High hydrostatic pressure (HHP) microbial kinetics in orange comminuted

Vinicio Serment-Morenoa, Zamantha Judith Escobedo-Avellanedab , Jorge Santos Welti-Chanesc

a
Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, México(vsermentm@gmail.com)
b
Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey,
México(zamantha17@yahoo.com.mx)
c
Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, México(jwelti@itesm.mx)

ABSTRACT
An orange derived paste can be obtained by mixing several of its components, where juice is the major
contributor. This product, known as comminuted, can be used as a refreshing drink base to enhance
nutritional and sensorial characteristics. However, as orange peel is used for comminuted elaboration,
microbial contamination may also be augmented. Orange fruit spoilage is frequently related to acidic
fermentative microorganisms which can be readily inhibited by freezing or applying heat treatment.
Nevertheless, orange peel is a great source of nutraceutic compounds which can easily be degraded by
extreme temperature exposure. The present study proposes a high hydrostatic pressure treatment (HHPT) on
orange comminuted. Natural flora inactivation followed a first order kinetic (R2=0.76-0.91) at certain time
intervals for most applied pressure levels (100-414 MPa). Weibull equation accurately described survival
curves (R2=0.74-0.99) at nonlinear portions for200-414 MPa. Pressures below 200 MPa were unable to
reduce more than 1.5 logarithmic cycles. Microorganism stress adaptation was observed for low pressure,
and extended treatment times (>4 min) resulted in no further growth inhibition. The highest pressure value
applied (414 MPa) was enough to assure microbial safety by reducing population to less than 100 CFU/g
within a two minute treatment. High pressure processing is definitely suitable for orange comminuted safety
requirements and may provide improved nutraceutic and sensorial quality.

Keywords: orange comminuted; high hydrostatic pressure; microorganism inactivation kinetics; food safety; novel food
technologies.

INTRODUCTION

Orange juice may be the most widely consumed plant derived juice. According to the Florida Department of
Citrus (FDOC) there was a worldwide orange production of 63,906 metric tons in 2009, where most of the
fruit is destined toward juice production. Meanwhile ≈700,000 tons/year of peel solids are discarded as waste
or used for byproduct processing [1, 2]. Several studies have demonstrated that orange peel is a rich source of
flavonoids which can be used as a preventive for several chronic degenerative diseases [2, 3, 4]. Therefore,
orange peel is suitable for human consumption. In the present work, orange peel is blended with juice
forming an orange-yellowish paste. This product, also known as orange comminuted, can be used as a
refreshing drink base.

Fruit juice decomposition is frequently associated with fermentative yeasts such as Saccharomyces
cerevisiae, causing ethanolic spoilage, carbonation, production of H2S and off-odors [5]. The processing of
industrial products obtained from citrus fruits, such as heat pasteurization, freezing and sterilization, allows
the reduction or elimination of microbial spoilage potential. Although effective, these processes can severely
damage the quality of food outcome. Losses in sensorial, nutritional, and physicochemical characteristics are
among the detrimental changes achieved. High hydrostatic pressure (HHP) is a relatively new technology
that offers microbial inactivation with less severe modifications in the end product. Pressure levels range
from 100-1000 MPa for experimental appliance and from 100-600 MPa for commercial use [6, 7].

Microorganisms can be affected in several ways with the use of HHP, being the cytoplasmic membrane the
key target. Several other effects can be accounted: changes in macromolecular (proteins, DNA) interactions
and structure; inducement of stress defense and carbohydrate genes; key enzyme inactivation; cell wall and
cytoskeleton changes are suggested by transmission electron microscopy [8, 9]. Microorganism resistance to
pressure varies widely depending on physical (pressure level, time of exposure, substrate) and morphological
(specie, strain, physiological state) factors [10].
Goals aim toward demonstrating that HHP are capable of significantly inactivating orange comminuted
microflorae. Kinetic data parameters such as microbial inactivation constant (k), decimal reduction time (D)
and the z value (amount of pressure required to increase the inactivation rate by ten-fold) obtained can
describe microorganism inhibition and utilized for an adequate process design.

MATERIALS & METHODS

Orange comminuted. Valencia oranges were purchased in local supermarket. Comminuted was prepared by
mixing orange components in the following proportions: juice 71.6% (w/w), pericarp 12.4% (w/w) and outer
layer 16.0% (w/w). All elements were liquefied for two different one minute cycles at maximum speed in
domestic mixer. Samples were stored in polyethylene bags and frozen at -80°C until used.

