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TECNOCIENCIA CHIHUAHUA, Vol. XVIII (4): e1683 (2024)
https://vocero.uach.mx/index.php/tecnociencia
ISSN-e: 2683-3360
Research Article
Effect of a black garlic extract combined with ascorbic
acid on the colour and lipid oxidative stability of
patties
Efecto de un extracto de ajo negro combinado con ácido ascórbico
sobre el color y la oxidación lipídica de hamburguesas
*Correspondencia: mmaria00@estudiantes.unileon.es (Mauricio Fernando Mariño-Almache)
DOI: https://doi.org/10.54167/tch.v18i4.1683
Recibido: 30 de septiembre de 2024; Aceptado: 16 de diciembre de 2024
Publicado por la Universidad Autónoma de Chihuahua, a través de la Dirección de Investigación y Posgrado.
Editor de Sección: Dr. Diego Eloy Carballo-Carballo
Abstract
Oxidation of meat products limits their quality and shelf life and is prevented by antioxidant
additives. Natural ingredients rich in antioxidants are proposed as an alternative to these additives.
In this study, the antioxidant effect of an aqueous extract of black garlic, as a natural ingredient rich
in polyphenols, was evaluated in pork patties. 20 ml of extract, obtained from a homogenate
prepared with one-part garlic and three parts water, was used per kg of patty in combination with
three levels of ascorbic acid (from 0.125 to 0.5 g/kg). The effect of different combinations to prevent
discolouration in raw patties and lipid oxidation stability in cooked patties during aerobic
refrigerated storage was tested. The use of black garlic extract darkened the patties and increased
the red and yellow indices. Ascorbic acid potentiated the antioxidant effect of black garlic in cooked
patties and vice versa. The black garlic extract combined with ascorbic acid, even in an amount four
times lower than that commonly used in the meat industry, was the treatment with the highest
antioxidant effect.
Keywords: natural antioxidant, patty, meat quality, black garlic, shelf life
Nuria Fernández Fernández-Valladares 1, Irma Caro2, Javier Mateo1 y Mauricio Fernando
Mariño-Almache1*
1 Departamento de Higiene y Tecnología de los Alimentos, Universidad de León. Campus Vegazana s/n,
24007 León, Spain
2 Facultad de Medicina, Universidad de Valladolid. Avenida Ramón y Cajal 7, 47005 Valladolid, Spain
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TECNOCIENCIA CHIHUAHUA, Vol. XVIII (4): e1683 (2024)
Resumen
La oxidación de los productos cárnicos limita su calidad y vida útil y se evita mediante aditivos
antioxidantes. Como alternativa a estos aditivos, se propone el uso de ingredientes naturales ricos
en antioxidantes. En este estudio, se evaluó el efecto antioxidante de un extracto acuoso de ajo negro,
como ingrediente natural rico en polifenoles, en hamburguesas de cerdo. Se utilizaron 20 ml de
extracto, obtenido de un homogeneizado preparado con una parte de ajo y tres de agua, por kg de
hamburguesa en combinación con tres diferentes niveles de ácido ascórbico (entre 0,125 a 0,5 g/kg).
Se comprobó el efecto de las distintas combinaciones para prevenir la decoloración en las
hamburguesas crudas y la estabilidad de la oxidación lipídica en las hamburguesas cocinadas
durante el almacenamiento aeróbico refrigerado. El uso de extracto de ajo negro oscureció las
hamburguesas y aumentó los índices de rojo y amarillo. El ácido ascórbico potenció el efecto
antioxidante del ajo negro en las hamburguesas cocidas y viceversa. El extracto de ajo negro
combinado con ácido ascórbico, incluso en una cantidad cuatro veces inferior a la utilizada
habitualmente en la industria cárnica, fue el tratamiento con mayor efecto antioxidante.
Palabras clave: antioxidante natural, hamburguesa, calidad de carne, ajo negro, vida útil.
