Original article

Effects of lysozyme on Alicyclobacillus acidoterrestris under

laboratory conditions

Antonio Bevilacqua,* Emanuela Ciuffreda, Milena Sinigaglia & Maria R. Corbo

Department of the Science of Agriculture, Food and Environment, University of Foggia, Via Napoli 25, 71122 Foggia, Italy

(Received 11 March 2013; Accepted in revised form 11 July 2013)

Summary This research was aimed at investigating the bioactivity of lysozyme towards Alicyclobacillus acidoterres-

tris. Lysozyme (0.1–20 ppm) was tested towards five different strains; the experiments were performed on

both spores and vegetative cells using a microdilution approach to assess the minimal inhibitory concen-

tration (MIC). Thereafter, spore viability of two selected strains was evaluated through the traditional

plate count. Finally, an experiment was run to assess the role of lysozyme on the complex phenomenon

of spore germination. The MIC of lysozyme towards vegetative cells varied from 0.1 to 6 ppm, while for

the spores, it was from 0.1 to 3 ppm. Concerning spore viability, the effect of lysozyme relied upon its

amount on the broth, as at MIC it caused a slight reduction in spores (approximately 1 log cfu ml�1)

after 24 h; otherwise, higher concentrations caused a decrease in spore level below the detection limit.

Concerning spore germination, lysozyme exerted a promoting effect on this phenomenon and reduced the

optical density by 66%.

Keywords Germination, inhibition index, minimal inhibitory concentration, spores, vegetative cells.

Introduction

The genus Alicyclobacillus was introduced in the bacterialtaxonomy by Wisotzkey et al. (1992), when they reclassi-fied Bacillus acidoterrestris, Bacillus acidocaldarius andBacillus cycloheptanicus as Alicyclobacillus acidoterrestris,Alicyclobacillus acidocaldarius and A. cycloheptanicus,because these species possess the x-fatty acids in themembrane. The isolation of Alicyclobacillus pomorum,Alicyclobacillus contaminans, Alicyclobacillus macrospo-rangiidus (Goto et al., 2007) and Alicyclobacillus aeris(Guo et al., 2009) amended the description of the genus,as they do not possess this kind of fatty acids.

Generally, alicyclobacilli are described as spore-formermicroorganisms, able to grow under thermophilic andacidic conditions, with an aerobic metabolism, and resistantto pasteurisation of fruit juices and acidic drinks (Bevilac-qua et al., 2008b). Alicyclobacillus acidoterrestris is thespecies with the strongest impact on food microbiology,due to its ability to produce guaiacol and halo-phenols andspoil fruit juices.

Spore inactivation could be achieved through physicaltreatments (thermal shock, homogenisation, microwave,

ultrasound, pulsed light; Giuliani et al., 2010; Jovettaet al., 2011; Bevilacqua et al., 2012; Chaine et al., 2012;Silva et al., 2012; Sokoowska et al., 2012) or usingsome chemicals (benzoate and organic acids, fattyacids, nisin, essential oils; Shearer et al., 2000; Bevilac-qua et al., 2008b, 2010). Lysozyme is a promising alter-native, as in the past, it showed a strong bioactivityagainst vegetative cells and spores (Buonocore et al.,2004; Bevilacqua et al., 2007). Bevilacqua et al. (2007)suggested that the strong bioactivity of enzymetowards vegetative cells could be attributed to itsclassical lytic activity, while they could not find anyevidence for a possible mode of action against spores.Setlow (2003) reported that lysozyme and other ‘non-nutrient factors’, as Ca-DPA (calcium dipicolinate),high pressure or alkylamines, influenced positivelyspore germination in Bacillus subtilis. Therefore, themain aim of this research was to study the effects oflysozyme on A. acidoterrestris through some intermedi-ate milestones:1 evaluating the minimal inhibitory concentration (MIC)

of lysozyme towards the vegetative cells and spores;

2 studying the kind of effect (biostatic or biocidal); and

3 focusing on the role of lysozyme on the complex phe-

nomenon of spore germination.*Correspondent: E-mails: antonio.bevilacqua@unifg.it;

abevi@libero.it

International Journal of Food Science and Technology 2014, 49, 224–229

doi:10.1111/ijfs.12302

© 2013 The Authors. International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

