Biochemical Changes During The Storage of High Hydrostatic Pressure - Jacobo Velázquez 2010

Biochemical Changes during the Storage of High
Hydrostatic Pressure Processed Avocado Paste

D.A. Jacobo-Velázquez and C. Hernández-Brenes
Abstract: High hydrostatic pressure (HHP) processing improves the shelf life of avocado paste without a significant
impact on flavor; however, scarce information is available on biochemical modifications during its extended storage
period. The present study focused on the changes in oxidative enzyme activities of pressurized avocado paste (600 MPa
for 3 min) during refrigerated storage (45 d at 4 ◦ C). Aerobic plate counts (APC), lactic acid bacteria counts (LAB), pH,
and instrumental color were also evaluated during storage.
Processing with HHP caused a decrease in polyphenol oxidase (PPO) and lipoxygenase (LOX) activities, resulting in
residual enzyme levels of 50.72% and 55.16%, respectively. Although instrumental color values didn’t change significantly
during the evaluated storage period, both enzymes (PPO and LOX) recuperated their activities at 10 to 15 d of storage,
reached the original values observed in the fresh paste, and then started a declining phase until the end of the storage
period. Pulp pH presented a consistent decline during the first 20 d of storage. LAB counts were very low during storage,
discarding lactic acid production as responsible for the observed pH decline. Enzyme reactivation, cell disruption, and a
gradual migration of intracellular components such as organic acids are herein proposed as the main mechanisms for the
deterioration of HHP treated avocado paste during its refrigerated storage.
Keywords: avocado, high hydrostatic pressure, lipoxygenase, polyphenol oxidase, stability
Practical Application: At the present, HHP is the most effective commercial nonthermal technology to process avocado
paste when compared to thermal and chemical alternatives. Although it has proven to be an excellent product-technology
match, little information is known on the biochemical changes that take place in the product during its refrigerated shelf
life. Biochemical reactions during storage are important, since they can influence avocado paste nutritional and flavor
qualities at the time of product consumption. The present study reports for the first time the re-activation of PPO and
LOX during storage of avocado paste under commercial and economically feasible processing conditions (600 MPa and
3 min). The reactivation of oxidative enzymes observed in the present study is relevant for future studies on the HHP
S: Sensory & Food
stability of food systems in general, and it is considered an important finding for the food industry and researchers seeking
to deliver products with superior nutritional and flavor characteristics.
Quality
Introduction research is needed on technologies that add value to the fruit

Avocado (Persea americana) production, processing and commer- and minimize postharvest losses. Quality losses of fresh avoca-
cialization are activities of very relevant socioeconomic impact dos have been mainly attributed to enzymatic oxidative reactions.
for Mexico. According to the Food and Agricultural Organiza- Avocado protein content is high (1.96% w/w) and a major frac-
tion of the United Nations statistical database (FAOSTAT) since tion of it has been reported to be composed of oxidative enzymes
1961 Mexico has been reported as the number one avocado pro- (Kahn 1977; Elez-Martı́nez and others 2005). When avocados are
ducer in the world. In 2007 the Mexican avocado production processed into paste the tissue undergoes partial disruption of cel-
exceeded 1 million tons, which represents approximately 34% of lular organelles releasing enzymes such as PPO and its substrates
the total world production (FAOSTAT 2009). A large percent- (phenolic compounds), causing the formation of o-quinones,
age of the production is exported to different continents, being which undergo further polymerization to form brown pigments
the United States the most important market with approximately (Weemaes and others 1999).
120000 tons/y (FAOSTAT 2009). Because of its significance, Pulp browning of avocados has also been associated with the
formation of undesirable flavors and nutritional losses (Weemaes
and others 1998). In addition to PPO, avocado fruit contains
MS 20090648 Submitted 7/6/2009, Accepted 3/30/2010. Authors are with Dept.
other enzymes such as LOX and hydroperoxy lyase, which can
of Biotechnology and Food Engineering, School of Biotechnology and Health, Tec- catalyze the degradation of lipids generating off-flavors and the
nológico de Monterrey-Campus Monterrey, E. Garza Sada 2501 Sur, C.P. 64849, coupled degradation of carotenoids and other nutrients during
Monterrey, N.L., México. Author Jacobo-Velázquez is also with Dept. of Horticultural storage (Robinson and others 1995; Jacobo-Velázquez and others
Sciences, Texas A&M Univ., HFSB Building, Room 202, MS 2133, College Sta- 2010).
tion, TX 77843-2133, U.S.A. Direct inquiries to author Hernández-Brenes (E-mail:
[email protected]). An additional factor that can affect the stability of fresh avo-
cado products is microbial growth. Minimally processed avocado
C 2010 Institute of Food Technologists