1. Introduction
One of the limiting factors in the quality of meat and meat products is lipid auto-oxidation
(Morrissey et al., 1998). This oxidation is an irreversible process that occurs by a chain reaction
mechanism mediated by the formation of free radicals. The initiation of this reaction and its extent
over a given time depends on a balance between pro-oxidant and antioxidant factors in the product
and the environment (Choe and Min, 2006). In the autoxidation process, peroxides and secondary
oxidation compounds of low molecular weight are generated, and chemical changes occur in proteins
such as cross-linking or oxidation of metals such as iron bound to myoglobin. As a result, the sensory
attributes of the meat are altered, and potentially toxic compounds such as free radicals or cholesterol
oxides are formed (Morrissey et al., 1998; Sampels, 2013). In response to this alteration process, meat
processing can resort to the use of antioxidant additives, which are substances that, at low
concentrations, prevent or avoid oxidative damage to specific molecules by scavenging reactive
oxygen species (ROS), enhancing biological antioxidant defences or inhibiting ROS production
(Khlebnikov et al., 2007).
Research and the food industry are actively working on incorporating antioxidant-rich natural
ingredients derived from condiments, spices, fruits, vegetables or seeds containing polyphenols or
other bioactive compounds (Karre et al., 2013; Falowo et al., 2014; Shahidi and Ambigaipalan, 2015).
Antioxidant compounds encompass a wide range of sources, including conventional and non-
conventional edible materials and by-products such as fruit peels, seeds, and medicinal herbs. The
most frequent bioactive compounds with antioxidant power present in plants are phenolic
compounds, although there are also other natural antioxidant compounds such as non-phenolic
terpenoids, vitamins, metal-chelating ions, proteins and peptides (Falowo et al., 2014). In addition to
their antioxidant properties, antioxidant-rich ingredients could contribute to the nutritional
improvement of foods, including meat products, by providing fibre, vitamins or minerals (Falowo
et al., 2014), as well as antimicrobial compounds (Hygreeva et al., 2014). However, on the other hand,
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Fernández-Valladares et.al
dietary phenolic compounds can exert a negative impact on the digestion processes such as enzyme
inhibition (Cirkovic Velickovic and Stanic-Vucinic, 2018).
Black garlic is the result of the transformation or blackening of common garlic through the Maillard
reaction. Its use as a food ingredient or for medical purposes dates to ancient times in regions of the
Asian continent. Kimura et al. (2017) reported that the consumption of black garlic as a condiment in
culinary preparations or for direct consumption is spreading and that it is associated with functional
antioxidant, anti-inflammatory, anti-cancer or anti-diabetic properties, indicating its beneficial
health properties.
To obtain the black garlic, fresh garlic is kept at a constant temperature of 45-70 °C with a relative
humidity of 70-80 % for a variable period of time, usually between 15-40 days, after which it is cooled
and dried for approximately one week (Dewi and Mustika, 2018). The process results in a product
with 40-50 % moisture content, a mild aroma, and a tender and creamy texture. It has been
determined that the optimal ageing period for garlic to maximise its antioxidant capacity is 21 days
(Choi et al., 2014). Black garlic is a stable product at room temperature, with a shelf life of about 18
months, provided the storage conditions are appropriate (dry, cool and away from light).
The use of black garlic as an antioxidant ingredient in meat derivatives has scarcely been studied. In
recent studies (Lee et al., 2019; Barido et al., 2022; Santos et al., 2024), it has been found that the use of
black garlic in meat patties revealed a significant lipid antioxidant effect. The antioxidant effect has
been attributed to active compounds such as reducing sugars resulting from enzymatic hydrolysis
flavonoids, polyphenols, alkaloids, S-allyl-cysteine, and some of the Maillard reaction intermediates,
i.e. 5-hydroxymethylfurfural (Choi et al., 2014; Yuan et al., 2016; Kimura et al., 2017). This effect could
be comparable or synergistic with that of ascorbic acid, an antioxidant additive commonly used in
the meat industry.
This study aimed to assess the impact of combining black garlic with ascorbic acid, a commonly used
antioxidant additive in the meat industry, on lipid oxidation and colour stability in pork patties
stored under aerobic conditions.
2. Materials and methods
2.1 Experimental design
The study comprised two main phases: an in vitro analysis and an in-situ application. In the in
vitro part, the antioxidant capacity of a black garlic aqueous extract was evaluated by determining
the total polyphenol content and performing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. The
antioxidant capacity of the garlic extracts obtained by the DPPH test was compared with that of two
antioxidant additives used in the food industry: ascorbic acid and butylated hydroxytoluene (BHT).