224

Materials and methods

Strains and spore production

This study focused on five strains of A. acidoterrestris,purchased from a Public Collection (Deutsche Samm-lung von Mikroorganismem und Zellkulturen’s collec-tion-DSMZ, Braunschweig, Germany; strains DSMZ2498 and 3922) or belonging to the Culture Collectionof Laboratory of Applied Microbiology (University ofFoggia; namely the strain c4, isolated from a spoiledpear juice, and the strains c8 and c10, isolated fromsoil). The strains were stored on malt extract agar(MEA) slants (Oxoid, Milan, Italy), adjusted to pH4.5; spores were produced as reported by Sinigagliaet al. (2003) on acidified malt extract agar, incubatedat 45 °C for 7 days until approximately 70–80% ofcells sporulated, as evaluated by microscopic examina-tion. Spores were removed by a mild agitation ofplates using a glass spreader after adding 5 mL of dis-tilled water; the spore suspension was centrifuged at1300 g for 10 min, after which the supernatant wasdiscarded and the pellet resuspended. Spores werecleaned by washing the pellets with sterile distilledwater, followed by centrifugation; then, spore suspen-sion was heated at 80 °C for 10 min to eliminate vege-tative cells and stored at 4 °C until use. Spore numberwas assessed immediately after the production throughthe spread plate count on acidified MEA, incubated at44 °C for 2–4 days and reported as log cfu mL�1.

The vegetative cells were produced as follows: themicroorganisms were inoculated in acidified maltextract broth, incubated at 45 °C for 18–24 h; then,the cultures were centrifuged at 1000 g for 10 min andwashed twice with sterile saline solution; spore absencein cell suspensions was checked through microscopeexamination. Cell suspensions were used immediately,and their concentration was assessed through the platecount.

Bioactivity of lysozyme towards vegetative cells andspores

Aliquots of acidified malt extract broth (20 mL), con-taining different amounts of lysozyme (Sigma-Aldrich,Milan, Italy; 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6,to 10 ppm) were inoculated individually with eithervegetative cells or spores to 3 log cfu mL�1 and thenincubated at 45 °C for 7 days. Alicyclobacilli growthwas evaluated through absorbance measurement at420 nm (Bevilacqua et al., 2008a). Aliquots of acidifiedmalt extract broth, not containing lysozyme, but inoc-ulated with either vegetative cells or spores, were usedas controls.

For each time of sampling, data were modelled asinhibition index, as reported by Altieri et al. (2009):

I:I: ¼ Absc �Abssð ÞAbsc

� 100

where Absc is the absorbance of the control and Abssthe OD values at 420 nm in the samples containinglysozyme.The lowest concentration of lysozyme able to main-

tain the inhibition index to 100% for the entire run-ning time (7 days) was assumed as the MIC.

Viability of Alicyclobacillus acidoterrestris DSMZ 2498and DSMZ 3922

Aliquots of acidified malt extract broth added withlysozyme (from 0.1 to 12 ppm) were inoculated withspores of either DSMZ 2498 or DSMZ 3922 strains to5 log cfu mL�1. Then, the samples were stored at45 °C; the level of alicyclobacilli was assessed periodi-cally (immediately after the inoculation and after 30,60, 90 min, 3, 5, 7, 24, 48 and 72 h) through the tradi-tional plate count on acidified malt extract agar, incu-bated at 45 °C for 2–4 days.Aliquots of malt extract broth, not containing lyso-

zyme, but inoculated with spores, were used as controls.

Spore germination

This assay was performed on the spores of the strainDSMZ 2498, using the protocol from Jagtap et al.(2006), modified as follows. The spores were inocu-lated to 7-log cfu mL�1 in the following samples:Control: distilled water acidified to pH 4.5.

L-alanine: acidified distilled water, containing L-alanine

50 mM (Sigma-Aldrich).

Lysozyme: acidified distilled water containing lysozyme

(0.1 ppm).

L-alanine + lysozyme: acidified distilled water containing

both L-alanine and lysozyme.The samples were heat-treated to 70 °C for 10 min (for

spore activation), added with lysozyme and L-alanine andstored at 45 °C; spore germination was assessed throughabsorbance measurement at 600 nm (after 30, 60 and90 min).Data were modelled as a decrease in absorbance,

using the following equation:

D ¼ 1� AbstAbs0

� �� �� 100

where Abst is the absorbance of the samples (control,L-alanine, etc.) and Abs0 the initial absorbance of eachsample.

Statistical analysis

The analyses were performed in triplicate over threedifferent batches; data were analysed through one-way

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2014

Lysozyme vs. A. acidoterrestris A. Bevilacqua et al. 225

analysis of variance (one-way ANOVA), using Tukey’stest as post hoc comparison method (P < 0.05).

Results and discussion

The first step of this research was aimed at the defini-tion of the MIC of lysozyme towards vegetative cellsand spores, using a microdilution method (Table 1);MIC towards vegetative cells varied from 0.1 ppm(strains DSMZ 2498 and c10) to 6 ppm (strain c8),while for the spores, it was from 0.1 ppm (strainDSMZ 2498) to 3 ppm (strain c8).