Aerobic bacteria counts (AB). Poured plate technique was applied. Ten grams of orange comminuted were
added to 90 ml of 0.1% (w/v) peptone water and homogenized for 90 sec in masticator. One milliliter of each
dilution was transferred to a petri dish. Standard count agar was (SCA) was poured until the bottom of the
dish was covered and incubated for 24h at 35±2 °C. Colonies were counted by duplicate and reported as
CFU/g of comminuted.

High Hydrostatic Pressure Treatment (HHPT). Fifty gram samples were processed in a Welch 2L (Avure
Technology) food processor using distilled water as the pressure transmitting fluid. The equipment does not
provide an inner vessel temperature control when pressurized, so temperature slightly rose (1-9°C) due to
water adiabatic compression. Treatments included pressure levels of 100, 200, 300 and 414 MPa, during 1, 2,
3 and 4 min at room temperature. HHPT efficiency was determined by the microbial logarithmic reduction.

RESULTS & DISCUSSION

Bacterial growth inhibition. Comminuted orange presented a moderate initial microbial load (103-105
CFU/g). Low pressures (100-200 MPa) barely accounted to reduce 1.5 log cycles. Bacterial populations were
stabilized as pressure and treatment duration levels increased and a “tail effect” trend can be observed (Figure
1). Meanwhile, treating comminuted samples with 300 and 414 HHP resulted in effectively reducing BC to
eliminate microbial load (<10 CFU/g) by treating samples with at least 4 and 2 min respectively. However,
[11] achieved complete E. coli inhibition by just applying CUT at 276 MPa and higher pressures.

Figure 1. Bacterial population for high pressure treated orange comminuted.

It is also important to denote the curvature concavity inversion when applying 300 MPa. This adjustment
may indicate population variation, or that their adaptability towards the stress conditions of high pressure
processing have gone beyond its limit and can no longer be accumulated. As a result, treatment efficiency
was remarkably increased as both pressure and time levels augmented.

Kinetic parameters for linear inactivation modelling are shown in Table 1. Data considered for this analysis
ranges from control samples through the highest inactivation values achieved. Kinetic constants (k [=] min-1)
values of 0.51 and 1.53 was reported by [12] to inactivate aerobic bacteria in orange juice at 300 and 400
MPa respectively. Decimal reduction times for 300 and 400 MPa were in accordance with those of [13] for
native microflorae in Hamlin orange juice treated at 350, 400 and 450 MPa (D = 1.32 min, 0.47 min and 0.18
min respectively).

Table 1. First order bacterial inactivation kinetic parameters

for pressure treated orange comminuted
P(MPa) k(min-1) D(min) R2
100 0.13 7.42 0.89
200 0.33 3.02 0.78
300 0.50 1.99 0.85
414 1.41 0.69 0.91

Bacterial population shows moderate resistance toward pressure exposure as noted by z = 317 MPa (Figure 3,
R2=0.98). However, z value calculated diverted largely when compared with 85-103 MPa obtained by [11,
13] or 556 MPa [12]. Result variation may be due to orange variety, processing conditions, peel addition or
come up time consideration for kinetic calculations, since D are similar between them.

Figure 3. Effect of pressure on D values for HHP treated orange comminuted

Nonetheless, the first order kinetics was only followed at 100 MPa, since all greater pressures exhibit a
resistant remaining population (Figure 2). Weibull equation (1) has been extensively used to describe
nonlinear survival curves [14] and applied to experimental data. This model adequately described kinetics
from 200-414 MPa, especially in the nonlinear data range. All curves presented downward concavities as
noted by n<1 (Table 2) with the exception of 300 MPa. This could indicate that damage is being accumulated
until a pressure threshold is reached (n=1, between 200-300 MPa). Extending treatments beyond this critical
value can result in diminished bacterial resistance and the already noticed curve concavity inversion. Both
parameters seem to be pressure dependant with no obvious tendency, manifesting peak values for the upward
concavity curve (300 MPa).
� log � � ���/��� (1)

Where:
S: survival fraction after pressure treatment (CFU/CFU0)
t: process time (min)
δ: first decimal reduction time (min)
n: shape factor

Figure 2. Bacterial counts for high pressure treated orange comminuted and Weibull adjusted modeling.