In the in-situ phase, the focus was on assessing the colour stability of raw pork patties and the
oxidative stability of lipids in cooked pork patties containing different levels of the black garlic
extract combined with different amounts of ascorbic acid, during a six-day period of aerobic
refrigerated storage. The colour stability was determined by measuring the change in instrumental
colour, and the oxidative stability of lipids was determined by changes in the concentration of
thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation.
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2.2 Preparation of the black garlic extract and patties
Two samples of black garlic were purchased at the local market. The garlic cloves were minced
with a domestic food processor, and 25 g of minced garlic was mixed with 75 g of distilled water
with an Ultraturrax T18 IKA Werke (Staufen, Germany) for 2 min at 13,500 revolutions per minute.
The pH was then adjusted with 0.1 M NaOH to 7.0 and centrifuged for 25 min at 12,000 rpm with a
Beckman J2-21 centrifuge (Palo Alto, U.S.A). The supernatant was first filtered through a Whatman
No. 1 filter (Darmstadt, Germany) and then through a 0.45 mm pore size polytetrafluoroethylene
filter (Membrane Solutions, LLC, Auburn, WA, USA).
Frozen pork loin meat purchased from a local market produced the patties. A 2 kg meat piece was
thawed for 48 h at 4 °C, minced in a Mainca mincer (Mainca PM-12; Granollers, Spain) with a 6 mm
plate, salt was added at a rate of 15 g/kg and mixed by hand for 6 min. The different patties were
prepared in duplicate (two batches, each prepared on different days). Two sets of patties were
prepared inside each replication, one with each sample of black garlic purchased. Five treatments
(formulations) were prepared for each set (Table 1): i) a control sample without added antioxidants;
ii) a formulation containing 2 % (v/w) black garlic extract, attempting to use a high quantity without
compromising sensory characteristics (Augustynska-Prejsnar et al. 2024); iii) patties with 2 % (v/w)
of an ascorbic acid solution (25 mg/mL), a level commonly used in the food industry (0.5 mg ascorbic
acid/kg patty); and iv-vi) formulations combining 2 % (v/w) black garlic extract with 2 % (v/w)
ascorbic acid with low (6.25 mg/mL), medium (12.5 mg/mL), and high (25 mg/mL) concentrations,
respectively.
Tabla 1. Formulaciones de hamburguesas y cantidades de antioxidantes para cada tratamiento experimental.
Table 1. Patty formulations and amounts of antioxidants for each experimental treatment
Amounts used
(g or ml/100 g of patty mix)
Concentration
(g/kg of patty mix)
Meat+salt
Water
Ascorbic acid
solution
Black garlic
extract
Black garlic
(g)*
Ascorbic
acid
CON
90
10
0
0
0
0
Asch
90
8
2
0
0
0.5
BG
90
8
0
2
5
0
BG+Ascl
90
6
2
2
5
0.12
BG+Ascm
90
6
2
2
5
0.25
BG+Asch
90
6
2
2
5
0.5
CON: control treatment; Asch: patty to which a 25 mg/mL ascorbic acid solution was added; BG: patty containing
black garlic; BG+Ascl: patty containing black garlic and a 6.25 mg/mL ascorbic acid solution: BG+Ascm: patty
containing black garlic and a 12.5 mg/mL ascorbic acid solution; BG+Asch: patty containing black garlic and a 25
mg/mL ascorbic acid solution.
* g equivalents, i.e. grams of black garlic to prepare the amount of garlic extract needed to produce 1 kg of patty
mixes.
Two patties, 80 g, 5-6 cm diameter and 1.3-1.6 cm thick patties covered with cellophane plastic were
prepared for each treatment and set. One of the patties was used for colour determination, which
was determined on the day of the patty forming (day 1) and after 6 days of refrigerated storage (day
7). The other patty was vacuum packaged, cooked for 40 min at 70 °C, removed from the packaging
and used for TBARS determination just after cooking (day 1) and after six days of storage (day 7).
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Fernández-Valladares et.al
Raw and cooked samples were stored in plastic trays covered with polyethylene cling film at 4 °C
under darkness.
