). measured. This result was in agreement with the reported pH values
They promoted the overall sensory quality by providing a caramel (2.3–5.7) of commercial liquid smoke products (Montazeri et al., 2013).
flavour to soften phenols’ heavy smoky aromas, and contribute to Liquid smoke contained many acidic compounds contributing to the
antimicrobial capacity (Maga, 2009). food flavour, colour, texture and antimicrobial capacity.
The TPC value of fast pyrolysis liquid smoke was 21.9 ± 1.5 mg GAE/
3.2.2. Chemical characteristics mL. A previous study reported TPC values of 5.3 and 4.4 mg GAE/mL for
The concentration of liquid smoke residual matters in oven drying two liquid smoke samples by using a slow pyrolysis method (Manu &
was found to be 109.0 ± 5.0 mg/mL. A previous study used the same Sangsrichan, 2009). Similarly, another study reported TPC values of
method and found that the concentration of a wood smoke condensate liquid smoke ranging from 0.94 to 4.74 mg GAE/mL (Ma et al., 2013). In
was 186 mg/mL (Putnam et al., 1999). They also found the concentra­ comparison, fast pyrolysis liquid smoke showed a much higher antiox­
tions of commercial liquid smokes were in the range of 44–74 mg/mL. idant capacity. This result agreed with the abundance of phenols shown
The commercial liquid smoke samples may be mixed with water and in Table 1. Therefore, fast pyrolysis process showed an advantage of
other flavouring ingredients to improve its sensorial properties. In elevating the antioxidant capacity of liquid smoke.
contrast, the fast pyrolysis liquid smoke and wood smoke condensate
were non-diluted samples.
The pH value of fast pyrolysis liquid smoke was 2.36 ± 0.01 as

4
X. Xin et al. Food Control 124 (2021) 107874

3.3. Cytotoxicity assay Table 2

Microbiological analysis of liquid smoked mussel meat during 30 days refrig­
The dose-response relationship for fast pyrolysis liquid smoke erated storage.
treated L cells is shown in Fig. 3. As can be seen, liquid smoke hardly Days Non-treated Liquid smoked
affected the cell viability (>0.90) at low concentrations (<26 μg/mL). 5 vol% 10 vol% 15 vol%
However, the result exhibited concentration-dependent behaviour, and
TBC, log CFU/g
cell viability was lower at higher concentrations. The acidic environ­
0 3.65 ± 0.04a,A 3.03 ± 0.00b,A 2.63 ± 0.04d,A 2.78 ± 0.01c,A
mental conditions of liquid smoke were mainly responsible for the 14 4.25 ± 0.04a,B 0.00 ± 0.00b,B 0.00 ± 0.00b,B 0.00 ± 0.00b,B
reduction of cell viability (Kim et al., 2011). The same cytotoxicity level 30 4.40 ± 0.03a,B 4.23 ± 0.01a,C 3.41 ± 0.02b,C 2.91 ± 0.15c,A
was previously reported in the studies of commercial liquid smokes Enterobacteriaceae, log CFU/g
(Putnam et al., 1999) and a rice hull-derived liquid smoke (Kim et al., 0 3.31 ± 0.05a,A 2.32 ± 0.01b,A 1.76 ± 0.08b,A 1.54 ± 0.15c,A
14 4.21 ± 0.05a,B 0.00 ± 0.00b,B 0.00 ± 0.00b,B 0.00 ± 0.00b,B
2011). 30 4.33 ± 0.00a,B 0.00 ± 0.00b,B 0.00 ± 0.00b,B 0.00 ± 0.00b,B
Cytotoxicity performance of liquid smoke can be expressed as IC50
value. The value of fast pyrolysis liquid smoke was 43.8 μg/mL. In Results are expressed as mean ± standard deviation. Means followed by lower
case letter within a row and means followed by same upper case letter within a
