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Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology
Food Microbiology 36 (2013) 320e326
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Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology Chedia Aouadhi a, b, c, d, *, Hélène Simonin b, c, Abderrazak Maaroufi a, Slah Mejri d a
Laboratory of Epidemiology and Microbiology, Bacteriology and Biotechnology Development Group, Pasteur Institute of Tunisia (IPT), BP 74, 13 Place Pasteur, Belvédère, 1002 Tunis, Tunisia b LUNAM Université, Oniris, UMR 6144, GEPEA, BP 82225, Nantes F-44322 Cedex 3, France c CNRS, Nantes F-44322, France d Laboratory of Animal Resources and Food, National Institute of Agronomy, Tunis (INAT), 43 Rue Charles Nicole, Cité Mahrajène, Le Belvédère, 1082 Tunis, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2013 Received in revised form 27 May 2013 Accepted 29 June 2013 Available online 8 July 2013
Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only D-glucose, L-alanine, and inosine were considered as strong independent germinants. Both inosine and L-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (L-alanine, D-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 C in presence of 9 and 60 mM of D-glucose and L-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Bacillus sporothermodurans Spore germination Amino acid Inosine Germinant Response surface methodology
1. Introduction Bacillus sporothermodurans is a non pathogenic Gram-positive bacterium (Hammer et al., 1995; Pettersson et al., 1996), predominated in UHT milk (Olivier et al., 2002) and can be isolated from raw milk and a non-milk source, namely silage and a feed concentrate for dairy cattle (Vaerewijck et al., 2001; Scheldeman et al., 2002). B. sporothermodurans, like many Bacillus and Clostridium species, is able of differentiation between two distinct cellular morphologies: the vegetative cell and the spore. The spore is metabolically dormant and provides to cell the ability to withstand environmental condition disadvantageous for vegetative life (Setlow, 2003). This bacterium is able to produce highly heat resistant
* Corresponding author. Laboratory of Epidemiology and Microbiology, Bacteriology and Biotechnology Development Group, Pasteur Institute of Tunisia (IPT), BP 74, 13 Place Pasteur, Belvédère, 1002 Tunis, Tunisia. Tel.: þ216 23 014 563; fax: þ216 71791833. E-mail address: [email protected] (C. Aouadhi). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.06.021
spores which enable it to survive to high-temperatures of UHT treatment (Pettersson et al., 1996). These spores can germinate and grow up to 105 CFU/mL in stored milk and therefore cause its nonsterility (Klijn et al., 1997). Despite their dormancy and resistance, spores retain a sensitive mechanism which is able of detecting and rapidly responding to the presence of specific germinative substances (Foerster and Foster, 1966; Gould and Dring, 1972; Moir and Smith, 1990) to return in life in as little as 20 min via germination process and outgrowth (Sarker et al., 2002). Germination can be triggered by a variety of factors, including nutrients as amino acids, ribosides (Rossignol and Vary, 1979; Moir et al., 1994; Clements and Moir, 1998; Ireland and Hanna, 2002), sugars (Racine et al., 1979; Irie et al., 1982) or mixtures like L-asparagine, D-glucose, D-fructose, and ions of potassium (AGFK) (Wax and Freese, 1968; Cabrera-Martinez et al., 2003). In addition to nutrients, spores can be germinated by other number of factors like surfactants (Paidhungat et al., 2001; Setlow et al., 2003) or physical treatments such as hydrostatic pressure (Aouadhi et al., 2012). Few studies have investigated the factors affecting spore
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germination with nutrient elements. For example, Clements and Moir (1998) have studied the effect of the temperature of incubation, the pH, and the cations on the germination of Bacillus cereus spores. The germination of spores by nutrient elements has been studied more extensively for some Bacillus species such as Bacillus subtilis (Irie et al., 1982; Paidhungat and Setlow, 2000), Bacillus anthracis (Ireland and Hanna, 2002), Bacillus megaterium (Racine et al., 1979; Rossignol and Vary, 1979), Bacillus licheniformis (White et al., 1974), B. cereus (Clements and Moir, 1998; Barlass et al., 2002) and some Clostridium species (Takeshi et al., 1988; Alberto et al., 2003; Paredes-Sabja et al., 2008). Nutrient-induced germination can be considered as the natural germination pathway, and this requires specific receptors. Germination is initiated when nutrient molecules activate the Ger receptors present in the spore’s inner membrane (Paidhungat et al., 2001). Shortly after, release of Ca2þDPA and rapid efflux of monovalent cations (Hþ, Naþ and Kþ) can be observed, while the core is partly rehydrated. Then, hydrolysis of the oppressing cortex layer, accomplished by cortex lytic enzymes CwlJ and SleB, becomes crucial to provide more space to the expanding core. After cortex degradation, the now fully rehydrated core allows the reactivation of enzymes and the synthesis of ATP from the 3-PGA (3-posphoglyceric acid) precursor. Degradation of the SASP proteins releases the spore’s DNA, and the spore initiates RNA, protein, and DNA syntheses in the further outgrowth phase (Setlow, 2003). There is much interest in this process because spores may affect the stability of food products, in particular dairy products, through germination. When spores germinate, they lose their resistance and become easy to inactivate (Paredes-Sabja et al., 2008). Therefore, the induction of spore germination following by inactivation of the germinated spores by other treatment can be used to inactivate Bacillus spores. In a previous work, we investigated the pressureinduced germination of B. sporothermodurans spores (Aouadhi et al., 2012). The present study is dedicated to the nutrientinduced germination of these spores that had not previously been studied. The aims of this study were to investigate the germination of B. sporothermodurans spores by nutrient elements (major naturally used amino acids, purine ribonucleosides (inosine), and sugars) and the effect of different factors (pH, temperature, and cations). The ultimate objectives of this work were to identify the specific germinant of these highly heat resistant spores and select optimal conditions to maximize their germination rate. 2. Materials and methods 2.1. Bacterial strain and spore preparation B. sporothermodurans strain LTIS27 described by Aouadhi et al. (2012) was used in this study. Cells were grown on brain-heart infusion agar (Biokar-Diagnostics) supplemented with 1 mg/L vitamin B12 (SigmaeAldrich) (BHIeB12) at 37 C. Twenty-four hours before use, cultures were held in BHIeB12 broth on a rotary shaker at 37 C to obtain a working culture. Spores were prepared according to the modified method of Ireland and Hanna (2002). A working culture (18 h, 37 C under agitation) was diluted 1:10 into sporulation medium (25 g/L nutrient broth (Pronadisa, Microbiological culture media and diagnostic reagents), 1 mg/L vit B12, 8 mg/ L MnS04 H20, and 1 g/L CaC12 H20) then incubated for 7 days at 37 C. This medium was then centrifuged at 6000 g for 10 min. The resulting pellet, containing spores and non-sporulated vegetative cells, was washed three times with sterile distilled water, resuspended in 5 mL sterile distilled water and treated at 100 C for 30 min to destroy vegetative cells. The endospore suspension was centrifuged and washed 4 times as described above. The cleaned
321
endospore preparation was stored at a concentration of 107 spores/ mL of distilled water at 4 C. 2.2. Spore germination assay The spore suspension was heat activated in distilled water at 100 C for 30 min and then suspended in germination buffer (10 mM TriseHCL and 10 mM NaCl, pH 8) for 15 min at 37 C (Barlass et al., 2002). The germination was initiated by addition of the following compounds: sugars (D-glucose and D-fructose) or all major naturally used amino acids or purine ribonucleosides (inosine), at different concentrations (10, 20, 50, 100, and 250 mM). For L-alanine germination, O-carbamyl-D-serine (5 mg/mL) (Sigmae Aldrich) was added to inhibit the alanine racemase activity (Clements and Moir, 1998; Barlass et al., 2002). All amino acids used for the germination assays were L-isomers (SigmaeAldrich) dissolved in sterile water at stock concentrations of 1 M. All compounds used were sterilized by filtration using 0.45 mm-pore-size filters. The optical density of the spore suspension was measured at 580 nm (OD580), after 60 min of incubation at 37 C. The germination rate was expressed as the maximum ratio of loss of OD580 of the spore suspension, relative to the initial value (Clements and Moir, 1998; Ireland and Hanna, 2002). The data were expressed as the percent reduction in OD (with the initial reading taken as 100%) which was calculated as [(ODi ODf)/DOi] 100, where ODi is the initial OD580 value measured for the sample and ODf is the OD580 value measured after 60 min incubation for the test sample. 