GREENING OF GARLIC IN MEAT MARINADES

Contents
General Main Root Causes ............................................................................................................................................2
1. Organosulfur compounds ................................................................................................................. 2
2. Non-sulfur garlic phytochemicals, .................................................................................................... 3
3. Aminoacid Interactions ..................................................................................................................... 4
4. Storage Temperature Conditions...................................................................................................... 4
5. Environment pH ................................................................................................................................ 5
6. Incidental Additives........................................................................................................................... 5
7. Active Alliinase (4.4.1.4) in Dehydrated Garlic ................................................................................. 6
Mechanism of Garlic Greening from organosulphur Compounds .................................................................................7
Thiosulphinate Base Pigments .................................................................................................................. 8
Reactants – Aminoacid Requirments for Pigmentation ........................................................................... 8
Enzyme - Alliinase (4.4.1.4) - Properties and Stability ............................................................................ 11

Summary

Main root causes of garlic greenings are listed based on the literature review, which involves
enzymatic process of Sulphur-organic compounds break down and their interaction with amino
acids, flavonoids and incidental additives. Based on the explained mechanism of pigmentation
evolvement, it can be lead to conclusion that the most common cause in green color
evolvement in meat product marinades may be failure to denature Alliinase during dehydration
process. In addition, it is worth mentioning that warehousing conditions become a factor too.
While operators prefer to store garlic in lower temperatures to stop germination, studies have
revealed that storing garlic under 20°C will favor greening. Alliinase activity may be added as
part of Quality Assessment for dehydrated granulated garlic or garlic juices used in meat
Marinades.

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 GREENING OF GARLIC IN MEAT MARINADES

General Main Root Causes

Based on the literature search the greening of the garlic may happen from the followings:

1. Organosulfur compounds
The discoloration is due to pigments that form between sulfur compounds in garlic and amino acids.
When the garlic tissue is disrupted, as happens in processing, an enzyme is liberated and reacts with it
to form thiosulfinates compounds that then react with the natural amino acids in the garlic to form blue
pigments.
S-allyl-L-cysteine sulfoxide (alliin) accounts for approximately 80% of cysteine sulfoxides in garlic (Figure
1). When raw garlic cloves are crushed, chopped, or chewed, an enzyme known as alliinase is released.
Alliinase catalyzes the formation of sulfenic acids from L-cysteine sulfoxides (Figure 2). Sulfenic acids
spontaneously react with each other to form unstable compounds called thiosulfinates. In the case of
alliin, the resulting sulfenic acids react with each other to form a thiosulfinate known as allicin (half-life
in crushed garlic at 23°C is 2.5 days). The formation of thiosulfinates is very rapid and has been found to
be complete within 10 to 60 seconds of crushing garlic. Color development in these situations was
extensively studied [Kubec, R., Hrbáčová,M., Musah,R and, and Velíšek,J (2004) : Allium Discoloration: 
Precursors Involved in Onion Pinking and Garlic Greening, Journal of Agricultural and Food Chemistry, 52
(16), 5089-5094].
The pH optimum for pigment formation has been found to be between 5.0 and 6.0 for all thiosulfinates
tested. [Kubec, R., Velíšek, J. (2007): Allium Discoloration:  The Color-Forming Potential of Individual
Thiosulfinates and Amino Acids:  Structural Requirements for the Color-Developing Precursors, J. Agric.
Food Chem., 55 (9), pp 3491–
3497]
Crushing garlic does not
change its γ-glutamyl-L-
cysteine peptide content. γ-
Glutamyl-L-cysteine peptides
include an array of water
soluble dipeptides, including
γ-glutamyl-S-allyl-L-cysteine, γ-glutamylmethylcysteine, and γ-glutamylpropylcysteine

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GREENING OF GARLIC IN MEAT MARINADES