Table 2. Weibull inactivation kinetic parameters

for pressure treated orange comminuted
P(MPa) δ(min) n R2
100 1.59 -0.84 0.25
200 1.00 0.31 1.00
300 8.74 1.28 0.74
414 0.74 0.77 0.80

CONCLUSION
High hydrostatic pressure processing is definitely suitable for orange comminuted pasteurization. Orange
peel addition for comminuted preparation may imply a greater microbial initial population, but does not
represent an important concern. Safe comminuted orange can be accomplished at 300 MPa within a 4 min
treatment. Bacterial counts pressure resistance apparently starts to critically decrease between 200-300 MPa.
Inactivation kinetics for comminuted do follow a first order inactivation model only a restricted range.
Nevertheless, calculated processing parameters for the linear model and seem very closely related to those
describing microbial reduction for different orange juice varieties. On the other hand, Weibull equation
adequately predicts the survival fraction from 200-414 MPa, the nonlinear range. The latter model is
preferable for process design and future kinetic studies in comminuted.

REFERENCES
[1] Espachs-Barroso A., Soliva-Fortuny R.C., & Martıín-Belloso O. 2005. A natural clouding agent by orange peels
obtained using galacturonase and cellulose. Journal of Food Chemistry 92(1), 55-61.
[2] Manthey J.A. & Grohman K. 2001. Phenols in Citrus Peel Byproducts. Concentrations of Hydroxycinnamates and
Polymethoxylated Flavones in Citrus Peel Molasses. Journal of Agricultural and Food Chemistry 49(7), 3268-3273.
[3] Manthey J.A. & Grohman K. 1996. Concentrations of Hesperidin and Other Orange Peel Flavonoids in Citrus
Processing Byproducts. Journal of Agricultural and Food Chemistry 44(3), 811-814.
[4] Tripoli E., La Guardia M., Gimmanco S., Di Majo D. & Giammanco M. 2007. Citrus flavonoids: Molecular structure,
biological activity and nutritional properties: A review. Journal of Food Chemsitry 104(4), 466-479.
[5] Zook C.D., Parish M.E., Braddock R. J. & Malaban M. O. 1999. High Pressure Inactivation Kinetics of
Saccharomyces cerevisiae Ascospores in Orange and Apple Juices. Journal of Food Science 64(3), 533-535.
[6] Balasubramanian V.M., Farkas D. & Turek E.J. 2008. Preserving Foods through High-Pressure Processing. Journal of
Food Technology 62(11), 32-38.
[7] Torres J.A. & Vázquez G. 2005. Commercial opportunities and research challenges in HHP of foods. Journal of Food
Engineering 67(1-2), 95-112.
[8] Fernandes P.M.B. 2005. How does yeast respond to pressure? Brazilian Journal of Medical and Biological Research
38(8), 1239-1245.
[9] Mañas P. & Pagán R. 2005. Microbial inactivation by new technologies of food preservation. A Review. Journal of
Applied Microbiology 98(6), 1387-1399.
[10] Patterson M.F. 2005. Microbiology of pressure-treated foods. A Review. Journal of Applied Microbiology 98(6),
1400-1409.
[11] Guerrero-Beltrán J.A., Barbosa-Canovas G. V. & Welti-Chanes J. 2011. High Hydrostatic Pressure Effect on Natural
Microflora, Saccharomyces cerevisiae, Escherichia coli, and Listeria Innocua in Navel Orange Juice. International
Jouranl of Food Engineering. Manuscript 2166.
[12] Dogan C. & Erkmen O. 2004. High pressure inactivation kinetics of Listeria monocytogenes inactivation in broth,
milk and peach and orange juices. Journal of Food Engineering 62, 47-52.
[13] Parish M. E. 1998. High Pressure Inactivation of Saccharomyces cerevisiae Endogenous Microflora and
Pectinmethylesterase in Orange Juice. Journal of Food Safety 18, 57-65
[14] Mafart P., Couvert O., Gaillard S. & Leguerinel I. 2002. On calculating sterility in thermal preservation methods:
application of the Weibull frequency distribution model. Journal of Food Microbiology 72, 108-113
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