Minimal inhibitory concentration definition wasbased on the inhibition index after 7 days; however,data analysis for a shorter contact time (for example2 days) could be of interest to highlight the amountsof lysozyme able to exert a partial and time-dependenteffect.

Figure 1 shows inhibition index (I.I.) towards thevegetative cells of the strains DSMZ 3922, c8 and c4.Concerning the collection isolate (DSMZ 3922), I.I.increased following the increase in the concentrationof lysozyme up to 100% with 0.4 ppm of lysozymeadded, with a significant effect of the contact time at0.2 ppm: at this concentration, in fact, I.I. was 31%after 2 days, and then it decreased to 3% at the end ofthe running time.

The strain c8 was more resistant as it was inhibitedafter 2 days by higher concentrations (4–6 ppm); then,I.I. decreased within the running time, and after7 days, lysozyme effect was significant only at 6 ppm.After 2 days, the strain c4 appeared completely inhib-ited also by the lowest amount of the enzyme; how-ever, data analysis after 7 days revealed that the effectof lysozyme from 0.1 to 0.5 ppm was partial and timedependent, as one could infer from the decrease in I.I.

The strains DSMZ 2498 and c10 showed a similartrend, being completely inhibited after 7 days by0.1 ppm of lysozyme (data not shown).

As an example, Fig. 2 shows inhibition profile ofalicyclobacilli when spores were used; the effect oflysozyme was mostly affected by the contact time at0.1–0.5 ppm for the strain DSMZ 3922 and at 0.2–1 ppm for the isolate c8. A similar effect was observed

for the other strains in the presence of 0.05 ppm oflysozyme (data not shown).Concerning spore viability over time, Fig. 3 shows

death kinetic for the strains DSMZ 2498; the effect oflysozyme relied upon its amount on the broth, theMIC (0.1 ppm) caused a slight reduction in spores(approximately 1 log cfu mL�1) after 24 h, withoutfurther changes within the remaining running time;otherwise, higher concentrations (for example anamount twentyfold higher than MIC) caused adecrease in spore level below the detection limit within72 h. A similar trend was observed for the strainDSMZ 3922: a slight reduction in the presence ofMIC (0.6 ppm) and spore reduction below the

Table 1 Minimal inhibitory concentration of lysozyme towards veg-

etative cells and spores of Alicyclobacillus acidoterrestris

Strains Vegetative cells (ppm) Spores (ppm)

DSMZ 3922 0.4 0.6

DSMZ 2498 0.1 0.1

c4 0.5 0.2

c8 6.0 3.0

c10 0.1 0.2

DSMZ 3922

0

20

40

60

80

100

120

Lysozyme (ppm)

Inhi

bitio

n in

dex

(%)

2 Days7 Days

c8

0

20

40

60

80

100

120

Lysozyme (ppm)

Inhi

bitio

n in

dex

(%)

2 Days7 Days

γ4

0

20

40

60

80

100

120

0 0.1 0.2 0.3 0.4 0.5

0 2 4 6 8

0 0.1 0.2 0.3 0.4 0.5 0.6Lysozyme (%)

Inhi

bitio

n in

dex

(%)

2 Days7 Days

Figure 1 Inhibition index of lysozyme towards Alicyclobacillus

acidoterrestris: inoculation of the samples with vegetative cells. Mean

values � standard deviation.

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2014

Lysozyme vs. A. acidoterrestris A. Bevilacqua et al.226

detection limit with higher amounts (12 ppm; data notshown).

The last step of this research was aimed to give aninsight in the complex phenomenon of spore germina-tion and try to understand which is the role of lysozyme;thus, spores of A. acidoterrestris DSMZ 2498 wereinoculated in the presence of L-alanine (a promoting ele-ment for spore germination; Løvdal et al., 2012), lyso-zyme or both of them. Spore germination was assessedby measuring absorbance fall, as some authors reportedthat spore germination caused (Fig. 4) a 60/70%decrease in absorbance at 600 nm (Ghosh & Setlow,2010).