R
S264 Journal of Food Science r Vol. 75, Nr. 6, 2010 doi: 10.1111/j.1750-3841.2010.01654.x
Further reproduction without permission is prohibited
Enzyme stability during storage of HHP processed avocados . . .
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products (that is, ready-to-eat/nonpasteurized slices and pastes High hydrostatic pressure processing
with food additives) are generally packaged in films or containers Avocado paste packages were commercially processed in
that provide conditions that are ideal for the growth of LAB. Avomex Inc. (Sabinas, Coahuila, Mexico). Samples were pres-
It is well known that avocados are usually consumed as a fresh surized at 600 MPa for 3 min, using a 215L ULTRA HHP pro-
product and that thermal processing of avocado pastes can generate cessing unit (Avure Technologies, Kent, Wash., U.S.A.). The time
bitter off-flavors (Ben-et and others 1973). Therefore, processing required to reach 600 MPa was 3.5 min. The decompression time
of avocado paste with HHP has been pointed as the most effec- was 2.75 min. Purified water was used as the pressurization media.
tive technology when compared to other thermal and chemical Samples were pressurized at a vessel temperature of 23 ◦ C. Pro-
alternatives (López-Malo and others 1998). In addition of being cessing conditions were closely monitored and recorded to assure
a good product-technology match, the industrial production of experimental reproducibility. After processing, the samples were
HHP avocado paste also triggered the commercial expansion and placed in a cold water bath (1 to 3 ◦ C) and transported under
improvement of HHP as an alternative pasteurization technol- refrigeration (4 ◦ C) to the Biotechnology Center at Tecnológico
ogy (Palou and others 2000). Valuable research was conducted by de Monterrey (Monterrey, Nuevo León, México).
López-Malo and others (1998) who worked on the optimization
of the pressurization operation for salted (1.5% NaCl) and acid- Storage study
ified (pH values of 3.9, 4.1, and 4.3) avocado pastes considering Avocado paste samples (60 plastic bags, 200 g each) were stored
various processing conditions (345, 517, 689 MPa for 10, 20, and under refrigerated conditions for 45 d at 4 ◦ C and sampled before
30 min). The researchers concluded that treatments with the lowest and after processing, and every 5 d during the storage period
pH and highest pressures applied, resulted in greater PPO inacti- (45 d). Samples for microbiological, pH, and color determinations
vation. Their study also considered the evaluation of changes in were analyzed immediately, and subsamples (2 mL) for enzymatic
microbial and instrumental color values for the duration of a stor- assays were placed in cryogenic vials and stored at −80 ◦ C until
age period of 60 d at 5 ◦ C, during which bacterial counts remained they were analyzed.
very low and color values acceptable by consumers for treatments
with residual PPO activities <45% after processing. Although it
is known that HHP treatments result in shelf life extension of Microbiological analyses
avocado paste, very little scientific information is available in the Lactic acid bacteria. LAB counts were performed with a
literature on the biochemical and enzymatic changes that take modification of the procedure described by Vedamuthu and oth-
place during the shelf life of the processed product. Furthermore, ers (1992). Avocado paste samples (11 g) were diluted with MRS
avocado pastes without food additives (salt or acidulants) are com- broth (Difco Laboratories, Detroit, Mich., U.S.A.) in sterile con-
mercially available and their mechanisms of deterioration during tainers to an initial dilution of 1 : 10. Additional serial dilutions
storage remain unknown. Understanding the factors that cause were performed as needed to enable colony enumeration. Plating
deterioration during storage of avocado paste and influence con- MRS-agar media as well as the MRS broth were prepared ac-
sumer rejection can provide new information for shelf life dating cording to manufacturer instructions and sterilized at 121 ◦ C for
and process/product improvements. 15 min. Sample aliquots (1 mL) were pipetted into each Petri dish
S: Sensory & Food