2. Non-sulfur garlic phytochemicals,

The flavonoids and saponins are the most important. [Lanzotti, V. (2006): The analysis of onion and
garlic, Journal of Chromatography, 1112 (2006) 3–22 ]. Typical property of Flavonoids, is they are water-
soluble pigments that can turn blue or purple under acidic conditions. This is a reported to be a variable
phenomenon that is more pronounced for immature garlic but can differ among cloves within a single
head of garlic. [Harris, L., GARLIC: Safe Methods to Store, Preserve, and Enjoy, University of California,
Davis]Some type of these phenolic base compounds like the one containing ferulic acid and tryosine are
reported to form polumers whichh exhibit phosphorescence. They absorbs light in the bluer region of
the spectrum and emit light in the green part of the spectrum [Noel, A., Borguet, Y. P., Raymond, J. E., &
Wooley, K. L. (2014). Poly(ferulic acid-co-tyrosine): Effect of the Regiochemistry on the Photophysical
and Physical Properties en Route to Biomedical Applications. Macromolecules, 47(20), 7109–7117. ].

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GREENING OF GARLIC IN MEAT MARINADES

3. Amino acid Interactions

Experiments with isotope-labeled precursors revealed that two molecules of an amino acid are involved
in the formation of each color compound. In the purple/blue species (λmax = 565–600 nm), both amino
acid molecules are incorporated into two 3,4-dimethylpyrrole-derived rings linked together by a
propenylidine bridge. On the other hand, the yellow compounds (λmax = 420–450 nm) contain only one
N-substituted 3,4-dimethylpyrrole ring, to which the second amino acid is bound via a propenylidine
side chain. [Kubec, R. , Curko, P , Urajová, P., Rubert, J. and Hajšlová, J. (2017): Allium Discoloration:
Color Compounds Formed during Greening of Processed Garlic, J. Agric. Food Chem., 65 (48), pp 10615–
10620]

4. Storage Temperature Conditions

Operators prefer storing garlic at 32°F (0°C) to prevent germination [Devinder Dhingra (2003):
OPTIMIZATION OF THE DRYING PROCESS OF GARLIC (Allium sativum L.), Dissertation Submitted to the
Punjab Agricultural University].
However, studies have found that storing garlic bulbs for a month at or above 23°C, prior to processing,
prevented the production of a green pigment in the garlic puree. Garlic stored for 2-4 weeks at 3 °C was
much more susceptible to greening than that stored at room temperature. [Lukes, T. M. Factors
governing the greening of garlic puree. J.Food Sci. 1986, 51, 1577, 1582.]. It was found an association
with presence of amino acid S‐(1‐propenyl) cysteine sulfoxide, as necessary component for the
development of the green color.
It was established that storage at low temperature (less than 10 °C) was required for garlic greening
occurring either during processing or in the course of “Laba” garlic preparation while storage at high
temperature (higher than 20 °C) inhibited its occurrence. This was explained with the γ-glutamyl
transpeptidase (GGT) activity correlated with garlic greening because the activity of this enzyme is very
sensitive to storage temperature. Cold storage facilitated the GGT activity whereas warm storage
inhibited the activity of this enzyme, just like the effect of storage temperature on greening, indicating
that the increase of GGT activity could be a direct factor resulting in garlic greening. Consistent with this
conclusion, the concentration of total thiosulfinates (the color developers) in garlic purees likewise
exhibited a reversible change by moving garlic bulbs from one low storage temperature to a higher one;
namely, it increased with increasing storage time during storage at 4 °C while decreasing as storage time
increased during storage at 35 °C. [Lei Li, Dan Hu, Ying Jiang, Fang Chen, Xiaosong Hu and Guanghua
Zhao (2008): Relationship between γ-Glutamyl Transpeptidase Activity and Garlic Greening, As

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Controlled by Temperature, J. Agric. Food Chem., 56 (3), pp 941–945]. Greening in crushed garlic cloves
increases with increasing GGT expression at low temperature, while alliinase expression is not affected.
[Jungeun Cho, Minkyu Park, Doil Choi, Seung Koo Lee (2011): Cloning and expression of γ‐glutamyl
transpeptidase and its relationship to greening in crushed garlic (Allium sativum) cloves, J. Foos Sci and
Agric., (92) 2, pp 253-257].