2.3. Effect of pH, temperatures and cations on spore germination After heating at 100 C for 30 min, spores were suspended in the germination buffer for 15 min at 37 C (Pettersson et al., 1996; Montanari et al., 2004). The following germinant (D-glucose (50 mM), L-alanine (100 mM), inosine (50 mM)) were then added. A control, without compounds, was included for each experiment. To study the effect of pH or temperature incubation, spores, after the addition of germinants, were incubated for 60 min at different pH (5, 6, 7, 8 and 9) at 37 C or at different temperatures (20, 30, 37, 40 and 50 C). The adjustment of pH was achieved by the addition of sodium hydroxide (2N NaOH) or hydrochloric acid (2N HCl). To study the effect of cations on spore germination, the chemical compounds used were KCl, CaCI2, MgCl2, and NaCl. The solutions (KCl, CaCl2, MgCl2, or NaCl (Fluka chemical)) at a concentration of 100 mM (White et al. 1974) were added to the medium containing the spore suspension in the presence or absence of L-alanine, inosine, or D-glucose. 2.4. Statistical analyses and experimental design Data were subjected to one-way analysis of variance to evaluate the significance of each independent factor on spore germination. A Fisher LSD test was used to discriminate the means with a significance level fixed at 5%. In order to maximize the germination rate, diverse factors was combined. In fact, the optimization of the nutrient-induced germination of B. sporothermodurans LTIS27 spores was realized using a central composite experimental design with three factors as described by Aouadhi et al. (2012). The factors investigated were Lalanine concentration (20e60 mM), D-glucose concentration (5e 10 mM), and incubation temperature (30e40 C). The experimental response (germination rate) was estimated, taking into account the influence of the experimental factors. An analysis of variance and an estimation of response surface by multiple linear regressions
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were performed using the software STATGRAPHICS Centurion XV version 15.2.06. 3. Results and discussion 3.1. Germination survey with amino acids, inosine or sugar In this study, the effect of the major naturally used amino acids, inosine, and sugars on the germination of B. sporothermodurans LTIS27 spores was evaluated. The progress of germination of spore suspensions was estimated as the percent reduction in initial OD. The germination rate of B. sporothermodurans LTIS27 spores induced by different concentrations of diverse compounds used is shown in Fig.1. In presence of some compounds (D-fructose, L-arginine, L-asparagine, L-aspartate, L-cysteine, L-glutamate, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-serine, L-tyrosine, or L-valine), the germination rate increases with increasing concentrations, reaching near maximal levels (between 22 et 26%) at concentrations as low as 20 or 50 mM; however, at concentration higher than 50 mM, the germination rate did not exceed 30%. So it seems that the germination rate reaches a plateau and that increasing the concentration of some compounds will not necessarily further increase the germination rate. While the germination rate in presence of L-alanine, inosine or D-glucose keep on increasing to 62% with increasing the concentrations as high as 50 mM for inosine and D-glucose and 100 mM for L-alanine. Fig. 2 summarizes the effect of the major naturally used amino acids, inosine, and sugars, with a concentrations of 100 mM, on the germination of B. sporothermodurans LTIS27 spores. Strong germination responses were defined as those causing 40% or greater germination in 15 min (Ireland and Hanna, 2002). Considering this definition, we noted that B. sporothermodurans LTIS27 spores respond differently to germinant and among 23 compounds studied only D-glucose (62%), L-alanine (60%), inosine (52%), and Ltyrosine (44%) were able to deliver a significant germination response. This result shows that the specific germinants (causing 50% or greater germination) of B. sporothermodurans LTIS27 spores were D-glucose, L-alanine, and inosine. This was consistent with the data from a variety of Bacillus species. For example, D-glucose was specific germinant of B. megaterium spores (Hyatt and Levinson, 1962; Racine et al., 1979). L-alanine was considered as a strong germinant for several species of Bacillus such as B. cereus (Barlass et al., 2002), B. subtilis (McCann et al., 1996; Cabrera-Martinez
et al., 2003) and some species of Clostridium (Broussolle et al., 2002; Paredes-Sabja et al., 2008). Inosine is potent independent germinant for B. cereus spores (Barlass et al., 2002; Hornstra et al., 2006), while it is not a strong independent germinant for B. anthracis spores (Ireland and Hanna, 2002). It is a strong cogerminant when it is associated with amino acids (Ireland and Hanna, 2002). Finally, because L-alanine, inosine, and D-glucose have induced a percentage of germination upper than 50%, we were interested in testing these compounds as co-germinant for amino-acid-induced germination. 3.2. Alanine, inosine and D-glucose as co-germinants for aminoacid-induced germination The effect of L-alanine (1 mM) or inosine (1 mM) as a cogerminant in combination with one of the amino acids tested is represented in Fig. 3. Two distinct effects of L-alanine or inosine were observed. In fact, these two germinants stimulated response of some amino acids (L-histidine, L-tryptophan and L-tyrosine for Lalanine and L-alanine, L-cysteine, L-histidine, L-tyrosine, L-tryptophan and L-serine for inosine) to trigger significant germination (between 42 and 82%). However, with the other amino acids, no change in the germination rate was observed (data not shown). The strongest response (82%) was observed in presence of both Lalanine and inosine. Hence, these data shows that L-alanine and inosine can be used either as independent germinants or as cogerminants for some amino acids. This result is consistent with previous works carried out with other Bacillus species (Ireland and Hanna, 2002; Hornstra et al., 2006). For example, B. cereus endospores germinated in response to inosine or L-alanine, but the most rapid germination was elicited by a combination of these two germinants (Clements and Moir, 1998; Barlass et al., 2002). Moreover, inosine was used as a co-germinant with L-alanine, L-cysteine, L-histidine, L-methionine, L-phenylalanine, L-proline, L-serine, Ltryptophan, L-tyrosine, and L-valine for B. anthracis (Ireland and Hanna, 2002) and with L-alanine, L-glutamine, and phenylalanine for B. cereus (Hornstra et al., 2006). Ireland and Hanna (2002) showed two independent types of germinant specificities depending upon inosine (amino acids inosine dependent (AAID) responses). The first, AAID1, was accomplished through inosine and non aromatic amino acids (L-alanine, L-cysteine, L-proline, L-serine, and L-valine). The second, AAID2, was obtained with inosine combined with aromatic amino acids (L-histidine, L-phenylalanine, L-
70
Germination rate (%)
60
L-alanine L-leucine
50
L-arginine L-cysteine
40
L-tyrosine L-glycine L-glutamine
30
L-tryptophan Inosine D-glucose
20
D-fructose 10 0
0.1 0,1
1
10
20
50
100
Concentration (mM) Fig. 1. Effect of various concentrations of different nutrient elements on the germination of B. sporothermodurans LTIS27 spores. Values given are means (error bars represent standard deviations) of three independent experiments.
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323
Germination rate (%)
70 60 50 40 30 20
D-glucose
L-tyrosine
L-alanine
Inosine
L-tryptophane
L-glycine
L-cysteine
L-arginine
L-leucine
L-aspartate
L-histidine
D-fructose
L-isoleucine
L-pheny
L-lysine
L-proline
L-asparagine
L-serine
L-threonine
L-glutamate
L-methionine
L-valine
0
No addition
10
Fig. 2. Response of B. sporothermodurans LTIS27 spores to diverse nutrient elements. Values given are means (error bars represent standard deviations) of three independent experiments.
tryptophan, and L-tyrosine). Given that the inosine may potentiate some aromatic and non aromatic acids in triggering a robust germination response, we can hypothesize that both AAIDI responses are observed for B. sporothermodurans LTIS27. Several authors reported that L-alanine may potentiate other products (amino acid, inosine, sugars) in triggering a robust germination response (Yasuda and Tochikubo, 1984; Barlass et al., 2002; Ireland and Hanna, 2002). In the literature, two distinct germination responses of spore were observed when using Lalanine (1 mM) as a co-germinant with different amino acids. For B. anthracis, it was reported that the addition of L-alanine can elicit amino acid-mediated germination. This was explained by the reaction of the aromatic side chains of some amino acids (L-histidine, L-tyrosine, and L-tryptophan) with L-alanine (Ireland and Hanna,
(a) Germination rate (%)
90 80
2002). For B. cereus, no significant germination was detected in the presence of L-alanine (1 mM) as a co-germinant (Hornstra et al., 2006). The effect of D-glucose (1 mM) on the germination of B. sporothermodurans LTIS27 spores initiated by amino acids was also tested. The strongest germination response was observed with a combination of D-glucose (1 mM) and L-alanine (100 mM); the germination rate being of 89%. In contrast, D-glucose didn’t show any affect on spore germination in presence of other products (data not shown). The stimulation of L-alanine-induced germination by Dglucose was observed for B. megaterium (Hyatt and Levinson, 1961, 1962; Racine et al., 1979) and B. subtilis (White et al., 1974). Moreover, D-glucose was used as a co-germinant with some amino acids (L-leucine, L-proline, L-alanine and L-valine) for germination of B. megaterium spores (Hyatt and Levinson, 1962); however, it didn’t affect the induced-L-alanine germination of B. licheniformis spores (White et al., 1974). It seems that there is a synergistic function of Lalanine receptor with D-glucose-sensitive components in the germination apparatus for B. sporothermodurans spores.