The present study focused on the evaluation of PPO and LOX (Ø10 cm), and then 15 mL of MRS-agar media (45 ± 1 ◦ C) were
enzymatic activities during storage (45 d at 4 ◦ C) of avocado paste poured into each plate. Inoculated samples were homogenized
Quality
without food additives and processed under common commercial into the plating media by means of slow circular motions. Once
conditions (600 MPa for 3 min). Changes in pH, APC, LAB the media solidified, the plates were incubated at 37 ◦ C for 48 h in
and instrumental color were also evaluated during storage with the presence of 5% CO2 . Sterility controls were also prepared by
the purpose of elucidating potential mechanisms of the product plating sterile MRS broth (1 mL) instead of the diluted samples.
deterioration during its shelf life. LAB counts were performed in triplicate for each dilution. Data
were reported as colony forming units (CFU) per gram of avocado
paste.
Materials and Methods Aerobic plate counts. Mesophilic bacteria APCs were ob-
Materials and processing tained using AOAC method 966.23C published by FDA (2009).
Avocados (Persea americana Mill, cv. Hass) used in the study were Avocado paste samples (11 g) were diluted with phosphate buffer
obtained from the Mexican region of Michoacan in the year 2005. (3.4% w/v, pH 7.2) in sterile containers to an initial dilution of
Fruits were washed, sanitized, peeled, and macerated into avocado 1 : 10. Additional serial dilutions were performed when needed.
paste. The paste (approximately 15 kg) was vacuum de-aerated Plating media was prepared by dissolving tryptone (5 g), yeast
(−88.2 kPa) prior to packaging using an Ultravac UV2100A pump extract (2.5 g), dextrose (1 g) and bacteriological agar (15 g) in
(Koch Equipment LCC, Kansas City, Mo., U.S.A.). double distilled water and adjusting to volume (1L). Plating media
Individual avocado paste samples (200 g) were packaged into was sterilized (121 ◦ C for 15 min) and 15 mL aliquots were poured
plastic bags (14 cm length × 12.6 cm width × 3 cm height) into Petri dishes (Ø10 cm). Sample dilutions (1 mL) were plated in
of a thickness of 0.018 cm that contained 7 layers of polymers the Petri dishes and incubated at 35 ◦ C for 48 h. Sterility controls
to make them practically impermeable to oxygen (WinpaK Ltd., were also prepared by plating phosphate buffer (1 mL). Bacterial
Winnipeg, MAN, Canada). To reduce the final dissolved oxygen enumeration was performed in triplicate for each sample dilution,
content of the product to approximately 0.15 ppm, vacuum pack- and data reported as CFU per gram of avocado paste.
aging (−67.9 kPa for 2 s) was performed using a Multivac R230
model 542 packaging machine (Multivac, Wolfertschwenden, pH determination
Germany). Following vacuum packaging and prior to process- A Beckman potentiometer (Fullerton, Calif., U.S.A.) was cali-
ing, the samples were placed in cold water (1 to 3 ◦ C for 20 min) brated using pH 4 and 7 buffer solutions and it was used to collect
to reach an internal temperature of approximately 5 ◦ C. pH values of avocado paste samples. The electrode was inserted
Vol. 75, Nr. 6, 2010 r Journal of Food Science S265

directly into the paste and allowed to stabilize before the measure- minute by the use of an extinction coefficient of 23000 M−1 cm−1
ment was taken. at 234 nm.
Polyphenol oxidase activity determination Color determination