5. Environment pH
Studies conducted by Chinese for producing of “Laba” Garlic, (Chinese pickled garlic where Garlic Cloves
are expected to be green), have indicated that greening is promoted in acid environment [Bing Bai, Fang
Chen, Zhengfu Wang, Xiaojun Liao, Guanghua Zhao, and Xiaosong Hu (2005): Mechanism of the
Greening Color Formation of “Laba” Garlic, a Traditional Homemade Chinese Food Product, J. Agric.
Food Chem., 2005, 53 (18), pp 7103–7107]. Soaking aged garlic in 5% (v/v, pH 2.33) acetic acid solution,
after 2 days, the garlic cloves turned green. On day 4, pigment(s) diffused from garlic cloves to the
pickling solution. The solution exhibits two maximal absorbances at ∼440 and ∼590 nm, corresponding
to yellow and blue species, respectively, the combination of which creates the green coloration. With
increasing time from 4 to 25 days, the concentration of both yellow and blue species increases at nearly
the same rate, while after 25 days, the concentration of the yellow species increases faster than that of
the blue species. They found that, most thiosulfinates (∼85%) in garlic cloves were converted within 4
days, suggesting that thiosulfinate conversion is proportional to the formation of the pigments. UV−vis
spectral measurements and pH results suggest that the color formation occurs by two kinds of
processes:  one enzymatic and the other nonenzymic. Low pH (2.0−3.0) favors nonenzymic reactions,
while high pH (6.0 or above) is conducive to enzymatic reactions. Thus, the ideal pH for the entire
process of garlic greening is between 4.0 and 5.0.

6. Incidental Additives
Copper from water source, or other can create green colored chelated complexes. The other sources of
copper might be the butter or lemon juice.
In addition, there is reported that Iodine from Iodized salts may turn garlic blue.

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7. Active Alliinase (4.4.1.4) in Dehydrated Garlic

Garlic used for pickling or dehydrated is thermally treated in order to destroy alliinase for two main
reasons: to eliminate pungent flavor and inhibit green discoloration. [Devinder Dhingra (2003):
OPTIMIZATION OF THE DRYING PROCESS OF GARLIC (Allium sativum L.), Dissertation Submitted to the
Punjab Agricultural University].
.

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GREENING OF GARLIC IN MEAT MARINADES

Mechanism of Garlic Greening from Sulphur Organic Compounds

It has been confirmed that the sequence of reactions leading to pigment formation includes
1. Upon disruption of the tissue, isoalliin and other S-alk(en)ylcysteine sulfoxides are enzymatically
cleaved, yielding 1-propenyl-containing thiosulfinates [CH3CHdCHS(O)SR; R ) methyl, allyl,
propyl, 1-propenyl] among others.
2. reaction of the thiosulfinates with amino acids to produce the pigments (Figure 3). Pigments of
very similar natures are produced, with (E)-S-(1-propenyl) cysteine sulfoxide (isoalliin) serving as
the primary precursor.
Whereas the propyl, 1-propenyl, and methyl derivatives form pink, pink-red, and magenta compounds,
those containing the allyl group (4) give rise to blue products after reacting with glycine at pH 5.0.

[Kubec, R., Hrbáčová,M., Musah,R and, and Velíšek,J (2004) : Allium Discoloration:  Precursors Involved in Onion Pinking and Garlic
Greening, Journal of Agricultural and Food Chemistry, 52 (16), 5089-5094]

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In garlic, abundant green-blue compounds formed from 19/20 and 23, which are the key color-
developing compounds occurring in crushed garlic.

Thiosulphinate Base Pigments

Model systems have been analyzed using synthetic Thiosulphonates, in order to analyses the chemical
substances responsible for the pigmentation. The one associated with garlic are highlighted below.
[Kubec, R., Hrbáčová,M., Musah,R and, and Velíšek,J (2004) : Allium Discoloration:  Precursors Involved
in Onion Pinking and Garlic Greening, Journal of Agricultural and Food Chemistry, 52 (16), 5089-5094]

Reactants – Amino acid Requirements for Pigmentation

It has been found that an unsubstituted primary amino group and a free carboxyl group are essential
structural features required for amino compounds to be able to generate the pigments.