comparison, the values of three commercial products were 25.6, 26.2,
column (TBC and Enterobacteriaceae separately) are not significantly different
and 44.3 μg/mL, respectively (Putnam et al., 1999). It indicated that the
at p < 0.05.
fast pyrolysis liquid smoke prepared in this study could meet the safety
requirements as food flavourings.
refrigerated storage. The viable Enterobacteriaceae in the smoked
samples decreased to 2.32 log CFU/g or lower at the beginning of
3.4. Liquid smoking of mussel meat
storage, and no Enterobacteriaceae colony was detected during the
whole storage time. These results are in agreement with a previous study
3.4.1. Microbiological analysis
(Estrada-Muñoz, Boyle, & Marsden, 1998): they observed high antimi­
The microbiological analysis results of liquid smoked mussel meat
crobial capacity of a diluted liquid smoke (8%) against E. coli O157:H7
were presented in Table 2. The total bacterial counts (TBC) were at low
by in vitro and in vivo analysis.
levels with an average value of 3.65 log CFU/g for the non-smoked
samples at the beginning of storage. The same initial TBC levels for
3.4.2. Image analysis
non-treated mussel meat were also observed in a previous study (Caglak
Fig. 4 presents the images of smoked mussel meat during storage,
et al., 2008). The TBC for non-smoked samples increased gradually
and Table 3 shows the changes in L*a*b* coordinates of the images. The
during the 30 days of storage, indicating that the mussel samples were
deviations in the coordinates were relatively high because of the vari­
undergoing spoilage.
ations in the mussel’s gender. Male mussels have a creamy white, while
To the contrary, TBC numbers of smoked samples were deceased to
female mussels have an orange colour (Alçiçek & Balaban, 2015a).
3.03 log CFU/g or lower at the beginning of storage. The three diluted
L* values in Table 3 decreased slightly indicating darker colours with
liquid smoke samples demonstrated significant antimicrobial capability.
the increase of liquid smoke concentration from 0 to 15%. This was due
TBC of treated samples maintained at low levels during the storage,
to the browning effect of liquid smoke via Milliard reaction (Lund & Ray,
except a striking increase (4.23 log CFU/g) was found in the samples
2017). The carbonyls in liquid smoke (Table 1) underwent reactions
treated with 5 vol% liquid smoke after 30 days of storage. Similarly,
with specific amino acids in the mussel meat, giving the brown pigment
Siskos et al. used a diluted liquid smoke (2 vol%) to treat trout fillets,
to the mussel meat samples. The increases of a* and L*values with the
and also observed an increase in TBC numbers after 25 days of storage
(Siskos et al., 2007). Highly diluted liquid smokes may not be able to
provide enough bactericidal activity to preserve food for a long period.
Although most strains of Enterobacteriaceae are innocuous, some
strains such as E. coli O157:H7 is recognized as a foodborne pathogen
(Jaeger & Acheson, 2000). Table 2 shows similar trends in the changes
of Enterobacteriaceae inactivation to those of TBC numbers. The
average colony-forming unit of non-smoked samples was initially 3.31
log CFU/g, and it gradually increased to 4.33 log CFU/g after 30 days of