Acetone insoluble solids (AIS) from each sample of avocado Instrumental color parameters were monitored during storage
paste were prepared, as described by Kahn (1977), to remove period using a Minolta CR-300 colorimeter (Minolta Co. Ltd.,
compounds that caused interference in the PPO enzymatic as- Osaka, Japan) in the CIE Lab scale with a D65 illumination source
say. Samples of avocado paste (50 g) were homogenized in cold and a standard observer placed at 10◦ . Data collected included de
acetone (200 mL, −20 ◦ C) and vacuum filtered through What- CIE color coordinates lightness values (L ∗ ), a∗ values (green-red
man nr 1 filter paper (Whatman Intl. Ltd., Maidstone, U.K.), scale), b∗ values (yellow-blue scale), and the calculation of hue
using a Savant Gel Pump 110 (Farmingdale, N.Y., U.S.A.). Pre- angles (Hue).
cipitates were reextracted 2 more times with the same volume
of acetone, followed by a final acetone wash (100 mL). The AIS Statistical analyses
extracts were placed in aluminum trays and allowed to dry in the Microbiological and physicochemical data represent the mean
dark, at room temperature for 24 h. PPO assay solutions were values of samples taken from three individual plastic bags collected
prepared by dissolving AIS (0.3 g) in McIlvaine buffer pH 6.5 prior to HHP processing, and at days 0, 5, 10, 15, 20, 25, 30,
(25 mL) prepared with citric acid 0.1 M and sodium dibasic phos- 35, 40, and 45 during the storage period. Analyses of variance
phate 0.2 M. PPO enzymatic assays were carried out according (ANOVA) were conducted using JMP software version 5.0 (SAS
to the conditions described by López-Malo and others (1998). Inst. Inc., Cary, N.C., U.S.A.), and mean separation performed
A fresh (+)-catechin assay solution (4 mM) in McIlvaine buffer using the LSD test (P < 0.05).
pH 6.5 was prepared daily. (+)-catechin assay solution (2.5 mL)
was added to the spectrophotometer quartz cuvette (3 mL) and Results and Discussion
enzymatic reaction started by the addition of PPO assay solutions
(0.5 mL). Changes in absorbance at 420 nm, were collected every Microbiological counts as affected by high hydrostatic
30 s for 3 min. Spectrophotometric determinations were done in a pressure processing and storage
BeckmanR model DU 650 spectrophotometer (Fullerton, Calif., HHP processing resulted in approximately 4 and 5 log reduc-
U.S.A.). In all cases absorbance change rate remained constant tions from the initial APC and LAB levels, respectively (Table 1).
during the enzymatic assay. Initial velocity data (A min−1 ) was López-Malo and others (1998) reported approximately 2 to 3 log
estimated from the slope of absorbance versus time curves consid- reductions in APC counts for salted (1.5% NaCl) and acidulated
ering at least 5 absorbance measurements. Enzymatic activity was (pH 3.9, 4.1, and 4.3) avocado purees treated under various pro-
reported in PPO Units (U) per gram (fresh weight basis), where a cessing conditions (345, 517, 689 MPa for 10, 20, and 30 min).
U was defined as 0.001 A420nm min−1 at pH 6.5 and 25 ◦ C. Although processing conditions used by López-Malo and others
(1998) do not match those used in the present study, it was ob-
Lipoxygenase activity determination served that under commercial processing conditions (600 MPa,
S: Sensory & Food
LOX extraction and activity determinations were performed 3 min) and without previous acidification it is possible to obtain
as described by Anthon and Barrett (2003) with minor modifi- significant microbial reductions that increase the safety and shelf
Quality
cations. Briefly, avocado paste samples (2 g) were homogenized, life of the product.
with a mortar and pestle, in cold 0.1 M sodium phosphate buffer Although the mechanism of microbial inactivation induced by
pH 6 (2 mL). A subsample of homogenized samples (1 mL) was high pressure is not fully understood, recent published data has re-
then transferred into centrifuge tubes (Eppendorf Scientific Inc., vealed that pressurization induces modification in cell membrane
Hamburg, Germany) and centrifuged at 16100 × g, for 5 min at
5 ◦ C using an Eppendorf Model 5415C Centrifuge (Eppendorf
Scientific Inc.). Supernatants were discarded and pellets were Table 1– Effects of high hydrostatic pressure (HHP) processing
resuspended in sodium phosphate buffer solution (0.1 M and (600 MPa for 3 min) and storage (4 ◦ C for 45 d) on the microbial
pH 6) in the presence of 0.1% Triton X-100 to facilitate the solubi- levels of avocado paste.
lization of the enzyme. Samples were centrifuged a 2nd time and Mesophilic
supernatants were collected as LOX assay solutions, maintained bacteria aerobic Lactic acid
under refrigeration, and promptly assayed. Substrate assay solution plate countsA bacteriaA
Storage time (d) (CFU/g)B (CFU/g)B
was prepared by mixing together linoleic acid (140 mg), Tween-20
(280 mg) and de-ionized water (5 mL). The mixture was clarified Unprocessed 240 × 104 ± 110 × 105 ±
by the addition of a 1N NaOH solution (1 mL) and adjusted to samples (day 0) 100 × 104 12 × 105
HHP processed 0 120 ± 30 b 52 ± 26 b
volume in a 25 mL volumetric flask. LOX assay was performed by samples 5 98 ± 34 b 80 ± 5 b
pipetting 0.1M phosphate buffer pH 6 (1 mL) and the substrate 10 115 ± 62 b 208 ± 90 a
assay solution (20 μL) into a 1.5 mL quartz cuvette. Reaction 15 63 ± 13 b 135 ± 12 ab
was started by the addition of the LOX extracts (25 μL) into the 20 150 ± 55 b 153 ± 70 ab
25 82 ± 11 b 115 ± 43 ab
cuvette containing a substrate concentration of 0.5 mM linoleic 30 98 ± 20 b 82 ± 6 b
acid in the total reaction volume. Spectrophotometric readings at 35 150 ± 27 b 190 ± 15 ab
234 nm were collected at 25 ◦ C for 3 min, and enzyme activities 40 142 ± 52 b 117 ± 37 ab
(A/min) were estimated by the initial velocities method from 45 638 ± 240 a 162 ± 26 ab
the linear portion of the curves. Enzymatic activity was reported A
Values represent the mean ± standard error of 3 replications.
B
CFU = colony forming units.
in LOX Units (U) per gram fresh weight basis, where a U was Different letters within a column indicate that values are significantly different by the LSD
defined as the mmol of linoleic acid conjugated dienes formed per test (P < 0.05).
S266 Journal of Food Science r Vol. 75, Nr. 6, 2010

