Lupine Publishers | Scholarly Journal of Food and Nutrition
Abstract
A post-column reaction method, which
involves use of the pH indicator BTB, was developed and applied to the analysis
of low molecular weight organic acids in commercial beers, wines, and fruit
juices. Organic acids identified in these beverages were formic, acetic,
citric, pyruvic, tartaric, malic, succinic, lactic, and pyroglutamic. Amounts
of total acids in samples ranged from 980.4 mg/L to 513.5 mg/L in beers, from
7,502.3 mg/L to 5,573.3 mg/L in wines, and from 11,162.8 mg/L (orange) to
2,995.8 mg/L (mango) in fruit juices. Citric acid was found in the greatest
amount-ranging from 286.7 ± 4.5 mg mg/L to 139.0 ± 5.3 mg mg/L in beers. The
greatest level of malic acid was in a wine with 4,248.4 mg/L. The method
developed is applicable to determine amounts of organic acids, formic and
acetic acids, in beverages quickly and accurately.
Keywords: Organic acids analysis; HPLC/post-column; Beers; Fruit
juices; Wines
Introduction
Low molecular weight organic acids
(LMWOAs), including formic, acetic, malic, citric, tartaric, lactic, succinic,
and oxalic acid, are found in various beverages, such as beers, wines and fruit
juices (citrus, apple, grape, and melon), as well as in some foods [1,2]. These
acids contribute characteristic tastes to beverages. For example, formic acid
possesses pungent odor and sour taste in proper dilution. It is particularly
adaptable to the pineapple flavor. Acetic acid also has a pungent and stinging
sour odor, but it gives a clean-sour and acid taste at dilute concentrations
(lower than 1%) in water and been used in flavor compositions, such as butter,
chocolate, grape strawberry and wine. Malic acid is present in plum, peach,
apricot and related fruits and used for imitation fruit flavors, such as maple
[3]. Citric acid is the major acid component of citrus species and possesses a
clean acid taste in an aqueous solution [3,4]. In addition to the role of organic
acids in taste and flavor of beverages, they also play an important role in
quality of beverages, including beers [5], wines [6] and fruit juices [7]. A
capillary electrophoresis (CE) with spectrophotometric method achieved
successfully analysis of a limited number of LMWOAs [8].
This method has been widely used for
LMWOAs analysis in foods and beverages [9]. However, the resolution and
detection of LMWOAs formic and acetic, achieved better by chromatographic
methods than by the spectrophotometric method [10]. In the case of the most
commonly used gas chromatography (GC), a tedious derivatization of organic
acids is required to prepare samples for analysis because they are highly
soluble in water [11]. On the other hand, HPLC, which can take aqueous samples
directly, has been widely used to analyze LMWOAs in water samples; such as
wine, beer [12], vinegar and fruit juices [13], coffee [14] and alcoholic
beverage meads [15]. A HPLC interfaced to enzyme reactor analyzed oxalic acid
in fruit and vegetable juices [16]. After the electronspray ionization system
was advanced to interface between HPLC and mass spectrometers (LC/MS), LC/MS
application for the analysis of water-soluble chemicals, including LMWOAs,
developed significantly [17]. However, separation of LMWOAs by HPLC, formic
acid and acetic acid, remains relatively difficult.
In the present study, a previously
reported simple and accurate post-column reaction method for LMWOAs [18] was
improved and successfully applied to analyze LMWOAs, including formic and
acetic acids, in beers, wines, and fruit juices.
Materials and Methods
Beverage
Samples
All commercial beverages-10 kinds of
beers, 6 types of wines (5 red and 1 white), and 25 kinds of fruit juices-were
bought from a local market in Seoul, Korea.
Chemicals
and Reagents
Bromothymol blue (BTB, 95%), sodium
phosphate dibasic anhydrous (98.5%), sodium hydroxide (98%), perchloric acid
(67– 72%), oxalic acid (99.5–100.2%), phosphoric acid (85%), citric acid
(99.5%), tartaric acid (99.5%), malic acid (95–100%), quinin acid (100%),
succinic acid (99%), fumaric acid (99%), lactic acid (85%), formic acid (95%),
acetic acid (99.7%), pyroglutamic acid (99%) were purchased from Sigma–Aldrich
Chemical Co. (St. Louis, MO, USA) or TCI Chemical Co. Ltd. (Tokyo, Japan). The
purified water was prepared with a Zener Power II (Human Co., Korea). A stock
solution of standard organic acids (10g/L) was prepared in purified water for
preparation of standard solutions and spike analysis.
Preparation
of Beer and Wine Samples
Beer and wine samples (2 mL each)
were filtered with an Arcadis Syringe Filter with 0.45 mm PVDF membrane (Waters
Co, Milford, MA).