70 60
3.3. Effect of different cations, pH, and temperature on spore germination
50 40 30 20 10 0 L-alanine
L-cysteine
L-tyrosine L-tryptophan
L-serine
L-histidine
(b) Germination rate (%)
60 50 40 30 20 10 0 L-tyrosine
L-tryptophane
L-histidine
Fig. 3. Germination of B. sporothermodurans LTIS27 spores in presence of major naturally utilized amino acids with co-germinant (L-alanine (a) and inosine (b)). Germination was initiated by the addition of 20 mM of amino acids with (solid bars) or without (shaded bars) 1 mM L-alanine (a) or 1 mM inosine (b) as indicated. Values given are means (error bars represent standard deviations) of three independent experiments.
The effect of diverse cations (NaCl, MgCl2, CaCl2 and KCl) on the response of B. sporothermodurans LTIS27 spores alone or in presence of some germinant (L-Alanine, inosine or D-glucose) is illustrated in Table 1.a. The cations enhanced the germination of B. sporothermodurans LTIS27 spores. Indeed, a strong germination response (49 and 43%) was induced by KCl and CaCl2, respectively. Nevertheless, in presence of L-alanine, inosine, or D-glucose, the different cations affected germination of B. sporothermodurans spores by decreasing their rate. The effect of different cations on spore germination is more contradictory in the literature. While Clements and Moir (1998) showed that potassium ions strongly inhibited inosine-induced germination of B. cereus spores, White et al. (1974) reported that potassium ions didn’t affect spore Lalanine-induced germination of B. licheniformis spores. The present study shows that salts reduced the D-glucoseinduced germination rate of B. sporothermodurans spores, whereas, for other species the effect of sugars and especially D-glucose was remarkably increased by salts (Hyatt and Levinson, 1962; Rode and Foster, 1962; Hyatt and Levinson, 1964). The effect of temperature on the spore germination of B. sporothermodurans LTIS27 in presence of L-alanine, inosine, or D-
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Table 1 Effects of different cations (a), incubation temperature (b) and pH (c) on the germination rate of B. sporothermodurans LTIS27 spores in presence of D-glucose, Lalanine or inosine. Germination ratea: No addition (a) Ion (100 mM) None 22.3 1.4a NaCl 31.6 1.8a KCL 49 1a CaCl2 43.3 1.8a MgCl2 29 1.5a (b) Incubation temperature 20 11.6 1.7a 30 20.3 1.2a 37 22.6 1.4a 40 19.6 1.6a 50 10 1.1a (c) pH 5 21.6 1.4a 6 32 1.5a 7 22 1.4a 8 18 1.5a 9 13.3 1.8a
D-glucose
L-alanine
(50 mM)
(100 mM)
Inosine (50 mM)
62.6 32.6 54.6 21.6 44.3 ( C) 35.3 45 59.6 51.6 30
1.4b 1.4a 1.4ab 1.7a 1.4a
60.6 36.6 42.6 42.6 45.6
1.2a 1.8a 1.4a 1.2a 1.4a
52 39 32 43.3 22.6
1.1b 1.5a 1.7ab 1.8a 1.4a
2a 1.1a 1.4a 1.6a 1.1a
21.6 51.3 56.6 44.6 20
1.2ab 1.6b 1.8ab 1.9b 1.1a
19.3 48.6 50.6 43 23.6
1.8a 1.2a 1.2a 1.5a 1.81a
40 54.3 60 53 30.3
1.1ab 1.2b 1.7ab 1.1ab 1.4a
29 57 52 30.3 20
0.5ab 0.5b 1.5ab 1.8ab 1.1a
22 51 52 25 19.7
1.1ab 1.5b 1.7ab 1.8ab 1.4a
a Results are the means standard deviations of three independent experiments. Means with different letters are different (P < 0.05).
glucose is shown in Table 1b. Spore germination was maximum in the temperature range of 30e40 C. In fact, spore germination decreased significantly at temperature above 40 C or below 30 C. For example, at 20 C, only 22% of loss of initial OD580 was observed in presence of inosine. The influence of pH on the spore germination of B. sporothermodurans is shown in Table 1c. Spore germination induced by L-alanine, inosine, or D-glucose increased with increasing of pH from 5.0 to 7.0. For pH above 8.0, the germination rate decreased significantly. It was clear that the optimal germination rate of B. sporothermodurans spores, in the presence of Dglucose or inosine or L-alanine, occurred in the pH range of 6.0e8.0. Some spores can germinate in a larger pH domain for B. licheniformis and Paenibacillus polymyxa or more restricted conditions for B. cereus (White et al., 1974; Huo et al., 2010). Furthermore, Ciarciaglini et al. (2000) reported that the germination of B. subtilis pSB357 spores, induced by L-alanine, was completely inhibited at pH 4.0. The low pH was thought to have altered the ionization state of amino acid side chains, thus changing their charge distributions, hydrogen bonding, and protein conformation. In our case, the germination was affected differently by pH in the presence of L-alanine or inosine or D-glucose. We can hypothesize that germinant receptors can be affected by pH and that extreme pH may inhibit expression of nutrient receptor genes or germinant binding (Broussolle et al., 2008; Huo et al., 2010). Moreover, it can be concluded that the pH conditions for optimal germination of Bacillus spores, depend on the microbial species and even strains within species. This could be due to differences in the nature of the enzyme systems which may be involved in their germination processes (White et al., 1974). For B. cereus, Clements and Moir (1998) showed that the differences in the optimum pH and temperature profiles and the effects of monovalent cations on germination in presence of L-alanine or inosine suggest that there are different germinant specific signal transductions mechanisms present in the spores. These differences would meant that either these germinants are detected by separate proteins in the spore or, possibly, by different binding sites in the same protein. In fact, Paredes-Sabja et al. (2011) showed that germinant receptors transduce the germination signal to downstream effectors, leading
Table 2 Test of significance for regression coefficients Model term
Coefficient estimate
Degree of freedom
Standard error
Probability
Intercept x1 (L-alanine) x2 (D-glucose) x3 (Temperature) x21 x22 x23 x1x2 x1x3 x2x3
380.3 1.91 2.99 23.44 0.0033 0.0955 0.301 0.0625 0.0437 0.01
1 1 1 1 1 1 1 1 1 1
0.0915 0.08 0.08 0.08 0.09 0.09 0.09 0.011 0.011 0.011
e 0.0000 0.0029 0.0022 0.1335 0.4764 0.0000 0.0128 0.0024 0.8982.
to the release of monovalent cations and ultimately DPA. The mechanism of this signal transduction is unclear, but proteine protein interactions between several SpoVA proteins and several germinant receptor subunits (Vepachedu and Setlow, 2007) suggest that the germinant receptors physically interact with at least some SpoVA proteins. Consequently, the triggering of spore germination would require that the germination signal be transduced from the low abundance germinant receptors to the SpoVA proteins (Vepachedu and Setlow, 2007). In addition, Huo et al. (2010) confirmed that the optimum temperature for spore germination is species-, strain-, and especially germinant-dependent. 3.4. Optimization nutrient-induced germination by response surface methodology (RSM) Throughout of this work, we noted that D-glucose was more effective than inosine on the L-alanine-induced germination. Moreover, the incubation temperature has a significant effect on the germination rate of B. sporothermodurans spores LTIS27. Therefore, the individual and interactive effects of L-alanine, Dglucose, and incubation temperature on the germination of B. sporothermodurans LTIS27 spores were studied using RSM. The response (Y) was measured in terms of the germination rate. The application of RSM provides an empirical relationship between the response variable and the test variable under consideration. Although this methodology has been used by some authors to evaluate the effect of various factors such as pressureetemperature on the inactivation of B. cereus (Ju et al., 2008) and pressure on the germination of B. sporothermodurans (Aouadhi et al., 2012) spores, to our knowledge it has never been used for the optimization of nutrient-induced spore germination. By analysis of the multiple regression application, the following quadratic model equation was obtained: Table 3 Experimental and predicted results for ten check-points of nutrient-induced germination of B. sporothermodurans LTIS27 spores. Trial no.