proteins resulting in the impairment of transportation mechanisms Gradual pH decline observed during the first 20 d of the present
that hamper the capability of the cells to actively intake nutrients storage study suggest that organic acids can be slowly leaching out
and eliminate unwanted metabolites (Ananta and others 2004; from vegetable cell organelles (Figure 1). This gradual liberation
Ananta and Knorr 2009). Additionally, it is known that enzymes of organic acids to the food matrix may be explained in terms of
due to their protein nature may be highly affected by pressuriza- physiological parameters such as microviscosity at storage temper-
tion, causing the impairment of essential biochemical routes and atures (4 ◦ C) and water content that may be affecting diffusion
generation of metabolic imbalances, including modifications in rates of intracellular components towards the food matrix. Ad-
gene expression related pathways (Hoover and others 1989; Smelt ditionally, the product acidification that takes place during stor-
and others 1994). HHP processing may also cause modifications age can also be attributed to an increase in free fatty acids being
in the permeability of bacterial cell membranes influencing mi- released from tryglicerides due to lipolysis (Castellanos-Dohnal
crobial inactivation (Shimada and others 1993; Pagán and Mackey 2005).
2000).
APC and LAB counts during storage for 45 d at 4 ◦ C are re- Avocado paste polyphenol oxidase and lipoxygenase
ported in Table 1. APC levels remained constant during the first activities as affected by high hydrostatic pressure
40 d of storage and presented and presented a sudden increase processing and storage
(P < 0.05) on day 45 (Table 1). Microbial inactivation extent HHP processing of avocado paste at 600 MPa for 3 min re-
achieved during HHP processing depends on the conditions se- sulted in an average residual PPO activity of 50.72% (Figure 2).
lected. Most cells are totally inactivated by disrupting their cell Residual PPO activities of approximately 12% and approximately
membrane and internal compartmentalization beyond reparabil-
ity. However, some cells may survive and stay on a latent state,
until cell damage is repaired and metabolic functions are gradu-
ally reestablished. Therefore, the residual aerobic microorganisms
present in HHP processed avocado pulps, were possibly able to
reinstate their functionality around day 40, generating a sudden
increase on their population between day 40 and 45. Mor-Mur
and Yuste (2005) indicated that high pressure treatments cause
sublethal injuries in a fraction of the microbial population, which
can recover and develop during the storage of processed foods.
LAB counts presented a slight increment after 10 d of storage and
remained constant thereafter until the end of the storage period.
APC and LAB determinations suggested that avocado paste pres-
surized under the conditions set for this study resulted in a product
that was safe for consumption and microbiologically stable during
the 45 d of duration of the storage period.
S: Sensory & Food

Avocado paste pH as affected by high hydrostatic pressure
Quality
processing and storage
The pH of a food matrix is a simple measurement that pro- Figure 1–Effects of high hydrostatic pressure (HHP) processing (600 MPa
for 3 min) and storage (4 ◦ C for 45 d) on the pH of avocado paste. Values
vides useful information on the potential chemical changes that represent the mean of 3 replications with their standard error bars. Data
are taking place in the product during processing and storage. Un- points with different letters indicate statistical difference by the LSD test
der the processing conditions evaluated, fresh avocado paste pH (P < 0.05). ∗ Unprocessed avocado paste.
was not significantly affected (P > 0.05) by the HHP treatment
itself (Figure 1). However during storage pH values presented a
constant decline during the first 20 d, and reached values that
were 10.87% lower than the initial pHs of pressurized samples at
day 0 (average pH = 6.35). Following the period of pH decline,
values remained stable at an average value of 5.8 until the end
of the storage time. Since LAB counts were low during storage,
bacterial production of lactic acid was not considered as the main
cause for the observed pH decline of avocado paste. Comparable
results have been observed by our group in previous storage stud-
ies with avocado paste processed under similar HHP conditions,
wherein the author observed a pH decline of avocado pulp from
6.49 to 5.42 during storage at 5 ◦ C for 45 d (Castellanos-Dohnal
2005). A possible explanation for the pH decline observed dur-
ing the first 2 wk of storage, in the present study, is the gradual
migration of organic acids from their intracellular locations to the
avocado pulp matrix. As discussed for bacterial cells, HHP treat-
ments also induce morphological changes of vegetable cells which Figure 2–Effects of high hydrostatic pressure (HHP) processing (600 MPa
for 3 min) and storage (4 ◦ C for 45 d) on polyphenol oxidase (PPO) activity
result in the rupture of membranes and leaching of intracellu- of avocado paste. Values represent the mean of 3 replications with their
lar constituents (Préstamo and Arroyo 1998; Gonzalez and others standard error bars. Data points with different letters indicate statistical
2008). difference by the LSD test (P < 0.05). ∗ Unprocessed avocado paste.