2.3 Polyphenol content and antioxidant activity of black garlic extract
The total polyphenol content of the garlic extracts was performed, in duplicate, by the Folin-
Ciocalteu test following the indications described by Musci and Yao (2017). 0.25 mL of a 1/50 (v/v)
aqueous dilution of garlic extract was added to 1 mL of 0.2 M Folin-Ciocalteu reagent, the mixture
was shaken and kept for 5 min at room temperature (20 °C) in the dark. Then 1 mL of a 10 % (w/v)
Na2CO3 solution was added, stirred and kept in the dark for 60 min. Finally, the absorbance of the
sample was determined at 765 nm in a Miltonroy spectrophotometer (Spectronic 40, Ivyland, USA).
Quantification was performed using gallic acid standard solutions. The results were expressed as
mg gallic acid equivalents/L of black garlic extract.
The DPPH test was carried out in duplicate, according to Habinshuti et al. (2019). A 1/800 dilution of
black garlic extracts (v/v) was prepared, and 1 mL of each dilution was mixed with 2.0 mL of a
solution of DPPH (0.1 M) in ethanol. The mixture was vortexed and kept in the dark for 20 min at
room temperature (22 °C). The absorbance was read at 517 nm using a Miltonroy spectrophotometer
(Spectronic 40, Ivyland, USA). The absorbance of control with 1 mL of distilled water and 2 mL of
DPPH solution was also determined. The colour reduction in percentage was calculated as a measure
of reducing activity (%) = [blank A517 - sample A517) / blank A517]×100. Sample A517 represents
the absorbance of the sample solution and blank A517 represents the absorbance of 1.0 mL of water
mixed with the DPPH solution. Furthermore, the antiradical capacity of ascorbic acid and BHT was
determined from 5 and 10 mg/L ethanol dilutions, respectively. One mL of each solution was mixed
with 2 mL of the DPPH solution, and the colour reduction was compared against the blank. Finally,
the antioxidant capacity of the extracts was compared with that of the ascorbic acid and BHT
solutions, obtaining the equivalent reducing capacity of the garlic extract (1 mL of extract) to 1 mg
of ascorbic acid or 1 mg of BHT.
2.4 Patty quality measurements
Colour determination was carried out in duplicate with a CM-700d colorimeter (Konica-Minolta,
Oxaca, Japan) in specular component included (SCI) mode, illuminant D65, measuring aperture 11
mm and observer angle 10°. Colour was expressed in the coordinates of lightness, L*; red index, a*;
and yellow index, b* (AMSA, 2012).
The TBARS test was carried out in duplicate following Nam and Ahn (2003). Half of each cooked
patty was minced with a domestic mincer, 2 g were taken, to which 20 mL of water was added,
homogenized for 1 min at 9,500 rpm with an IKA Ultra-turrax (Staufen, Germany) and filtered with
a strainer into a beaker. Then 1 mL of homogenate was placed into a tube, 50 μL of a solution of BHT
in 7.2 % (w/v) ethanol was added, and the mixture was shaken, and 2 mL of 20 mM thiobarbituric
acid in a 15 % (w/v) trichloroacetic acid solution was added and shaken again. The tubes were then
placed in a water bath for 20 minutes at 80 °C. After this time, the tubes were cooled in a water bath
and then centrifuged for 20 minutes at 3000 rpm and 5 °C using a Beckman centrifuge model J2-21
(Palo Alto, U.S.A). Finally, the absorbance of the supernatant was measured at 531 nm in a
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spectrophotometer. In addition, a series of standards were prepared to make a calibration curve from
a stock solution of tetraethoxypropane (TEP) 3 x 10-5 M, which were analyzed following the steps
described above, omitting filtration and centrifugation.
2.5 Statistical analysis
The mean values obtained from the analytical replicates of patty characteristics (colour and
TBARS) were analyzed by univariate analysis of variance (general linear model; software SPSS v.26;
IBM, Somers, NY, USA). Treatment and time were used as fixed factors, and garlic type as a random
factor. When the analysis was significant (P<0.05), a pairwise analysis was carried out with the
posthoc Tukey test to assess differences between treatments within the same day or days within a
treatment (P<0.05).