1 2 3 4 5 6 7 8 9 10
Factors
Germination rate (%) D-glucose (mM)
Temperature ( C)
Observed
Predicted
(mM) 35 30 25 40 50 40 35 45 55 60
7 7 6 6 5 8 9 8 8 5
38 35 30 35 40 30 40 30 35 30
76.5 94.1 57.5 71.3 82.5 72 75.2 78 94.9 92.8
78.88 93.14 58.404 69.32 84.46 72.034 74.15 77.529 95.587 91.7
L-alanine
C. Aouadhi et al. / Food Microbiology 36 (2013) 320e326
Germination rate (%)
(a)
Temperature (35°C)
110
90
70
50 20
30
40
50
60
5
6
7
8
9
10
D-glucose (mM)
L-alanine (mM)
Germination rate (%)
(b)
D-glucose (9 mM)
110
90
70 40 50
35
20
40
30
60
Temperature (°C)
L-alanine (mM)
Germination rate (%)
(c)
L-alanine (60 mM)
110
325
different combinations of process parameters (Table 3). The experimental values were found to be significantly in agreement with the predicted values with a correlation coefficient (R2) of 0.986 and a statistical significance level of P < 0.0001. Therefore, the model was proven to be quite reliable method for predicting the nutrient-induced germination of B. sporothermodurans spores. Response surface plots in Fig. 4 shows the effects of L-alanine, Dglucose, and incubation temperature on the germination rate. In each figure, one factor is maintained constant at its optimal value determined by Statgraphics. The germination rate of B. sporothermodurans spores increased with increasing temperature between 30 and 35 C, and decreased at temperature above 35 C. Furthermore, the germination rate increased with increasing L-alanine and D-glucose concentrations. As already shown with the interaction coefficients of the model, a significant synergistic effect was observed with LalanineeD-glucose and L-alanine-Temperature. In this work, the process parameters for 100% nutrientinduced germination of B. sporothermodurans LTIS27 spores were optimized with Statgraphics and the optimal germination rate was obtained in presence of L-alanine (60 mM), and D-glucose (9 mM) after incubation of spores for 60 min at 35 C. The optimal conditions inducing the germination of B. sporothermodurans spores obtained from this study slightly differ from other studies. For example, Ireland and Hanna (2002) reported that an important germination of B. anthracis spores (98%) was obtained in presence of L-alanine (100 mM). For B. cereus, various amino acids (L-alanine, L-cysteine, L-threonine, and L-glutamine) and inosine have been reported to trigger a robust germination response (between 90 and 100%) (Hornstra et al., 2006). For B. megaterium and B. subtilis, the strong and rapid germination response was achieved in presence of Lalanine and L-proline, respectively (Rossignol and Vary, 1979; McCann et al., 1996). These results show that the germination process depends on the species.
90
4. Conclusion
70
The effect of different nutrient elements (sugars (D-glucose and all major naturally utilized amino acids and inosine) on the spore germination of B. sporothermodurans has been studied here for the first time. These spores were shown to have a specific behavior which differs from that of other Bacillus species. Indeed, the specific germinants needed for B. sporothermodurans LTIS27 spores were D-glucose, L-alanine, and inosine and the optimal germination rate was obtained after 60 min of incubation of spore at 35 C in presence of 9 mM of D-glucose and 60 mM of L-alanine. Furthermore, we have found that inosine and L-alanine can be used as co-germinants at low concentration (1 mM) in presence of some other amino acids.
40
50 5
D-fructose),
6
35 7
8
9
10
30
Temperature (°C)
D-glucose (mM)
Fig. 4. Response surfaces plots showing the effects of L-alanine and D-glucose (a), Lalanine and temperature (b), and temperature and D-glucose (a) on the germination of B. sporothermodurans LTIS27 spores.
Y1 ¼ 380:306 þ 1:91x1 2:99x2 þ 23:44x3 0:003x21 þ 0:09x22 0:3x23 þ 0:06x1 x2 0:04x1 x3 þ 0:01x2 x3
(1)
where x1 is L-alanine concentration, x2 is D-glucose concentration and x3 is temperature levels. The analysis of variance (ANOVA) of the quadratic model was performed with Statgraphics and the statistical significance of each coefficient value was determined using the Fisher F-test (Table 2). The R2 value was 0.964 indicating that the derived model fitted the experimental data. The linear coefficients (x1, x2 and x3) and the interaction coefficients (x1x2 and x1x3) and x23 were significant with small P values (P < 0.05). The quadratic term coefficients (x21 and x22 ) and x2x3 were not significant. In order to validate the qualification of the polynomial equation, ten experimental checks were carried out under
Acknowledgments The authors gratefully acknowledge the financial support provided by the Tunisian Ministry of Higher Education and Scientific Research. Abbreviations list
DPA: HRS: RSM: UHT:
Dipicolinic Acid Highly Heat-Resistant Spores Response Surface Methodology Ultra-High Temperature
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References Alberto, F., Broussolle, V., Mason, D.R., Carlin, F., Peck, M.W., 2003. Variability in spore germination response by strains of proteolytic Clostridium botulinum types A, B and F. Letters in Applied Microbiology 36, 41e45. Aouadhi, C., Simonin, H., Prevost, H., De Lamballerie, M., Maaroufi, A., Mejri, S., 2012. Optimization of pressure-induced germination of Bacillus sporothermodurans spores in water and milk. Food Microbiology 30, 1e7. Barlass, P.J., Houston, C.W., Clements, M.O., Moir, A., 2002. Germination of Bacillus cereus spores in response to L-alanine and to inosine: the roles of gerL and gerQ operons. Microbiology 148, 2089e2095. Broussolle, V., Alberto, F., Shearman, C.A., Mason, D.R., Botella, L., Nguyen-The, C., Peck, M.W., Carlin, F., 2002. Molecular and physiological characterization of spore germination in Clostridium botulinum and Clostridium sporogenes. Anaerobe 8, 89e100. Broussolle, V., Gauillard, F., Nguyen-The, C., Carlin, F., 2008. Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group. Journal of Applied Microbiology 105, 1081e1090. Ciarciaglini, G., Hill, P.J., Davies, K., McClure, P.J., Kilsby, D., Brown, M.H., Coote, P.J., 2000. Germination induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores. Applied and Environmental Microbiology 66, 3735e3742. Cabrera-Martinez, R.M., Tovar-Rojo, F., Vepachedu, V.R., Setlow, P., 2003. Effects of over expression of nutrient receptors on germination of spores of Bacillus subtilis. Journal of Bacteriology 185, 2457e2464. Clements, M.O., Moir, A., 1998. Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants. Journal of Bacteriology 180, 6729e6735. Foerster, H.F., Foster, J.W., 1966. Response of Bacillus spores to combinations of germinative compounds. Journal of Bacteriology 91, 1168e1177. Gould, G.W., Dring, G.J., 1972. Biochemical mechanisms of spore germination. In: Halvorson, H.O., Hanson, R., Campbell, L.L. (Eds.), Spores V. The American Society of Microbiology, Washington, DC, pp. 401e408. Hammer, P., Lembke, F., Suhren, G., Heeschen, W., 1995. Characterization of a heat resistant mesophic Bacillus species affecting quality of UHT-milk e a preliminary report. Kiel Milchwirtsch Forschungsber 47, 303e311. Hornstra, L.M., De Vries, Y.P., Wells-Bennik, M.H., De Vos, W.M., Abee, T., 2006. Characterization of germination receptors of Bacillus cereus ATCC 14579. Applied and Environmental Microbiology 72, 44e53. Huo, Z., Yang, X., Raza, W., Huang, Q., Xu, Y., Shen, Q., 2010. Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR21. Applied Microbiology and Biotechnology 87, 527e536. Hyatt, M.T., Levinson, H.S., 1961. Interaction of heat, glucose, L-alanine, and potassium nitrate in spore germination of Bacillus megaterium. Journal of Bacteriology 81, 204e211. Hyatt, M.T., Levinson, H.S., 1962. Conditions affecting Bacillus megaterium spore germination in glucose or various nitrogenous compounds. Journal of Bacteriology 83, 1231e1237. Hyatt, M.T., Levinson, H.S., 1964. Effect of sugars and other carbon compounds on germination and post-germination development of Bacillus megaterium spores. Journal of Bacteriology 88, 1403e1415. Ireland, J.A., Hanna, P.C., 2002. Amino acid- and purine ribonucleoside- induced germination of Bacillus anthracisD Sterne endospores: gerS mediates responses to aromatic ring structures. Journal of Bacteriology 84, 1296e1303. Irie, R., Okamoto, T., Fujita, Y., 1982. A germination mutant of Bacillus subtilis deficient in response to glucose. Journal of Genetic Applied Microbiology 28, 345e354. Ju, X.R., Gao, Y.L., Yao, M.L., Qian, Y., 2008. Response of Bacillus cereus spores to high hydrostatic pressure and moderate heat. LWT: Food Science and Technology 41, 2104e2112. Klijn, N., Herman, L., Langeveld, L., Vaerewijck, M., Wagendorp, A., Huemer, I., Weerkamp, A., 1997. Genotypical and phenotypical characterization of Bacillus sporothermodurans strains, surviving UHT sterilization. International Dairy Journal 7, 421e428.