Preparation
of Fruit Juice Samples
Well-mixed juice samples were
centrifuged at 10,000 rpm for 5min at 4 ºC to collect the supernatant. The
supernatant was filtered with an Arcadis Syringe Filter with 0.45mm PVDF
membrane (Waters Co, Milford, MA) and were analyzed with HPLC.
Analysis
of Organic Acids in Beverages
Figure 1 shows the systematic
diagram of the post-column method used in the present study. A prepared
beverage sample was injected (10mL) into an Agilent 1100 model HPLC equipped
with a 5cm x 8.0 mm i.d. Shorex RS pack KC-LG guard column, which was connected
to two 30 cm x 8.0 mm i.d. KC-811 separation columns in series. The mobile
phase was a 3mmol perchloric acid solution with a 0.7 mL/min flow rate.
Temperature of the separation columns was 80ºC. The eluate from HPLC was mixed
with a reaction solution containing 0.2mmol bromothymol blue (BTB), 15 mmol Na2HPO4,
and 2 mmol NaOH. The reaction solution was purged at a 0.7mL/ min flow rate in
a T-way connector as shown in Figure 1. Adducts formed from the acids and BTB
in a 50 cm x 0.25 mm i.d. stainless reaction coil was monitored with a UV
detector at l = 440 nm.
Results and Discussion
As mentioned above, analysis of
LMWOAs is a difficult process. There have been advances in HPLC/MS methods recently
but some problems, such as insufficient resolution of formic acid and acetic
acid, remain. In order to resolve these problems, we developed a post-column
reaction method, which involved use of pH indicator BTB. This method and
related theory were originally advanced nearly three decades ago [18]. Figure 2
shows an HPLC postcolumn chromatogram of standard acids. This chromatogram
shows satisfactory resolution of low molecular weight acids for quantitation.
Phosphoric acid peak in this chromatogram may come from a phosphate in a
reaction solution. However, phosphoric acid is not organic acid, which is out
of scope of this study. The limits of detection (LOD) of acids were 25.0mg/L
for citric, malic and quinin, lactic; 12.5mg/L for formic and acetic; and 50mg/L
for pyroglutamic. The limits of quantitation (LOQ) of each acid were 75.0mg/L
for citric, malic, quinin, and lactic; 30.0 mg/L for formic; 50.0 mg/L for
acetic; and 150 mg/L for pyroglutamic.
Organic
Acids Found in Beer Samples
Table 1 shows the results of LMWOAs
analysis in beer samples. There are three monoprotic acids (lactic, acetic and
pyroglutamic), two diprotic acids (malic and succinic), and one triprotic acid
(citric acid). Amounts of total acids in beer samples varied considerably,
ranging from 1,801.3 mg/L (brand E) to 776.0 mg/L (brand D). Among the acids
identified in the beer samples, citric acid was generally found in the greatest
amount-ranging from 286.7 ± 4.5 mg/L (brand E) to 139.0 ± 5.3 mg/L (brand G),
followed by lacticranging from 274.3 ± 5.6 mg/L (brand E) to 19.3 ± 2.2 mg/L
(brand G), and pyroglutamic acid-ranging from 264.9 ± 1.4 mg/L (brand E) to
105.5 ± 2.4 mg/L (brand D. ) Previously, the electrophoresis method was
successfully used to analyze LMWOAs in beers. Klampft [19] found citric acid
with level of 193 ± 2.4 mg/L in Chinese rice beer, 171 ± 1.5 mg/L in white beer
and 178 ± 1.1 mg/L in Lager beer. Cordeiro-Ramirez et al. [20] reported citric
acid (ND – 59 ± 2 mg/L), fumaric acid (ND – 5.9 ± 0.7 mg/L), malic acid (106 ±
12 mg/L), pyroglutamic acid (92 ± 6 mg/L) in 6 different beers. Citric acid
exhibited synergistic effects with lactic acid-producing bacteria toward
inhibition of pathogenic bacteria grow [21]. The amounts found in these acids
in the present study were like these reports. For example, the present study
found citric acid with amounts ranging from 146.2 ± 3.8 mg/L brand D to 286.7 ±
4.5 mg/L in brand E. In addition, the present study found pyroglutamic acid
with levels ranging from 105.5 ± 2.4 mg/L in brand D to 264.9 ± 1.7 mg/L in
brand E. The level of acetic acid ranged from 98.2 ± 3.0 mg/L (brand C) to not
detected (brand H) in the present study, whereas acetic acid was not reported
from the studies conducted with the electrophoresis method.