25% have been previously reported by Weemaes and others (1998) stability of LOX in acidulated and salted avocado paste, observing
and Weemaes and others (1999), respectively, for partially purified that processing conditions of 689 MPa for 15 min at 21 ◦ C were
avocado PPO solutions adjusted to pH 5, 25 ◦ C and pressurized necessary to achieve complete enzyme inactivation.
at 650 MPa for 3 min. Although previously used processing con- Most of the existent studies on the effects of HHP processing on
ditions for pressure and time may be considered similar to the enzyme structure and stability have used experimental designs that
ones used in the present study (600 MPa, 3 min), the obtained seek the understanding of the influence of pressure and time as
residual PPO activities differ considerably. This can be possibly variables applied to food matrixes. Since pressurization can cause
due to differences in pH, temperature conditions during process- reversible and irreversible modification in protein structure (Cano
ing, sample differences, and complex enzyme stabilization effects and others 1997; Del Pozo-Insfran and others 2007) the present
within the nonpurified avocado matrix. Likewise, Weemaes and study included storage time as an additional variable, which can
others (1998, 1999) evaluated the inactivation of avocado PPO on influence the levels of enzymatic activity that may be present in the
partially purified avocado PPO model systems and reported that processed product at the time of consumption and consequently
PPO has a pressure sensitive isoenzyme which can be inactivated at affect its quality and nutritional composition.
pressures of 450 MPa and above as well as a pressure resistant isoen- Results from the present pasteurized avocado paste storage study
zyme that in a purified form requires pressures above 700 MPa to indicated that both PPO and LOX activities increased in the sub-
cause its inactivation. Similarly, it is possible that in the present sequent days following pressurization, peaked at a maximum value,
study (Figure 2), only the pressure sensitive PPO isoenzyme was and then started a declining phase until the end of the storage pe-
affected. riod (Figure 2 and 3). PPO activity reached a maximum average
Previous avocado study by López-Malo and others (1998) and value of 39050 ± 4500 U/g, which was not significantly differ-
Palou and others (2000) reported a PPO residual activity of ent than the average activity present in the unprocessed avocado
approximately 24% and 36.74%, respectively, when acidulated paste (Figure 2). Similarly, LOX activity levels also increased in
(pH 4.1 to 4.3) avocado paste was pressurized at 689 MPa and subsequent days after processing and reached a maximum average
21 ◦ C for 10 min. A well known strategy used to decrease the ac- activity of 0.31 ± 0.02 U/g, which was also nonsignificantly dif-
tion of detrimental enzymes in food matrixes consists on lowering ferent (P > 0.05) than the average LOX activity in unprocessed
the pH to values afar from their activity and stability optimums. samples (Figure 3). PPO reactivation has also been observed in
As demonstrated by prior avocado studies the combination of a HHP processed avocado paste in previous studies conducted in
decrease in pH and pressure results in higher PPO inactivation. our group (Castellanos-Dohnal 2005) and by Guerrero-Beltrán
However, decreasing the pH to inactivate enzymes may increase and others (2005) in peach. A slight increase in LOX activity dur-
the perception of sour flavor in the product. ing refrigerated storage has also been observed by our research
LOX is another relevant avocado enzyme, due to its potential group in mango paste processed at 600 MPa for 3 min (Ramos-
impact on the nutritional and sensory properties of avocado paste; Parra 2006). Processing reactivation of enzymes in model systems
and it is generally less stable to pressure than PPO (Seyderhelm treated with HHP has been mainly explained as being caused by
and others 1996). Pressurization of avocado paste at the processing the unfolding and refolding of enzymes back to the initial or other
conditions of the present study (600 MPa for 3 min), resulted in active structures (Gomes and Ledward 1996; Mozhaev and oth-
S: Sensory & Food
average residual LOX activities of 55.16% (Figure 3). In a similar ers 1996; Sun and others 2002). However, the increased activity
study conducted by Castellanos-Dohnal (2005) using also avocado observed in PPO and LOX during storage of the HHP-treated
Quality
paste without food additives and the same processing conditions, avocado paste, might be also due to the leaching of both enzymes
the author obtained slightly lower residual LOX levels (approx- from the intracellular compartment to the matrix. This increased
imately 43%). Palou and others (2000) also studied the pressure concentration of both proteins in the media may be influencing
the amount of both proteins recovered by the analytical procedures
used for their extraction from avocado paste and subsequent en-
zymatic analysis. Additionally, the application of stress conditions
such as wounding and high pressure to plant tissues may induce
the activation of defense mechanisms that trigger the biosynthesis
of LOX and PPO (Orozco-Cárdenas and others 2001). However,
subsequent experiments analyzing the HHP effect on the gene
expression and protein levels of avocado pulp would be needed to
support these explanations.
The subsequent decrease in LOX and PPO activities during
storage are thought to be related to the matrix pH, oxygen avail-
ability and protease release from tissue through time. It is well
known that surrounding (solvent) conditions such as pH and ionic
strength affect enzyme stability. Figure 1 shows that pH of the
HHP processed avocado paste decrease during storage. A signif-
icant decrease on pH to values below 6 was observed between
days 10 and 15, concurring with the decrease of PPO and LOX
residual activity. As mentioned before, the pH has a significant ef-
fect on enzyme activity and stability. Attractive ionic interactions
Figure 3–Effects of high hydrostatic pressure (HHP) processing (600 MPa needed to preserve the active form of the enzyme are perturbed
for 3 min) and storage (4 ◦ C for 45 d) on lipoxygenase (LOX) activity of
avocado paste. Values represent the mean of 3 replications with their
as pH changes (Fersht 1998). Consequently, long exposition to
standard error bars. Data points with different letters indicate statistical nonoptimal pH conditions may cause the unfolding of PPO and
difference by the LSD test (P < 0.05). ∗ Unprocessed avocado paste. LOX, adopting an inactive conformation. The behavior observed