As expected, the thermal treatment (70 °C for10 min) determined a reduction in absorbance

(approximately 15%), as it is well known that a slightheat shock can activate spores (Byun et al., 2011).A promoting effect on spore germination was alsoachieved by L-alanine (absorbance was reduced by25%), but this was an expected result as it is wellknown that L-alanine induces spore germination(Barlass et al., 2002). An absorbance fall, that is, apromoting effect on the germination, was found alsofor lysozyme, which decreased the absorbance by66%. Then, lysozyme was combined with L-alanine ora mild heat shock or both of them to study the possi-bility of enhancing the promoting effect of the enzymeon the germination; the results were quite interesting,

DSMZ 3922

0

20

40

60

80

100

Lysozyme (ppm)

Inhi

bitio

n in

dex

(%)

2 Days7 Days

c8

–20

0

20

40

60

80

100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.5 1 1.5 2 2.5 3 3.5Lysozyme (ppm)

Inhi

bitio

n in

dex

(%)

2 Days7 DaysFigure 2 Inhibition index of lysozyme

towards Alicyclobacillus acidoterrestris: inoc-

ulation of the samples with spores. Mean

values � standard deviation.

DSMZ 2498

012345678

0 20 40 60 80Time (h)

Log

cfu/

ml

Control

0.1 ppm2 ppm

Figure 3 Viability of Alicyclobacillus acidoterrestris (log cfu mL�1)

in a laboratory medium containing lysozyme (inoculation with spores).

Mean values � standard deviation.

0

10

20

30

40

50

60

70

80

90

Control L-alanine Lysozyme L-ala+Lys

Abs

orba

nce

decr

ease

(%)

No-heat

Heat

Figure 4 Decrease in absorbance of a spore suspension of Alicycloba-

cillus acidoterrestrisDSMZ 2498, containing lysozyme and/or L-alanine

after 30 min. Mean values � standard deviation.

© 2013 The Authors

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International Journal of Food Science and Technology 2014

Lysozyme vs. A. acidoterrestris A. Bevilacqua et al. 227

as the differences were not significant (lysozyme vs.lysozyme + heat shock) or the effect was slight (lyso-zyme vs. lysozyme + L-alanine), thus suggesting themajor role of lysozyme in this phenomenon.

The bioactivity of lysozyme towards A. acidoterres-tris confirmed literature reports (Buonocore et al.,2004; Conte et al., 2006; Bevilacqua et al., 2007;Sokoowska et al., 2012). Some preliminary experi-ments performed in the past (Bevilacqua et al., 2007)showed that higher concentrations of enzyme (from250 to 1000 ppm) reduced vegetative cells and sporesbelow the detection limit in few hours, as it was notpossible to estimate MIC.

The use of lower amounts of the lysozyme pointedout its strong bioactivity towards vegetative cells andspores, thus confirming the data of Takahashi et al.(2007), who reported that alicyclobacilli could beinhibited in orange juice with 0.001% of enzyme (cor-responding to 10 ppm). Moreover, MICs highlightedthe enhanced strain variability among the different iso-lates, being the strain c8 (the most resistant isolate)inhibited by amounts of enzyme 30–60-fold higherthan those requested to control the strain DSMZ 2498(the most sensitive isolate).

Concerning the effect of lysozyme on alicyclobacilli,the results pointed out that it could act as a control-ling or a biocidal agent, depending on its amount;namely, at MIC, the effect was biostatic, as only twen-tyfold higher concentrations reduced spore level belowthe detection limit.

Another topic of great concern is the possible modeof action of enzyme. Preliminary experiments pointedout that vegetative cells were inactivated by the classi-cal lytic action; otherwise, the results of the last stepof the research highlighted a promoting effect on sporegermination, similar to that reported for L-alanine andfor mild heat treatment. This promoting effect waspreviously postulated by Setlow (2003), but the noveltyof the result relied upon the extent of the promotion,as L-alanine decreased absorbance by 25%, whereasthe fall with lysozyme was 66%. Based on this evi-dence and by combining the results of the last assaywith the strong effect of lysozyme on spores (step 2),we could suggest a possible mode of action: lysozymeacts on the spores of A. acidoterrestris in two differentsteps: it promotes spore germination; thereafter, it actson the outgrown spores and causes a strong reduction.

In conclusion, the data of this research confirmedthe strong bioactivity of lysozyme towards A. acidoter-restris. MIC values were strain dependent, rangingfrom 0.1 to 6.0 ppm; moreover, the effect of lysozymecould be both biostatic and biocidal. In fact, when theamount of the enzyme in the broth was the MIC, itseffect was biostatic (i.e. control of alicyclobacilli withinthe running time without a significant change in sporenumber); on the other hand, higher amounts of the

compound caused a strong decrease in spore number.The last step of the research suggested a possible modeof action of lysozyme towards the spore; the use ofL-alanine as a positive control (this amino acid is apromoting element for spore germination) showed thatlysozyme could promote spore germination; thus, wecould suggest that the enzyme promotes spore germi-nation and then acts on the outgrown spores.

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