3. Results and discussion
3.1 Polyphenol content and antioxidant activity of black garlic extract
The antioxidant potential of natural antioxidants depends on the polyphenol content. However,
total polyphenol content has limitations in predicting antioxidant capacity, as the antioxidant
activity of polyphenolic compounds will depend on their chemical structure (Sang et al., 2002). Table
2 shows the polyphenol content of the black garlic used in the experiment. In addition, several
studies have shown how the ageing process to which fresh garlic is subjected to obtain black garlic
has an impact on the polyphenol content, leading to an increase in polyphenols (Kimura et al., 2017,
Choi et al., 2014). The amounts obtained in this study are lower than those of Toledano-Medina et al.
(2016), who found 2-4 times higher amounts. The variability between studies may be due not only
to the variety or maturity of garlic (Škrovánková et al., 2018), but also to the extraction method.
Tabla 2. Contenido en polifenoles solubles y capacidad antioxidante de un extracto acuoso de ajo negro (BG)
comparado con el del ácido ascórbico (Asc) y el hidroxitolueno butilado (BHT).
Table 2. Soluble polyphenol content and antioxidant capacity of a black garlic aqueous extract (BG) compared
to that of ascorbic acid (Asc) and butylated hydroxytoluene (BHT)
Mean ± standard deviation
(n = 2)
Polyphenol content$
408 ± 116
Antioxidant capacity of BG extract compared to Asc&
0.480 ± 0.100
Antioxidant capacity of BG extract compared to BHT&
0.169 ± 0.036
$ expressed as mg gallic acid equivalents/L black garlic extract (prepared from 25 g of garlic and 75 ml of
water)
& expressed as mL of garlic extract with an antioxidant capacity equivalent to 1 mg of ascorbic acid as determined
with the 2,2-diphenyl-1-picrylhydrazyl method.
Table 2 also shows the antioxidant (antiradical) capacity of garlic extracts relative to the antioxidant
capacity shown by ascorbic acid and BHT, two antioxidants used in the food industry. Overall, the
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Fernández-Valladares et.al
results show that 0.48 and 0.17 mL of garlic extract showed an anti-radical effect equivalent to 1 mg
of ascorbic acid and BHT, respectively. The antioxidant effect of black garlic has been mainly
attributed to polyphenols and the compound S-allyl-cysteine, derived from alliin (Toledano-Medina
et al., 2016). These authors compared the antiradical activity of fresh and black garlic using the DPPH
method and found that the activity of black garlic was about 10 times higher than that of fresh garlic.
3.2 Patty quality measurements (colour and lipid oxidation)
The CIELAB colour parameters of the patties with different formulations are shown in Table 3.
Treatment significantly affected the brightness of the patties, but time showed no significant effect.
Considering the mean L* values of both days, the use of black garlic decreased the brightness
compared to the control patties, with significant differences being observed when the addition of
garlic was accompanied by medium or high values of ascorbic acid. This tendency can be attributed
to the black hue of black garlic.
The redness index (a*) of the patties was significantly affected by treatment, storage and the
interaction between the above factors was also significant. On day 1 of storage, the addition of garlic
together with medium and high amounts of ascorbic generated a more intense red colour than the
control patties. It appears that black garlic has some synergy with ascorbic acid in the reddening of
the patties, which could be considered advantageous from a colour acceptance point of view. In
potential agreement, in a previous study, Augustynska-Prejsnar et al. (2024) found that the addition
of black garlic in amounts of 2 % in meat products made with minced meat (higher than the amount
used in this study) improved their sensory acceptability. However, sensory acceptability in terms of
colour, flavour and overall acceptability dropped with amounts higher than 4 %.
Overall, with storage time, the value of a* in the patties tended to increase. This increase might be
attributed to water increased oxymyoglobin concentration due to water evaporation from the patty
surface during storage or increased oxygen penetration (Callejas-Cárdenas et al., 2014). This increase
in a* indicates that the eventual oxidation of the meat pigment due to oxidation reactions leading to
discolouration was more than compensated by water evaporation or oxygen diffusion. However, the
reddening was not as marked in either patty, being more marked and significant in the CON patties,
those to which black garlic was added and those to which black garlic with high ascorbic acid was
added. After seven days of storage, however, no significant differences were observed between the
control and any of the other treatments, but it was observed that the addition of ascorbic acid to the
patties with black garlic made the patties redder than the patty with black garlic and no ascorbic
acid.