Table 2–Changes CIE color values following high hydrostatic pressure (HHP) processing of avocado paste at 600 MPa, 3 min and
during storage (4 ◦ C for 45 d).
Storage Net color
time (d) L∗ a∗ b∗ Hue angle difference (E)
Unprocessed 65.7 ± 0.6A abcB −17.1 ± 0.2 g 41.5 ± 0.2 a 112.4 ± 0.1 ab
samples (day 0)
HHP processed 0 65.8 ± 0.2 abc −17.2 ± 0.2 fg 40.9 ± 0.1 abc 112.8 ± 0.3 a 0.9 ± 0.5 d
samples 5 65.6 ± 0.5 abcd −17.0 ± 0.2 efg 41.4 ± 0.4 ab 112.4 ± 0.4 ab 0.8 ± 0.2 d
10 65.4 ± 0.1 abcd −16.5 ± 0.0 def 40.3 ± 0.2 bcd 112.3 ± 0.1 abc 1.7 ± 0.0 cd
15 66.5 ± 0.5 a −15.8 ± 0.2 bc 40.6 ± 0.5 abcd 111.2 ± 0.5 bcd 2.3 ± 0.5 bc
20 64.1 ± 0.3 e −16.4 ± 0.3 cde 38.7 ± 0.2 e 113.0 ± 0.5 a 3.5 ± 0.3 a
25 64.4 ± 0.6 de −17.0 ± 0.2 efg 40.6 ± 0.1 abcd 112.7 ± 0.2 a 2.1 ± 0.6 bc
30 64.8 ± 0.2 cde −16.1 ± 0.2 cd 39.7 ± 0.7 cde 112.1 ± 0.6 abc 2.5 ± 0.5 abc
35 66.3 ± 0.3 ab −15.4 ± 0.5 ab 41.1 ± 0.8 ab 110.5 ± 0.9 d 2.2 ± 0.3 bc
40 65.5 ± 0.2 abcd −15.5 ± 0.1 ab 40.1 ± 0.3 bcd 111.0 ± 0.2 cd 2.3 ± 0.1 bc
45 65.1 ± 0.4 bcde −14.9 ± 0.2 a 39.5 ± 0.4 de 110.6 ± 0.4 d 3.1 ± 0.3 ab
A
Valuesrepresent the mean ± standard error of 3 replications.
B
Different letters within a column indicate that values are significantly different by the LSD test (P < 0.05).
on Figure 2 and 3 may also be explained in terms of oxygen activities lower than 45%, should remain acceptable by con-
concentration. Although prior to packing avocado paste was vac- sumers for a period of 60 d. However, the increased activities
uum de-aereated, entrapped residual oxygen within the food ma- for LOX and PPO observed herein during the storage of HHP
trix was present. Therefore, PPO and LOX may have used that treated/nonacidulated avocado paste indicate that relevant enzy-
residual oxygen on early storage stages (<10 d) as co-substrate to matic changes are taking place during the shelf life of the product.
oxidize phenolics and polyunsaturated fatty acids. However, once Their residual enzymatic activities can potentially influence prod-
residual oxygen was entirely used, both enzymes may have been ar- uct flavor and nutritional composition, aspects that are relevant to
rested on oxygen-depleted conditions. Protease decompartmental- consumer acceptability and health.
ization and release from intracellular compartments through time In an additional study with guacamole paste (prepared with
may also explain the reduction on PPO and LOX residual activity onions, salt, and acidulants), conducted by Palou and others
at latter storage stages (>10 d). (2000), the researchers observed a decline in hue angle during
storage, which the researchers attributed to a decrease in the green
tones due to chlorophyll degradation. In agreement with their
Avocado paste color as affected by high hydrostatic previous research, in the present study Hue angles also presented a
pressure processing and storage slight declining trend during the storage period (Table 2), however
Results from instrumental color measurements indicated that the overall statistical difference for Hue data during storage was
avocado paste samples did not present significant color varia- nonsignificant (P < 0.05).
S: Sensory & Food