Organic
Acids Found in Wine Samples
Table
2 shows the results of LMWOAs analysis in wine samples. The total amounts of
acids were similar in all wine samples, ranging from 7,052.3mg/L (brand B) to
5,573.3mg/L (brand C). Tartaric acid was generally found in the highest
concentration, ranging from 2,599.9 ± 7.5mg/L (brand F) to 1,495.1 ± 5.6mg/L
(brand B). Amounts of citric acid, malic acid and lactic acid varied
significantly among brands. Lactic acid was found at high levels in brands A
(2,497.1 ± 2.8mg/L), C (2,271.1 ± 5.7 mg/L), and E (2,954.6 ± 4.2mg/L), but at
relatively low levels in brands B (169.8 ± 1.9mg/L), D (170.4 ± 6.3 mg/L), and
F (101.5 ± 4.9mg/L). On the other hand, high levels of malic acid were found in
brands B (4,248.4 ± 13.8 mg/L), D (2,403.2 ± 8.0mg/L), and F (2,248.8 ±
5.3mg/L), whereas its concentrations in A was only 35.6 ± 3.8 mg/L in, C was
17.3 ± 1.4 mg/L and in E was 58.7 ± 4.2mg/L. A similar trend was observed in
the case of citric acid. A previous study found lactic and tartaric acids in
Ribeiro Sacra wines as the predominant organic acid components [9]. Lactic acid
and tartaric acid levels ranged from 3,784mg/L to 452mg/L and 1978mg/L 866mg/L.
These results are consistent to the ones from the present study. As mentioned
above, the quality of wines depends on, in part, acidity associated with the
composition of acids [22]. The results in the present study demonstrate that
the composition of acids in different brands of wines varies. Therefore, the
analytical method developed can be useful in evaluating wine quality as well as
a guide to improvement in the winemaking process.
Organic
Acids Found in Fruit Juice Samples
Table
3 shows the results of LMWOAs analysis in fruit juice samples. Formic acid and
acetic acid, which are the lowest and 2nd lowest molecular weight
acids among LMWOAs and difficult to determine by a CE method [10], were
satisfactorily analyzed in the present study. The levels of formic and acetic
acids found ranged from 19.8 ± 1.0mg/L (mulberry) to 4.0 ± 0.2mg/L (mandarin)
and from 27.7 ± 2.7mg/L (tomato) to 17.7 ± 2.6mg/L (grape), respectively.
Orange juice samples had the greatest total concentration of acids, ranging
from 11,162.8 mg/L (brand D) to 8,469.5 mg/L (brand C), followed by mandarin
juice samples, ranging from 10,683.1mg/L (brand C) to 5,560.6 mg/L (brand B)
and grape juice samples, ranging from 5,208.2 mg/L (brand E) to 4,016.2 mg/L
(brand C). Citric acid and malic acid were generally detected in higher levels
than other acids, ranging from 10,270.1 ± 2.9mg/L (mandarin) to 36.9 ± 12.0mg/L
(mulberry) and from 3,990.3 ± 11.6mg/L (Grape) to 66.9 ± mg/L (mango),
respectively. The pear juice sample contained the lowest level of total acid
with 596.2 mg/L. The total amounts of acids in mixed juices ranged from
7,237.2mg/L (brand F) to 4,105.8 mg/L (brand A), which are comparable to those
of grape juices.
One previous report demonstrated
satisfactory analysis of organic acids in fruit juices (apple, peach, pear and
apricot) using HPLC with an ion- exclusion column [23]. Another previous study
reported the amounts of citric, malic, quinin and tartaric acids in apple,
orange, cranberry, white/red grapes, and pomegrante juices analyzed using
LC/MS/MS [24]. Effect of malic acid for the inactivation of common food
pathogens on fresh-cut lettuce was reported, suggesting that malic acid possesses
some biological activity [25]. The presence of formic and acetic acids was,
however, not reported in these studies. On the other hand, the present study
found 9.5 ± 3.6 mg/L formic acid in pear juice, 4.0 ± 0.2 – 10.0 ± 0.8 mg/L in
mandarin juice, 12.5 ± 0.5mg/L – 15.2 ± 2.4mg/L in orange juice, 13.4 ± 0.3
mg/L in grape fruit, 5.8 ± 0.5 – 10.7 ± 0.3 mg/L in grape juice, 6.6 ± 1.5mg/L
in mango juice, 10.1 ± 0.7 mg/L in tomato juice, and 19.8 ± 1.0 mg/L in
mulberry juice. In addition, acetic acid was determined in pear juice (8.4 ±
1.3 mg/L), grape juice (17.7 ± 2.6 mg/L – 21.1 ± 1.0 mg/L), mango (23.2 ± 0.5
mg/L), and tomato (27.7 ± 2.7 mg/L). However, it is difficult to compare the
results from the present study to those from the previous study because the
compositions of the juices vary in the different brands.
Conclusion
The present study demonstrates that
the method developed was useful to determine LMWOAs, formic acid and acetic
acid, present in samples with complex matrices of beverages. Acids composition
are important to evaluate quality of beverages. Therefore, the method developed
in the present study would useful to analyze LMWOAs levels for evaluating
quality of beverages. The CE method is also well-established method for LMWOAs
analysis. Therefore, it is recommended to use both methods, the present method
for formic acid and acetic acid
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