McCann, K.P., Robinson, C., Sammons, R.L., Smith, D.A., Corfe, B.M., 1996. Alanine germination receptors of Bacillus subtilis. Letters in Applied Microbiology 23, 290e294. Moir, A., Smith, D.A., 1990. The genetics of bacterial spore germination. Annual Review of Microbiology 44, 531e553. Moir, A., Kemp, E.H., Robinson, C., Corfe, B.M., 1994. The genetic analysis of bacterial spore germination. Journal of Applied Microbiology 76, 9Se16S. Montanari, G., Borsari, A., Chiavari, C., Ferri, G., Zambonelli, C., Grazia, L., 2004. Morphological and phenotypical characterisation of Bacillus sporothermodurans. Journal of Applied Microbiology 97, 802e809. Olivier, G.G., Scheldeman, P., Joey, M., Herman, L., Joosten, H., Heyndrick, M., 2002. Genetic heterogeneity in Bacillus sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic-PCR fingerprinting. Applied and Environmental Microbiology 68, 4216e4224. Paidhungat, M., Setlow, P., 2000. Role of Ger proteins in nutrients and non-nutrient triggering of spore germination in Bacillus subtilis. Journal of Bacteriology 182, 2513e2519. Paidhungat, M., Ragkousi, K., Setlow, P., 2001. Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca2þ-dipicolinate. Journal of Bacteriology 183, 4886e4893. Paredes-Sabja, D., Torres, J.A., Setlow, P., Sarker, M.R., 2008. Clostridium perfringens spore germination: characterization of germinants and their receptors. Journal of Bacteriology 190, 1190e1201. Paredes-Sabja, D., Torres, J.A., Setlow, P., Sarker, M.R., 2011. Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved. Trends in Microbiology 19, 85e94. Pettersson, B., Lembke, F., Hammer, P., Stackebrand, E., Fergus, G., 1996. Bacillus sporothermodurans a new species producing highly heat resistant endospores. International Journal of Systematic Bacteriology 46, 759e764. Racine, F.M., Dills, S.S., Vary, J.C., 1979. Glucose-triggered germination of Bacillus megaterium spores. Journal of Bacteriology 138, 442e445. Rode, L.J., Foster, J.W., 1962. Ionic germination of spores of Bacillus megaterium QM B 1551. Archives of Microbiology 43, 183e200. Rossignol, D.P., Vary, J.C., 1979. Biochemistry of L-proline-triggered germination of Bacillus megaterium spores. Journal of Bacteriology 138, 431e441. Sarker, M.R., Shivers, R.P., Sparks, S.G., Juneja, V.K., McClane, B.A., 2002. Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolate carrying plasmid genes versus chromosomal enterotoxin genes. Journal of Applied Microbiology 66, 3234e3240. Scheldeman, P., Herman, L., Goris, J., De Voe, P., Hendrickx, M., 2002. Polymerase chain reaction identification of Bacillus sporothermodurans from dairy source. Journal of Applied Microbiology 92, 983e991. Setlow, B., Cowan, A.E., Setlow, P., 2003. Germination of spores of Bacillus subtilis with dodecylamine. Journal of Applied Microbiology 95, 637e648. Setlow, P., 2003. Spore germination. Current Opinion in Microbiology 6, 550e556. Takeshi, K., Ando, Y., Oguma, K., 1988. Germination of spores of Clostridium botulinum type G. Journal of Food Protection 51, 37e38. Vaerewijck, M.J.M., DeVos, P., Lebbe, L., Scheldeman, P., Hoste, B., Heyndrickx, M., 2001. Occurrence of Bacillus sporothermodurans and other aerobic sporeforming species in feed concentrate for dairy cattle. Journal of Applied Microbiology 91, 1074e1084. Vepachedu, V.R., Setlow, P., 2007. Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores. FEMS Microbiology Letters 274, 42e47. Wax, R., Freese, E., 1968. Initiation of the germination of Bacillus subtilis spores by a combination of compounds in place of L-alanine. Journal of Bacteriology 95, 433e438. White, C.H., Chang, R.R., Martin, J.H., Loewznstein, M., 1974. Factors affecting Lalanine induced germination of Bacillus spores. Journal of Dairy Science 57, 1309e1314. Yasuda, Y., Tochikubo, K., 1984. Relation between D-glucose and L- and D-alanine in the initiation of germination of Bacillus subtilis spore. Microbiology and Immunology 28, 197e207.
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Food Microbiology journal homepage: www.elsevier.com/locate/fm
Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology Chedia Aouadhi a, b, c, d, *, Hélène Simonin b, c, Abderrazak Maaroufi a, Slah Mejri d a
Laboratory of Epidemiology and Microbiology, Bacteriology and Biotechnology Development Group, Pasteur Institute of Tunisia (IPT), BP 74, 13 Place Pasteur, Belvédère, 1002 Tunis, Tunisia b LUNAM Université, Oniris, UMR 6144, GEPEA, BP 82225, Nantes F-44322 Cedex 3, France c CNRS, Nantes F-44322, France d Laboratory of Animal Resources and Food, National Institute of Agronomy, Tunis (INAT), 43 Rue Charles Nicole, Cité Mahrajène, Le Belvédère, 1082 Tunis, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2013 Received in revised form 27 May 2013 Accepted 29 June 2013 Available online 8 July 2013
Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only D-glucose, L-alanine, and inosine were considered as strong independent germinants. Both inosine and L-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (L-alanine, D-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 C in presence of 9 and 60 mM of D-glucose and L-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Bacillus sporothermodurans Spore germination Amino acid Inosine Germinant Response surface methodology
1. Introduction Bacillus sporothermodurans is a non pathogenic Gram-positive bacterium (Hammer et al., 1995; Pettersson et al., 1996), predominated in UHT milk (Olivier et al., 2002) and can be isolated from raw milk and a non-milk source, namely silage and a feed concentrate for dairy cattle (Vaerewijck et al., 2001; Scheldeman et al., 2002). B. sporothermodurans, like many Bacillus and Clostridium species, is able of differentiation between two distinct cellular morphologies: the vegetative cell and the spore. The spore is metabolically dormant and provides to cell the ability to withstand environmental condition disadvantageous for vegetative life (Setlow, 2003). This bacterium is able to produce highly heat resistant
* Corresponding author. Laboratory of Epidemiology and Microbiology, Bacteriology and Biotechnology Development Group, Pasteur Institute of Tunisia (IPT), BP 74, 13 Place Pasteur, Belvédère, 1002 Tunis, Tunisia. Tel.: þ216 23 014 563; fax: þ216 71791833. E-mail address: [email protected] (C. Aouadhi). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.06.021
spores which enable it to survive to high-temperatures of UHT treatment (Pettersson et al., 1996). These spores can germinate and grow up to 105 CFU/mL in stored milk and therefore cause its nonsterility (Klijn et al., 1997). Despite their dormancy and resistance, spores retain a sensitive mechanism which is able of detecting and rapidly responding to the presence of specific germinative substances (Foerster and Foster, 1966; Gould and Dring, 1972; Moir and Smith, 1990) to return in life in as little as 20 min via germination process and outgrowth (Sarker et al., 2002). Germination can be triggered by a variety of factors, including nutrients as amino acids, ribosides (Rossignol and Vary, 1979; Moir et al., 1994; Clements and Moir, 1998; Ireland and Hanna, 2002), sugars (Racine et al., 1979; Irie et al., 1982) or mixtures like L-asparagine, D-glucose, D-fructose, and ions of potassium (AGFK) (Wax and Freese, 1968; Cabrera-Martinez et al., 2003). In addition to nutrients, spores can be germinated by other number of factors like surfactants (Paidhungat et al., 2001; Setlow et al., 2003) or physical treatments such as hydrostatic pressure (Aouadhi et al., 2012). Few studies have investigated the factors affecting spore
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germination with nutrient elements. For example, Clements and Moir (1998) have studied the effect of the temperature of incubation, the pH, and the cations on the germination of Bacillus cereus spores. The germination of spores by nutrient elements has been studied more extensively for some Bacillus species such as Bacillus subtilis (Irie et al., 1982; Paidhungat and Setlow, 2000), Bacillus anthracis (Ireland and Hanna, 2002), Bacillus megaterium (Racine et al., 1979; Rossignol and Vary, 1979), Bacillus licheniformis (White et al., 1974), B. cereus (Clements and Moir, 1998; Barlass et al., 2002) and some Clostridium species (Takeshi et al., 1988; Alberto et al., 2003; Paredes-Sabja et al., 2008). Nutrient-induced germination can be considered as the natural germination pathway, and this requires specific receptors. Germination is initiated when nutrient molecules activate the Ger receptors present in the spore’s inner membrane (Paidhungat et al., 2001). Shortly after, release of Ca2þDPA and rapid efflux of monovalent cations (Hþ, Naþ and Kþ) can be observed, while the core is partly rehydrated. Then, hydrolysis of the oppressing cortex layer, accomplished by cortex lytic enzymes CwlJ and SleB, becomes crucial to provide more space to the expanding core. After cortex degradation, the now fully rehydrated core allows the reactivation of enzymes and the synthesis of ATP from the 3-PGA (3-posphoglyceric acid) precursor. Degradation of the SASP proteins releases the spore’s DNA, and the spore initiates RNA, protein, and DNA syntheses in the further outgrowth phase (Setlow, 2003). There is much interest in this process because spores may affect the stability of food products, in particular dairy products, through germination. When spores germinate, they lose their resistance and become easy to inactivate (Paredes-Sabja et al., 2008). Therefore, the induction of spore germination following by inactivation of the germinated spores by other treatment can be used to inactivate Bacillus spores. In a previous work, we investigated the pressureinduced germination of B. sporothermodurans spores (Aouadhi et al., 2012). The present study is dedicated to the nutrientinduced germination of these spores that had not previously been studied. The aims of this study were to investigate the germination of B. sporothermodurans spores by nutrient elements (major naturally used amino acids, purine ribonucleosides (inosine), and sugars) and the effect of different factors (pH, temperature, and cations). The ultimate objectives of this work were to identify the specific germinant of these highly heat resistant spores and select optimal conditions to maximize their germination rate. 2. Materials and methods 2.1. Bacterial strain and spore preparation B. sporothermodurans strain LTIS27 described by Aouadhi et al. (2012) was used in this study. Cells were grown on brain-heart infusion agar (Biokar-Diagnostics) supplemented with 1 mg/L vitamin B12 (SigmaeAldrich) (BHIeB12) at 37 C. Twenty-four hours before use, cultures were held in BHIeB12 broth on a rotary shaker at 37 C to obtain a working culture. Spores were prepared according to the modified method of Ireland and Hanna (2002). A working culture (18 h, 37 C under agitation) was diluted 1:10 into sporulation medium (25 g/L nutrient broth (Pronadisa, Microbiological culture media and diagnostic reagents), 1 mg/L vit B12, 8 mg/ L MnS04 H20, and 1 g/L CaC12 H20) then incubated for 7 days at 37 C. This medium was then centrifuged at 6000 g for 10 min. The resulting pellet, containing spores and non-sporulated vegetative cells, was washed three times with sterile distilled water, resuspended in 5 mL sterile distilled water and treated at 100 C for 30 min to destroy vegetative cells. The endospore suspension was centrifuged and washed 4 times as described above. The cleaned