Concerning the yellowness index (b*), the addition of black garlic produced an increase compared
to the CON patty and with ascorbic acid, which was significant when the addition of black garlic
was accompanied by ascorbic acid and more evident after 7 days of refrigerated storage. The storage
time had no influence on this colour coordinate.
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Tabla 3. Efecto del tratamiento y del tiempo de almacenamiento refrigerado en el color de las hamburguesas
crudas (medias y desviaciones estándar).
Table 3. Effect of treatment and refrigerated storage time on the colour of raw patties (means and standard
deviations)
Storage
P-level
Day 1
(n = 4)
Day 7
(n = 4)
Mean value
Treatment
Day
Treatment
x day
L*
CON
45.2±0.6
45.4±0.1
45.28±2.50a
0.022
0.246
0.230
Asch
45.6±0.9
45.7±0.4
45.68±2.44a
BG
41.7±3.5
43.6±5.4
42.65±2.28ab
BG+Ascl
41.6±2.0
42.8±4.3
42.21±3.30ab
BG+Ascm
40.8±1.0
41.4±2.0
41.14±2.28b
BG+Asch
40.9±0.4
41.2±0.2
41.04±2.21b
a*
CON
5.86±0.03b,y
9.20±0.06ab,x
7.71±1.57
<0.001
0.046
<0.001
Asch
6.43±1.01ab,y
9.00±0.65ab,x
7.53±1.83
BG
7.10±0.81ab
7.32±1.70b
7.21±1.09
BG+Ascl
7.28±0.13ab
9.69±1.40a
8.27±1.32
BG+Ascm
7.62±1.19a
8.92±1.33ab
8. 49±1.53
BG+Asch
7.46±0.54a,y
10.18±0.40a,x
8.82±1.57
b*
CON
14.97±0.42
15.38±0.70b
15.17±0.95b
<0.001
0.166
0.0.30
Asch
15.28±2.07
15.64±0.19b
15.43±1.43b
BG
16.88±0.94
16.40±0.78ab
16.64±1.13ab
BG+Ascl
17.80±2.51
18.22±0.37a
18.02±1.60a
BG+Ascm
17.57±0.58
18.40±0.35a
17.98±1.00a
BG+Asch
17.12±1.06
18.46±1.11a
17.79±1.44a
CON: control treatament; Asch: patty to which a 25 mg/mL ascorbic acid solution was added; BG: patty
containing black garlic; BG+Ascl: patty containing black garlic and a 6.25 mg/mL ascorbic acid solution: BG+Ascm:
patty containing black garlic and a 12.5 mg/mL ascorbic acid solution; BG+Asch: patty containing black garlic
and a 25 mg/mL ascorbic acid solution.
ab Mean values followed by different superscript letters in the same columns are statistically significant (P <
0.05). xy Mean values followed by different superscript numbers in the same row are statistically significant (P
≤ 0.05).
The values of TBARS, an indicator of lipid oxidation, in the patties cooked at the beginning and end
of aerobic refrigerated storage, are shown in Table 4. On day 1 of storage, TBARS values were
generally lower than 0.5 mg of malondialdehyde/kg of patty in all treatments and significant
differences were observed. The patty with ascorbic acid showed the lowest oxidation rate, with
significant differences compared to the patty with black garlic and high levels of ascorbic acid.
Ascorbic acid, as a reducing agent, can act as an electron donor in free radical-mediated oxidative
processes by stabilizing, to some extent, the lipid oxidation of meat (Min et al., 2008) during the patty-
making and cooking processes. On the other hand, black garlic may contain coloured substances
such as Maillard compounds or phenolics, which could interfere in TBARS analysis, increasing the
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Fernández-Valladares et.al
absorbance and, thus, the concentration (Pérez and Estévez, 2020). Maybe that is why the average
TBARS values of black garlic samples on day 1 of storage are, on average, somewhat higher than
patties without black garlic.
Refrigerated aerobic storage of the cooked patties clearly caused lipid oxidation in all treatments, a
characteristic phenomenon of cooked meat during aerobic refrigerated storage (Min et al., 2008).
However, results showed that the intensity of oxidation depended on the treatment. The prevention
of oxidation by ascorbic acid and black garlic was also observed after seven days of storage. The
control patties were the most oxidized; in the second position, the patties with ascorbic acid or black
garlic as the only antioxidants and in the third position were the patties with both antioxidants, black
garlic and ascorbic acid.