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Out of the thiosulfinate analogs tested, only those containing at least a three-carbon chain with the ß-
carbon bearing a hydrogen atom yielded the pigments. Thiosulfonates, sulfoxides, sulfides, and di
Sulfides did not form any colored products.
The pH optimum for pigment formation has been found to be between 5.0 and 6.0 for all thiosulfinates
tested (Kubec R, Velisek J (2007) Allium discoloration: The color-forming potential of individual
thiosulfinates and amino acids: Structural requirements for the color-developing precursors. Journal of
Agricultural and Food Chemistry 55, 3491-3497).
Investigation of the variation in thiosulfinates and related compounds between fresh bulbs and cloves,
and commercial garlic products, indicated that thiosulfinates were released only from garlic
cloves and garlic powder products. (Lawson LD, Wang Z-Y, Hughes BG (1991): Identification and HPLC
quantitation of the sulfides and dialk(en)yl thiosulfinates in commercial garlic products. Planta Medica
57, 363-370).

The analysis indicated that thiosulfinates were released only from garlic cloves and garlic powder
products.

The vinyl dithiins (10,11) and ajoene (12) were

found only in products containing garlic
macerated in vegetable oil.

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The disulfide compounds containing two allyl (9a),

or methyl allyl (9c), or two methyl (9d) residues
were found only in products containing the oil of
steam distilled garlic.

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Enzyme - Alliinase (4.4.1.4) - Properties and Stability

Alliinase in garlic has been studied extensively and its properties are summarized below.
[http://lem.ch.unito.it/didattica/infochimica/2006_aglio/pagine/when.html#terzo]

Purified alliinase loses its activity after 14

days storage at 3°C (pH 6.4) and cannot be
stored at all at 10°C, particularly in diluted
concentration. However, the addition of
glycerol to dilute concentrations (final
concentration 10% v/v) stabilizes activity
completely for at least 30 days.60

It has been suggested that there are two different alliinase activities for garlic, one being specific for
substrate (+)-S-2-propenyl-L-cysteine sulphoxide and (+)-S-1-propenyl-L-cysteine sulphoxide at an
optimum pH of 4.5 and the other specific for substrate (+)-S-methyl-L-cysteine sulphoxide at an
optimum pH of 6.5. Both of these alliinases are irreversibly deactivated at pH 1.5-3 and >9.
Although alliinase can act on both (+) and (-) sulphoxides, it shows a preference for the (+) isomers. 3
The transformation rate of thiosulphinates from their relative precursors is also different while
maximum formation of diallyl thiosulphinate (allicin) can reach 100% within 1 minute, it takes more than
5 minutes for allyl methyl thiosulphinate to reach 100%.
The activity of alliinase is also pH dependent. Below pH 4, alliinase is partially deactivated whilst at pH
4.5-7, alliinase has very high activity. The effects of pH adjustment during the blending of garlic cloves on
the formation of flavor compounds were
studied by Yu and Wu 1 who showed that
maximum allicin formation occurred around
pH 6.5.

[Jing Wang, Yanping Cao, Baoguo Sun,

Chengtao Wang, Yingjie Mo, (2011): Effect of
ultrasound on the activity of alliinase from
fresh garlic, Ultrasonic Sonochemistry, (18) 2,
pp 534-540]

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Thermal destruction of Alliinase follows the first order kinetics with Z-value=9.4 °C. The effectiveness of
thermal treatment to eliminate pungent flavor and inhibit green discoloration increased with increasing
temperatures. Optimum blanch treatment was reached at 80 °C for 1.1 min. [Rejano, L., de Castro, A.,
Sánchez, A., Casado, J., Montaño, A. (2004): Thermal kinetics of pungency loss in relation to the quality
of pickled garlic, International Journal of Food Science & Technology 39(3):311 – 317]

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