tions as an effect of the application of the pressurization treatment
(Table 2). Similarly, López-Malo and others (1998) also reported
Quality
nonsignificant (P > 0.05) variations of instrumental color values of Conclusions
salted and acidulated avocado pastes subjected to HHP processing The present study generated new information on the enzymatic
treatments. Pressure treated avocado pastes also retained fresh-like and microbiological changes that take place in HHP treated avo-
color attributes. cado paste during refrigerated storage. Data from microbiological
In the present study, instrumental color values presented only analyses indicated that the product was safe for consumption dur-
slight variations during the evaluated storage period (45 d at ing the evaluated storage period (45 d at 4 ◦ C) and also indicated
4 ◦ C). The only parameter that increased significantly (P < 0.05) that LAB and APC counts did not change significantly during
was the instrumental CIE color a∗ -value (Table 2). López-Malo storage. Experimental observations of a continuous pH decline of
and others (1998) also collected color values of HHP processed the pressurized avocado paste during storage appear to be rele-
avocado pastes during storage, and similarly observed an increase vant as one of the major deteriorative changes that can influence
in Hunter a-values. In their study the same samples were also consumer acceptability of the product during storage. Another
evaluated by consumers for their color acceptability; with the relevant outcome of the present study was the observation of PPO
responses generated the authors established a Hunter a-value of and LOX increases in enzyme activity during the storage of pres-
−0.47 ± 0.3 (3.04 when converted to CIE a∗ -value) as the upper surized paste, suggesting their potential role in the generation of
acceptability limit for avocado paste color by consumers. In the undesirable flavors and nutritional losses (vitamin C, carotenoids,
present shelf life study, the observed CIE a∗ values remained within and monounsaturated fatty acids) in the product. Results from this
the consumer acceptability limits previously reported for avocado study also suggested that the observed changes in oxidative en-
paste. zymes during storage are relevant, and should also be considered
Although PPO activities during storage were not evaluated in in future experimental designs to select adequate HHP processing
prior avocado study (López-Malo and others 1998; Palou and conditions, which can improve the nutritional and flavor charac-
others 2000), the researchers established a regression equation teristics of pressurized products during their whole shelf life.
between the after processing residual PPO activities and their
respective color a∗ -values. Using instrumental Hunter a-values Acknowledgments
collected during storage, after processing residual PPO activi- This research was possible thanks to the research funds from
ties and consumer acceptability data, the researchers estimated the Tecnologico de Monterrey-Research Chair Initiative (CAT-
that HHP treated acidulated avocado pastes with residual PPO 05) and from the joint Research Program of The Univ. of

California Inst. for Mexico and the United States (UC MEXUS) Jacobo-Velázquez DA, Hernández-Brenes C, Cisneros-Zevallos L, Benavides J. 2010. Partial pu-
rification and enzymatic characterization of avocado (Persea americana Mill, cv. Hass) lipoxy-
and CONACYT. We also would like to thank Fresherized Foods, genase. Food Res Int 43(4):1079–85.
A Div. of Avomex Inc. for providing avocados, the use of HHP Kahn V. 1977. Some biochemical properties of polyphenoloxidase from two avocado varieties
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López-Malo A, Palou E, Barbosa-Cánovas GV, Welti-Chanes J, Swanson BG. 1998. Polyphe-
the advancement of avocado science. noloxidase activity and color changes during storage of high hydrostatic pressure treated
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Biochemical Changes during the Storage of High

Hydrostatic Pressure Processed Avocado Paste

Introduction research is needed on technologies that add value to the fruit

C 2010 Institute of Food Technologists

S: Sensory & Food

Vol. 75, Nr. 6, 2010 r Journal of Food Science S265

Polyphenol oxidase activity determination Color determination

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