321
endospore preparation was stored at a concentration of 107 spores/ mL of distilled water at 4 C. 2.2. Spore germination assay The spore suspension was heat activated in distilled water at 100 C for 30 min and then suspended in germination buffer (10 mM TriseHCL and 10 mM NaCl, pH 8) for 15 min at 37 C (Barlass et al., 2002). The germination was initiated by addition of the following compounds: sugars (D-glucose and D-fructose) or all major naturally used amino acids or purine ribonucleosides (inosine), at different concentrations (10, 20, 50, 100, and 250 mM). For L-alanine germination, O-carbamyl-D-serine (5 mg/mL) (Sigmae Aldrich) was added to inhibit the alanine racemase activity (Clements and Moir, 1998; Barlass et al., 2002). All amino acids used for the germination assays were L-isomers (SigmaeAldrich) dissolved in sterile water at stock concentrations of 1 M. All compounds used were sterilized by filtration using 0.45 mm-pore-size filters. The optical density of the spore suspension was measured at 580 nm (OD580), after 60 min of incubation at 37 C. The germination rate was expressed as the maximum ratio of loss of OD580 of the spore suspension, relative to the initial value (Clements and Moir, 1998; Ireland and Hanna, 2002). The data were expressed as the percent reduction in OD (with the initial reading taken as 100%) which was calculated as [(ODi ODf)/DOi] 100, where ODi is the initial OD580 value measured for the sample and ODf is the OD580 value measured after 60 min incubation for the test sample. 2.3. Effect of pH, temperatures and cations on spore germination After heating at 100 C for 30 min, spores were suspended in the germination buffer for 15 min at 37 C (Pettersson et al., 1996; Montanari et al., 2004). The following germinant (D-glucose (50 mM), L-alanine (100 mM), inosine (50 mM)) were then added. A control, without compounds, was included for each experiment. To study the effect of pH or temperature incubation, spores, after the addition of germinants, were incubated for 60 min at different pH (5, 6, 7, 8 and 9) at 37 C or at different temperatures (20, 30, 37, 40 and 50 C). The adjustment of pH was achieved by the addition of sodium hydroxide (2N NaOH) or hydrochloric acid (2N HCl). To study the effect of cations on spore germination, the chemical compounds used were KCl, CaCI2, MgCl2, and NaCl. The solutions (KCl, CaCl2, MgCl2, or NaCl (Fluka chemical)) at a concentration of 100 mM (White et al. 1974) were added to the medium containing the spore suspension in the presence or absence of L-alanine, inosine, or D-glucose. 2.4. Statistical analyses and experimental design Data were subjected to one-way analysis of variance to evaluate the significance of each independent factor on spore germination. A Fisher LSD test was used to discriminate the means with a significance level fixed at 5%. In order to maximize the germination rate, diverse factors was combined. In fact, the optimization of the nutrient-induced germination of B. sporothermodurans LTIS27 spores was realized using a central composite experimental design with three factors as described by Aouadhi et al. (2012). The factors investigated were Lalanine concentration (20e60 mM), D-glucose concentration (5e 10 mM), and incubation temperature (30e40 C). The experimental response (germination rate) was estimated, taking into account the influence of the experimental factors. An analysis of variance and an estimation of response surface by multiple linear regressions
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were performed using the software STATGRAPHICS Centurion XV version 15.2.06. 3. Results and discussion 3.1. Germination survey with amino acids, inosine or sugar In this study, the effect of the major naturally used amino acids, inosine, and sugars on the germination of B. sporothermodurans LTIS27 spores was evaluated. The progress of germination of spore suspensions was estimated as the percent reduction in initial OD. The germination rate of B. sporothermodurans LTIS27 spores induced by different concentrations of diverse compounds used is shown in Fig.1. In presence of some compounds (D-fructose, L-arginine, L-asparagine, L-aspartate, L-cysteine, L-glutamate, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-serine, L-tyrosine, or L-valine), the germination rate increases with increasing concentrations, reaching near maximal levels (between 22 et 26%) at concentrations as low as 20 or 50 mM; however, at concentration higher than 50 mM, the germination rate did not exceed 30%. So it seems that the germination rate reaches a plateau and that increasing the concentration of some compounds will not necessarily further increase the germination rate. While the germination rate in presence of L-alanine, inosine or D-glucose keep on increasing to 62% with increasing the concentrations as high as 50 mM for inosine and D-glucose and 100 mM for L-alanine. Fig. 2 summarizes the effect of the major naturally used amino acids, inosine, and sugars, with a concentrations of 100 mM, on the germination of B. sporothermodurans LTIS27 spores. Strong germination responses were defined as those causing 40% or greater germination in 15 min (Ireland and Hanna, 2002). Considering this definition, we noted that B. sporothermodurans LTIS27 spores respond differently to germinant and among 23 compounds studied only D-glucose (62%), L-alanine (60%), inosine (52%), and Ltyrosine (44%) were able to deliver a significant germination response. This result shows that the specific germinants (causing 50% or greater germination) of B. sporothermodurans LTIS27 spores were D-glucose, L-alanine, and inosine. This was consistent with the data from a variety of Bacillus species. For example, D-glucose was specific germinant of B. megaterium spores (Hyatt and Levinson, 1962; Racine et al., 1979). L-alanine was considered as a strong germinant for several species of Bacillus such as B. cereus (Barlass et al., 2002), B. subtilis (McCann et al., 1996; Cabrera-Martinez
et al., 2003) and some species of Clostridium (Broussolle et al., 2002; Paredes-Sabja et al., 2008). Inosine is potent independent germinant for B. cereus spores (Barlass et al., 2002; Hornstra et al., 2006), while it is not a strong independent germinant for B. anthracis spores (Ireland and Hanna, 2002). It is a strong cogerminant when it is associated with amino acids (Ireland and Hanna, 2002). Finally, because L-alanine, inosine, and D-glucose have induced a percentage of germination upper than 50%, we were interested in testing these compounds as co-germinant for amino-acid-induced germination. 3.2. Alanine, inosine and D-glucose as co-germinants for aminoacid-induced germination The effect of L-alanine (1 mM) or inosine (1 mM) as a cogerminant in combination with one of the amino acids tested is represented in Fig. 3. Two distinct effects of L-alanine or inosine were observed. In fact, these two germinants stimulated response of some amino acids (L-histidine, L-tryptophan and L-tyrosine for Lalanine and L-alanine, L-cysteine, L-histidine, L-tyrosine, L-tryptophan and L-serine for inosine) to trigger significant germination (between 42 and 82%). However, with the other amino acids, no change in the germination rate was observed (data not shown). The strongest response (82%) was observed in presence of both Lalanine and inosine. Hence, these data shows that L-alanine and inosine can be used either as independent germinants or as cogerminants for some amino acids. This result is consistent with previous works carried out with other Bacillus species (Ireland and Hanna, 2002; Hornstra et al., 2006). For example, B. cereus endospores germinated in response to inosine or L-alanine, but the most rapid germination was elicited by a combination of these two germinants (Clements and Moir, 1998; Barlass et al., 2002). Moreover, inosine was used as a co-germinant with L-alanine, L-cysteine, L-histidine, L-methionine, L-phenylalanine, L-proline, L-serine, Ltryptophan, L-tyrosine, and L-valine for B. anthracis (Ireland and Hanna, 2002) and with L-alanine, L-glutamine, and phenylalanine for B. cereus (Hornstra et al., 2006). Ireland and Hanna (2002) showed two independent types of germinant specificities depending upon inosine (amino acids inosine dependent (AAID) responses). The first, AAID1, was accomplished through inosine and non aromatic amino acids (L-alanine, L-cysteine, L-proline, L-serine, and L-valine). The second, AAID2, was obtained with inosine combined with aromatic amino acids (L-histidine, L-phenylalanine, L-
70
Germination rate (%)
60
L-alanine L-leucine
50
L-arginine L-cysteine
40
L-tyrosine L-glycine L-glutamine
30
L-tryptophan Inosine D-glucose
20
D-fructose 10 0
0.1 0,1
1
10
20
50
100
Concentration (mM) Fig. 1. Effect of various concentrations of different nutrient elements on the germination of B. sporothermodurans LTIS27 spores. Values given are means (error bars represent standard deviations) of three independent experiments.