Fig. 3. Dose-response relationship for fast pyrolysis liquid smoke treatment of L Fig. 4. Images of liquid smoked mussel meat during 30 days refriger­
cells for 24 h from MTT assay result (mean ± SD, n = 3). ated storage.

5
X. Xin et al. Food Control 124 (2021) 107874

Table 3 Table 4
Colour coordinates in image analysis of liquid smoked mussel meat during 30 Texture analysis profile of liquid smoked mussel meat during 30 days refriger­
days refrigerated storage. ated storage.
Days Non-treated Liquid smoked Days Non-treated Liquid smoked

5 vol% 10 vol% 15 vol% 5 vol% 10 vol% 15 vol%

L Hardness (g)
0 78 ± 5a,A 77 ± 4a,A 76 ± 3a,A 70 ± 5b,A 0 509 ± 54a,A 251 ± 102 ab,A 314 ± 137 ab,A 208 ± 123b,A
14 74 ± 6a,A 70 ± 5b,B 70 ± 5b,A 65 ± 5c,A 14 293 ± 58a,B 334 ± 129a,A 334 ± 116a,A 439 ± 138a,A
30 74 ± 5a,A 69 ± 6 ab,B 70 ± 5 ab,A 65 ± 5b,A 30 299 ± 85a,B 290 ± 135a,A 198 ± 30a,A,A 168 ± 60a,A
a Cohesiveness
0 12 ± 1a,A 12 ± 1a,A 12 ± 1a,A 14 ± 2a,A 0 0.32 ± 0.04a,A 0.39 ± 0.05a,A 0.35 ± 0.03a,A 0.42 ± 0.02a,A
14 14 ± 1a,B 14 ± 2a,A 13 ± 1a,A 15 ± 2a,A 14 0.31 ± 0.02a,A 0.34 ± 0.02 ab,A 0.41 ± 0.04 bc,A 0.48 ± 0.04c,A
30 12 ± 1a,AB 14 ± 1b,A 15 ± 1b,B 15 ± 1b,A 30 0.50 ± 0.07a,B 0.39 ± 0.04 ab,A 0.38 ± 0.03b,A 0.45 ± 0.02 ab,A
b Springiness (mm)
0 8 ± 4a,A 12 ± 4a,A 14 ± 3a,A 16 ± 3a,A 0 3.7 ± 0.9a,A 3.3 ± 0.2a,A 3.4 ± 0.3a,A 3.1 ± 0.5a,A
14 15 ± 3a,A 13 ± 3a,A 15 ± 4a,A 15 ± 2a,A 14 2.3 ± 0.3a,A 3.2 ± 0.5 bc,A 2.9 ± 0.2 ab,AB 3.9 ± 0.3c,A
30 15 ± 3 ab,A 12 ± 4a,A 18 ± 2b,A 14 ± 3 ab,A 30 3.6 ± 0.2a,A 2.7 ± 0.2 ab,A 2.6 ± 0.2b,B 3.2 ± 0.3 ab,A
Chewiness (mJ)
Results are expressed as mean ± standard deviation. Means followed by lower 0 8 ± 1a,A 4 ± 2a,A 5 ± 3a,A 3 ± 2a,A
case letter within a row and means followed by same upper case letter within a 14 4 ± 3a,A 6 ± 3a,A 5 ± 2a,A 11 ± 5a,A
column (L, a and b separately) are not significantly different at p < 0.05. 30 6 ± 3a,A 4 ± 3a,A 2 ± 1a,A 3 ± 1a,A

Results are expressed as mean ± standard deviation. Means followed by lower

increase of storage time in some cases implied that the mussel meat
underwent spoilage. The breakdown of tissue components of mussel
meat occurred in an autolytic stage of spoilage due to the enzyme ac­
tivity and oxidation of proteins and fats (Tan, Wu, Wang, & Teng, 2017).
3.4.3. Texture profile analysis
The results of texture profile analysis (hardness, cohesiveness,
springiness and chewiness) are presented in Table 4. The deviations of
the results are mainly due to the texture variances among different
mussel samples (Alçiçek & Balaban, 2015b). The hardness values of
control samples in Table 4 were slightly decreased with the increased
storage time. The mussel meat structure was weakened during spoilage
leading to the decrease of hardness. As proteins including collagen,
elastin and gelatine contributed to the textural properties to keep the
mussel structure intact and firm (Tan et al., 2017). The hardness values
at day 0 were also decreased by liquid smoke treatment, which agreed
with a previous study (Martinez, Salmerón, Guillén, & Casas, 2007). The
smoking treatment increased the activity of endogenous proteases, and
these enzymes (e.g. collagenase) hydrolysed different proteins and
broke down the connective tissue (Lakshmanan, Piggott, & Paterson,
2003).
Other texture characteristics did not show apparent trends in
Table 4, but they were varied in certain ranges. Similarly, the results of
water activity analysis fluctuated in the range of 0.85–0.98 without
showing any trend. Hence, the texture profile and water activity of
liquid smoke treated mussel meat were generally stable during the 30
days of the storage period, which was favourable to preserve the
freshness of mussel meat.
4. Conclusion
Fast pyrolysis and fractional condensation were employed to pro­
duce liquid smoke and other valuable products, including bio-oil and
biochar. The study showed that the yield and antioxidant capacity of fast
pyrolysis liquid smoke were higher compared with conventional
methods. The cytotoxicity analysis showed that the fast pyrolysis liquid
smoke had an equivalent safety level to the commercially available
liquid smoke products. Fast pyrolysis liquid smoke effectively preserved
green lipped mussel meat during the 30-day refrigerated storage. The
diluted liquid smoke at a concentration of 15 vol% inhibited microbial
growth in the mussel meat without significant changes in the colour and
textural properties, except a slight decrease in the hardness.
CRediT authorship contribution statement
Xing Xin: Methodology, Investigation, Validation, Writing - original
case letter within a row and means followed by same upper case letter within a
column (hardness, cohesiveness, springiness and chewiness separately) are not
significantly different at p < 0.05.
draft. Amy Bissett: Investigation, Validation. Joyce Wang: Investiga­
tion, Validation. Andrew Gan: Investigation, Validation. Kiri Dell:
Funding acquisition, Writing - review & editing. Saeid Baroutian:
Conceptualization, Funding acquisition, Supervision, Writing - review &
editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors acknowledge the University of Auckland FRDF Grant
3719621 and Health and Food Programme Seed Fund 48422.
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