Plant extracts, antimicrobials and ultraviolet light as hurdle technology in controlling Alicyclobacillus acidoterrestris

概要

The fruit juice industry has been driven by the consistent demands of consumers for
products that cater to their health and well-being. Nonetheless, a persistent spoilage
microorganism Alicyclobacillus acidoterrestris has plagued the industry in recent years and
has caused considerable losses for some. This spore-forming bacterium is considered as the
most important spoiler for fruit juices and acidic beverages due to its thermophilic and
acidophilic nature, which means that it can resist and survive typical thermal pasteurization
regimes that are applied in fruit juice production. Its survival and contamination ultimately
result in products that have unacceptable quality due to the off-flavors and off-odors that are
produced, resulting in substantial economic losses due to food wastage. Hurdle technology is
a novel concept that involves use of multiple, mild physical or preservation methods that can
result in process schedules with optimal efficacies. In this work, a hurdle technology
combining the use of UV-C processing with traditional and natural antimicrobials in order to
control A. acidoterrestris. was investigated and proposed.

Study 1: Effects of diluents, temperature and pH on the enumeration and growth kinetics of
Alicyclobacillus acidoterrestris in standard growth media
The efficacies of distilled water, saline solution, peptone water and phosphate buffered
saline as diluents on the enumeration and viability of A. acidoterrestris were investigated. In
typical microbiological experiments, diluted samples may be kept for a short period of time
prior to plating during intensive experimental runs, which could possibly affect the outcome
of enumerated counts. Distilled water proved to be the most ideal diluent as it was economic
yet effective enough to maintain organism viability. Interestingly, it was observed that the
common peptone water is actually unfavorable as a diluent for A. acidoterrestris. It must be
noted that there have been very little studies that have reported the same observation. In the
succeeding phase, the influences of temperature and pH on the growth behavior of A.

acidoterrestris vegetative cells in yeast starch glucose broth (YSGB) were also determined and
quantified. Variations in growth behavior included the presence or absence of growth lag as
well as differing growth rates and population changes. It is important to note that some
temperature-pH combinations of the experimental design even resulted in the inactivation of
vegetative cells, even though the operable region of the experimental design was based on
optimum ranges in published literature. Out of the three growth kinetic response parameters,
the model for lag time was not significant. Model fitting and analysis showed temperature and
pH were factors that directly influence the other growth kinetic parameters. Model validation
ultimately revealed acceptable performance for the population change model and limited
success for the growth rate model. The results established in the first study may be used in the
preparation of specific YSG growth media with the desired temperature and pH conditions that
will allow for the control of the growth kinetic parameters of A. acidoterrestris vegetative cells.
Also, the results obtained in this study inadvertently provided additional information as to how
A. acidoterrestris vegetative cells might be inhibited by environmental control and product
formulations. Based on the results of this study, it is believed that different strains of A.
acidoterrestris ought to have different optimum temperature and pH growth parameters.

Study 2: Antimicrobial efficacies of plant extracts and antimicrobials against Alicyclobacillus
acidoterrestris
In this study, 27 ethanolic plant extracts were screened for potential antimicrobial
activity against Alicyclobacillus spp. Licorice and sage extracts were found to be the two most
effective ones. The combined efficacies of the two chosen plant extracts with glycine and
sodium acetate were also investigated. Positive additive effects were observed for licorice
extracts with glycine or sodium acetate and sage extract with glycine. However, sage extract
with sodium acetate exhibited adverse effects, even up to antagonism. Prior to survival studies

in apple juice, germination studies of A. acidoterrestris spores were conducted, and the results
showed that A. acidoterrestris spores could germinate and grow in diluted apple juice even up
to concentrations of 90%, but not at the undiluted level. 50% apple juice was chosen as it was
without unwanted inhibitory effects or stresses at the onset of incubation. Based on calculated
FIC indices, the tested ideal combination of licorice extract with glycine or sodium acetate
resulted in sporicidal activity against A. acidoterrestris in apple juice. For sage extract with
glycine, only a temporary sporostatic effect was observed prior to eventual germination,
growth and proliferation of A. acidoterrestris spores. The results presented in this study may
be used in further research dealing with the shelf-life extension of fruit juices through the
application of similar hurdle technologies with natural antimicrobials.

Study 3: Influence of plant extracts and antimicrobials on the subsequent UV resistance of
Alicyclobacillus acidoterrestris
The effects of physicochemical stress exposures and their resulting physiologies on the
UV-C resistance of A. acidoterrestris were investigated. Microorganisms are exposed to
different environments during food production, and these unwanted exposures to different
physicochemical stresses can induce adaptive mechanisms that could enhance their survival
and further affect their resistance to the kill step during processing. In this study, inactivation
behaviors of A. acidoterrestris mainly followed first-order kinetics or a log-linear trend for
both vegetative cells and spores. It was also observed that the UV-C resistance of cells treated
in YSGB was higher than those treated in apple juice. Heterologous adaptation was observed
for heat-stressed cells but was limited to those treated in the YSGB suspending medium. More
importantly, spore cells were deemed to be the most resistant physiology as it was the least
susceptible to UV-C radiation. This study also investigated the possible hurdle technology
between UV-C processing and the application of antimicrobials such as plant extracts, glycine

and sodium acetate. It was observed that spore cells were rendered to be more susceptible to
UV-C in all treatment combinations of these antimicrobials. While it is generally believed that
using plant extracts may impart adverse effects on the sensory qualities of a food product,
colorimetry findings revealed that the treatment of plant extracts and antimicrobials have
indeed significantly changed the color profile of apple juice, but the color changes brought
about by the treatments were still within acceptable limits. Sensory analysis further showed
that the developed hurdle technology resulted in apple juice with acceptability ratings that were
generally a bit lower but did not significantly differ from the control. The results obtained from
this work may be used in the establishment of UV-C process schedules for better shelf-life
extension of apple juice.

Considering the economic importance of fruit juices as well as the challenges posed by
A. acidoterrestris to the fruit and beverage industry, along with the implications of various
food-, process-, and microorganism-related factors pertinent to food processing, food quality
and food safety, the present work has proposed a hurdle technology that makes use of natural
and traditional antimicrobials in line with a novel food processing technique in controlling the
spoilage microorganism and shows promise as a possible alternative to currently employed
thermal processing methods.