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Germination rate (%)
70 60 50 40 30 20
D-glucose
L-tyrosine
L-alanine
Inosine
L-tryptophane
L-glycine
L-cysteine
L-arginine
L-leucine
L-aspartate
L-histidine
D-fructose
L-isoleucine
L-pheny
L-lysine
L-proline
L-asparagine
L-serine
L-threonine
L-glutamate
L-methionine
L-valine
0
No addition
10
Fig. 2. Response of B. sporothermodurans LTIS27 spores to diverse nutrient elements. Values given are means (error bars represent standard deviations) of three independent experiments.
tryptophan, and L-tyrosine). Given that the inosine may potentiate some aromatic and non aromatic acids in triggering a robust germination response, we can hypothesize that both AAIDI responses are observed for B. sporothermodurans LTIS27. Several authors reported that L-alanine may potentiate other products (amino acid, inosine, sugars) in triggering a robust germination response (Yasuda and Tochikubo, 1984; Barlass et al., 2002; Ireland and Hanna, 2002). In the literature, two distinct germination responses of spore were observed when using Lalanine (1 mM) as a co-germinant with different amino acids. For B. anthracis, it was reported that the addition of L-alanine can elicit amino acid-mediated germination. This was explained by the reaction of the aromatic side chains of some amino acids (L-histidine, L-tyrosine, and L-tryptophan) with L-alanine (Ireland and Hanna,
(a) Germination rate (%)
90 80
2002). For B. cereus, no significant germination was detected in the presence of L-alanine (1 mM) as a co-germinant (Hornstra et al., 2006). The effect of D-glucose (1 mM) on the germination of B. sporothermodurans LTIS27 spores initiated by amino acids was also tested. The strongest germination response was observed with a combination of D-glucose (1 mM) and L-alanine (100 mM); the germination rate being of 89%. In contrast, D-glucose didn’t show any affect on spore germination in presence of other products (data not shown). The stimulation of L-alanine-induced germination by Dglucose was observed for B. megaterium (Hyatt and Levinson, 1961, 1962; Racine et al., 1979) and B. subtilis (White et al., 1974). Moreover, D-glucose was used as a co-germinant with some amino acids (L-leucine, L-proline, L-alanine and L-valine) for germination of B. megaterium spores (Hyatt and Levinson, 1962); however, it didn’t affect the induced-L-alanine germination of B. licheniformis spores (White et al., 1974). It seems that there is a synergistic function of Lalanine receptor with D-glucose-sensitive components in the germination apparatus for B. sporothermodurans spores.
70 60
3.3. Effect of different cations, pH, and temperature on spore germination
50 40 30 20 10 0 L-alanine
L-cysteine
L-tyrosine L-tryptophan
L-serine
L-histidine
(b) Germination rate (%)
60 50 40 30 20 10 0 L-tyrosine
L-tryptophane
L-histidine
Fig. 3. Germination of B. sporothermodurans LTIS27 spores in presence of major naturally utilized amino acids with co-germinant (L-alanine (a) and inosine (b)). Germination was initiated by the addition of 20 mM of amino acids with (solid bars) or without (shaded bars) 1 mM L-alanine (a) or 1 mM inosine (b) as indicated. Values given are means (error bars represent standard deviations) of three independent experiments.
The effect of diverse cations (NaCl, MgCl2, CaCl2 and KCl) on the response of B. sporothermodurans LTIS27 spores alone or in presence of some germinant (L-Alanine, inosine or D-glucose) is illustrated in Table 1.a. The cations enhanced the germination of B. sporothermodurans LTIS27 spores. Indeed, a strong germination response (49 and 43%) was induced by KCl and CaCl2, respectively. Nevertheless, in presence of L-alanine, inosine, or D-glucose, the different cations affected germination of B. sporothermodurans spores by decreasing their rate. The effect of different cations on spore germination is more contradictory in the literature. While Clements and Moir (1998) showed that potassium ions strongly inhibited inosine-induced germination of B. cereus spores, White et al. (1974) reported that potassium ions didn’t affect spore Lalanine-induced germination of B. licheniformis spores. The present study shows that salts reduced the D-glucoseinduced germination rate of B. sporothermodurans spores, whereas, for other species the effect of sugars and especially D-glucose was remarkably increased by salts (Hyatt and Levinson, 1962; Rode and Foster, 1962; Hyatt and Levinson, 1964). The effect of temperature on the spore germination of B. sporothermodurans LTIS27 in presence of L-alanine, inosine, or D-
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Table 1 Effects of different cations (a), incubation temperature (b) and pH (c) on the germination rate of B. sporothermodurans LTIS27 spores in presence of D-glucose, Lalanine or inosine. Germination ratea: No addition (a) Ion (100 mM) None 22.3 1.4a NaCl 31.6 1.8a KCL 49 1a CaCl2 43.3 1.8a MgCl2 29 1.5a (b) Incubation temperature 20 11.6 1.7a 30 20.3 1.2a 37 22.6 1.4a 40 19.6 1.6a 50 10 1.1a (c) pH 5 21.6 1.4a 6 32 1.5a 7 22 1.4a 8 18 1.5a 9 13.3 1.8a
D-glucose
L-alanine
(50 mM)
(100 mM)
Inosine (50 mM)
62.6 32.6 54.6 21.6 44.3 ( C) 35.3 45 59.6 51.6 30
1.4b 1.4a 1.4ab 1.7a 1.4a
60.6 36.6 42.6 42.6 45.6
1.2a 1.8a 1.4a 1.2a 1.4a
52 39 32 43.3 22.6
1.1b 1.5a 1.7ab 1.8a 1.4a
2a 1.1a 1.4a 1.6a 1.1a
21.6 51.3 56.6 44.6 20
1.2ab 1.6b 1.8ab 1.9b 1.1a
19.3 48.6 50.6 43 23.6
1.8a 1.2a 1.2a 1.5a 1.81a
40 54.3 60 53 30.3
1.1ab 1.2b 1.7ab 1.1ab 1.4a
29 57 52 30.3 20
0.5ab 0.5b 1.5ab 1.8ab 1.1a
22 51 52 25 19.7
1.1ab 1.5b 1.7ab 1.8ab 1.4a
a Results are the means standard deviations of three independent experiments. Means with different letters are different (P < 0.05).