The antioxidant activity of ascorbic acid in the patties after storage is attributable to electron donation
or free radical inhibition (Min et al., 2008). This activity may be limited because it decreases over time
due to ascorbic acid degradation or exposure to adverse environmental conditions such as light and
oxygen (Bellucci et al., 2022). The antioxidant effect observed in the patties with black garlic could be
exerted by a range of antioxidant substances, including polyphenols or Maillard reaction products,
which may act by anti-radical mechanism and by mechanisms other than hydrogen donation (Choi
et al., 2014; Kimura et al., 2017). Interestingly, the results show a clear synergistic effect of ascorbic
acid and black garlic independent of the amount of ascorbic acid used. The patties with black garlic
and ascorbic acid, unlike the rest and regardless of the level of ascorbic acid, showed TBARS values
below 2 mg/kg on day 7, which, according to Domínguez et al. (2017) can be considered a TBARS
threshold indicative of rancid flavour in meat.
Tabla 4. Efecto del tratamiento y del tiempo de almacenamiento refrigerado sobre la estabilidad oxidativa
lipídica de las hamburguesas cocidas determinada por las sustancias reactivas del ácido tiobarbitúrico
expresadas en mg de malondialdehído/kg de hamburguesa (medias y desviaciones estándar).
Table 4. Effect of treatment and refrigerated storage time on the lipid oxidative stability of cooked patties
determined by the thiobarbituric acid reactive substances expressed as mg of malondialdehyde/kg patty (means
and standard deviations)
Storage
P-level
Day 1
(n = 4)
Day 7
(n = 4)
Mean value
Treatment
Day
Treatment
x day
CON
0.30±0.07ab,x
3.97±0.69c,x
2.13±1.95
0.002
0.023
0.005
Asch
0.23±0.07b,x
2.45±0.20b,x
1.34±1.16
BG
0.42±0.07a,x
2.78±0.97b,x
1.60±1.42
BG+Ascl
0.39±0.04ab,x
1.72±0.38a,x
1.06±1.42
BG+Ascm
0.38±0.10ab,x
1.77±0.50a,x
1.07±0.81
BG+Asch
0.43±0.17a,x
1.66±0.50a,x
1.04±0.74
CON: control treatment; Asch: patty to which a 25 mg/mL ascorbic acid solution was added; BG: patty containing
black garlic; BG+Ascl: patty containing black garlic and a 6.25 mg/mL ascorbic acid solution: BG+Ascm: patty
containing black garlic and a 12.5 mg/mL ascorbic acid solution; BG+Asch: patty containing black garlic and a 25
mg/mL ascorbic acid solution.
abc Values followed by different superscripts in the same columns are statistically significant (P < 0.05).
xy Values followed by different superscripts in the same row are statistically significant (P ≤ 0.05).
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The synergy of ascorbic acid with other antioxidants, such as tocopherols, in the prevention of
oxidation of minced meat has been previously demonstrated (Bruun-Jensen et al., 1996), but no
previous studies on the synergistic effect of black garlic and ascorbic acid in meat products have
been found. According to Day et al. (2008), the ascorbic acid could have increased the stability of
black garlic polyphenols by reducing the phenoxyl radicals formed during oxidation, i.e.
regenerating polyphenols.
4. Conclusions
The addition of the aqueous black garlic extracts in the conditions used in this experiment
influences the colour of the patties, decreasing their lightness and increasing the red and yellow
indices. The combination of ascorbic acid with black garlic extract increases the redness and
yellowness after seven days of storage. The effect of black garlic extract on colour acceptability could
be positive, although sensory analysis is needed to confirm the latter. Adding aqueous black garlic
extract shows synergy with ascorbic acid in preventing lipid oxidation in cooked patties. The
combination of 0.5 % black garlic extract and ascorbic acid amounts of 0.12 g/kg in the patty
formulation showed optimal antioxidant activity in this study. Further research could test whether
this antioxidant activity is maintained by decreasing the amount of black garlic and the effect of
black garlic on the patty sensory properties.
Conflict of interest
There is no conflict of interest for the publication of these research results.
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