glucose is shown in Table 1b. Spore germination was maximum in the temperature range of 30e40 C. In fact, spore germination decreased significantly at temperature above 40 C or below 30 C. For example, at 20 C, only 22% of loss of initial OD580 was observed in presence of inosine. The influence of pH on the spore germination of B. sporothermodurans is shown in Table 1c. Spore germination induced by L-alanine, inosine, or D-glucose increased with increasing of pH from 5.0 to 7.0. For pH above 8.0, the germination rate decreased significantly. It was clear that the optimal germination rate of B. sporothermodurans spores, in the presence of Dglucose or inosine or L-alanine, occurred in the pH range of 6.0e8.0. Some spores can germinate in a larger pH domain for B. licheniformis and Paenibacillus polymyxa or more restricted conditions for B. cereus (White et al., 1974; Huo et al., 2010). Furthermore, Ciarciaglini et al. (2000) reported that the germination of B. subtilis pSB357 spores, induced by L-alanine, was completely inhibited at pH 4.0. The low pH was thought to have altered the ionization state of amino acid side chains, thus changing their charge distributions, hydrogen bonding, and protein conformation. In our case, the germination was affected differently by pH in the presence of L-alanine or inosine or D-glucose. We can hypothesize that germinant receptors can be affected by pH and that extreme pH may inhibit expression of nutrient receptor genes or germinant binding (Broussolle et al., 2008; Huo et al., 2010). Moreover, it can be concluded that the pH conditions for optimal germination of Bacillus spores, depend on the microbial species and even strains within species. This could be due to differences in the nature of the enzyme systems which may be involved in their germination processes (White et al., 1974). For B. cereus, Clements and Moir (1998) showed that the differences in the optimum pH and temperature profiles and the effects of monovalent cations on germination in presence of L-alanine or inosine suggest that there are different germinant specific signal transductions mechanisms present in the spores. These differences would meant that either these germinants are detected by separate proteins in the spore or, possibly, by different binding sites in the same protein. In fact, Paredes-Sabja et al. (2011) showed that germinant receptors transduce the germination signal to downstream effectors, leading
Table 2 Test of significance for regression coefficients Model term
Coefficient estimate
Degree of freedom
Standard error
Probability
Intercept x1 (L-alanine) x2 (D-glucose) x3 (Temperature) x21 x22 x23 x1x2 x1x3 x2x3
380.3 1.91 2.99 23.44 0.0033 0.0955 0.301 0.0625 0.0437 0.01
1 1 1 1 1 1 1 1 1 1
0.0915 0.08 0.08 0.08 0.09 0.09 0.09 0.011 0.011 0.011
e 0.0000 0.0029 0.0022 0.1335 0.4764 0.0000 0.0128 0.0024 0.8982.
to the release of monovalent cations and ultimately DPA. The mechanism of this signal transduction is unclear, but proteine protein interactions between several SpoVA proteins and several germinant receptor subunits (Vepachedu and Setlow, 2007) suggest that the germinant receptors physically interact with at least some SpoVA proteins. Consequently, the triggering of spore germination would require that the germination signal be transduced from the low abundance germinant receptors to the SpoVA proteins (Vepachedu and Setlow, 2007). In addition, Huo et al. (2010) confirmed that the optimum temperature for spore germination is species-, strain-, and especially germinant-dependent. 3.4. Optimization nutrient-induced germination by response surface methodology (RSM) Throughout of this work, we noted that D-glucose was more effective than inosine on the L-alanine-induced germination. Moreover, the incubation temperature has a significant effect on the germination rate of B. sporothermodurans spores LTIS27. Therefore, the individual and interactive effects of L-alanine, Dglucose, and incubation temperature on the germination of B. sporothermodurans LTIS27 spores were studied using RSM. The response (Y) was measured in terms of the germination rate. The application of RSM provides an empirical relationship between the response variable and the test variable under consideration. Although this methodology has been used by some authors to evaluate the effect of various factors such as pressureetemperature on the inactivation of B. cereus (Ju et al., 2008) and pressure on the germination of B. sporothermodurans (Aouadhi et al., 2012) spores, to our knowledge it has never been used for the optimization of nutrient-induced spore germination. By analysis of the multiple regression application, the following quadratic model equation was obtained: Table 3 Experimental and predicted results for ten check-points of nutrient-induced germination of B. sporothermodurans LTIS27 spores. Trial no.
1 2 3 4 5 6 7 8 9 10
Factors
Germination rate (%) D-glucose (mM)
Temperature ( C)
Observed
Predicted
(mM) 35 30 25 40 50 40 35 45 55 60
7 7 6 6 5 8 9 8 8 5
38 35 30 35 40 30 40 30 35 30
76.5 94.1 57.5 71.3 82.5 72 75.2 78 94.9 92.8
78.88 93.14 58.404 69.32 84.46 72.034 74.15 77.529 95.587 91.7
L-alanine
C. Aouadhi et al. / Food Microbiology 36 (2013) 320e326
Germination rate (%)
(a)
Temperature (35°C)
110
90
70
50 20
30
40
50
60
5
6
7
8
9
10
D-glucose (mM)
L-alanine (mM)
Germination rate (%)
(b)
D-glucose (9 mM)
110
90
70 40 50
35
20
40
30
60
Temperature (°C)
L-alanine (mM)
Germination rate (%)
(c)
L-alanine (60 mM)
110
325
different combinations of process parameters (Table 3). The experimental values were found to be significantly in agreement with the predicted values with a correlation coefficient (R2) of 0.986 and a statistical significance level of P < 0.0001. Therefore, the model was proven to be quite reliable method for predicting the nutrient-induced germination of B. sporothermodurans spores. Response surface plots in Fig. 4 shows the effects of L-alanine, Dglucose, and incubation temperature on the germination rate. In each figure, one factor is maintained constant at its optimal value determined by Statgraphics. The germination rate of B. sporothermodurans spores increased with increasing temperature between 30 and 35 C, and decreased at temperature above 35 C. Furthermore, the germination rate increased with increasing L-alanine and D-glucose concentrations. As already shown with the interaction coefficients of the model, a significant synergistic effect was observed with LalanineeD-glucose and L-alanine-Temperature. In this work, the process parameters for 100% nutrientinduced germination of B. sporothermodurans LTIS27 spores were optimized with Statgraphics and the optimal germination rate was obtained in presence of L-alanine (60 mM), and D-glucose (9 mM) after incubation of spores for 60 min at 35 C. The optimal conditions inducing the germination of B. sporothermodurans spores obtained from this study slightly differ from other studies. For example, Ireland and Hanna (2002) reported that an important germination of B. anthracis spores (98%) was obtained in presence of L-alanine (100 mM). For B. cereus, various amino acids (L-alanine, L-cysteine, L-threonine, and L-glutamine) and inosine have been reported to trigger a robust germination response (between 90 and 100%) (Hornstra et al., 2006). For B. megaterium and B. subtilis, the strong and rapid germination response was achieved in presence of Lalanine and L-proline, respectively (Rossignol and Vary, 1979; McCann et al., 1996). These results show that the germination process depends on the species.
90
4. Conclusion
70
The effect of different nutrient elements (sugars (D-glucose and all major naturally utilized amino acids and inosine) on the spore germination of B. sporothermodurans has been studied here for the first time. These spores were shown to have a specific behavior which differs from that of other Bacillus species. Indeed, the specific germinants needed for B. sporothermodurans LTIS27 spores were D-glucose, L-alanine, and inosine and the optimal germination rate was obtained after 60 min of incubation of spore at 35 C in presence of 9 mM of D-glucose and 60 mM of L-alanine. Furthermore, we have found that inosine and L-alanine can be used as co-germinants at low concentration (1 mM) in presence of some other amino acids.
40
50 5
D-fructose),
6
35 7
8
9
10
30
Temperature (°C)
D-glucose (mM)
Fig. 4. Response surfaces plots showing the effects of L-alanine and D-glucose (a), Lalanine and temperature (b), and temperature and D-glucose (a) on the germination of B. sporothermodurans LTIS27 spores.
Y1 ¼ 380:306 þ 1:91x1 2:99x2 þ 23:44x3 0:003x21 þ 0:09x22 0:3x23 þ 0:06x1 x2 0:04x1 x3 þ 0:01x2 x3
(1)
where x1 is L-alanine concentration, x2 is D-glucose concentration and x3 is temperature levels. The analysis of variance (ANOVA) of the quadratic model was performed with Statgraphics and the statistical significance of each coefficient value was determined using the Fisher F-test (Table 2). The R2 value was 0.964 indicating that the derived model fitted the experimental data. The linear coefficients (x1, x2 and x3) and the interaction coefficients (x1x2 and x1x3) and x23 were significant with small P values (P < 0.05). The quadratic term coefficients (x21 and x22 ) and x2x3 were not significant. In order to validate the qualification of the polynomial equation, ten experimental checks were carried out under
Acknowledgments The authors gratefully acknowledge the financial support provided by the Tunisian Ministry of Higher Education and Scientific Research. Abbreviations list
DPA: HRS: RSM: UHT:
Dipicolinic Acid Highly Heat-Resistant Spores Response Surface Methodology Ultra-High Temperature
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