EFFECT OF DIFFERENT HERBS ON ALICYCLOBACILLUS ACIDOTERRESTRIS CULTURES

Aleksandra Duda-Chodak, Tomasz Tarko, Joanna Gajny, Tadeusz Tuszyński
Department of Fermentation Technology and Technical Microbiology, Food Technology Institute, Agricultural University in Cracow, Poland

ABSTRACT

The aim of the study was to evaluate the effect of fifteen herbs on the growth of Alicyclobacillus acidoterrestris (A.a.). Contrary to our expectations, the majority of the selected herbs stimulated the A.a. growth. The most efficient in inhibition of A.a. growth was extract of dog rose, characterized by low antioxidant activity. Exposition on 10% extract of dog rose fruit or dwarf everlast flower enabled the reduction of A.a. cells number by about 80% (when compared with control without herbal extract) after 60 h of culturing. In the same concentration extracts from nettle leaves diminished the growth by 70%, while roots of liquorice and angelica by 50%. Although the extracts concentrations applied in the experiments were too high to become competitive (economic considerations) with chemical disinfectants, in the many of cases the inhibitory effect was stronger for lower doses of herbs. It suggest that the further research on the impact of herbal extracts (of concentration below 1%) on A. acidoterrestris growth is needed. In the presented study, no correlation between antioxidant potential of examined herbs and their inhibitory effect on A.a. growth was shown.

Key words: Alicyclobacillus acidoterrestris, antioxidants, growth inhibition, herbal extract, juice spoilage, polyphenols.

INTRODUCTION

Alicyclobacillus acidoterrestris is a gram-positive, aerobic, spore-forming bacterium, resistant to high temperature (optimum 40–60°C) and acidic environment (pH 3.5-4.5) [18]. Its occurrence in fruit juices is the cause of heavy losses in food industry. The main cause of deterioration of the fruit juices quality is the production of chemical compounds with unpleasant odor, such as guaiacol, 2,6-dibromophenol and 2,6-dichlorophenol, as well as changes in juice pH, color and consistency [14,18]. Members of Alicyclobacillus genus occur in soil, but they were detected also in pulp and fruits, fresh and concentrated fruit juices, and even in citrus essence and condensation water (from the evaporator) [13]. The Alicyclobacillus spores are not inactivated by the pasteurizing conditions generally applied to juice concentrates and juice-containing beverages, so the pasteurization is not sufficient in prevention of fruit juices spoilage [9,10,11]. The presence of ω-cyclohexyl or ω-cycloheptyl fatty acids is thought to be the factor that enables these organisms to survive under acidic and thermal conditions [18]. Alicyclobacillus acidoterrestris, the most frequent odor producing species, are the main target microorganisms of this quality concern. The critical step during fruit juices production is the quality of washing water and the efficiency of the wash. In order to reduce Alicyclobacillus contamination, the raw material must firstly be washed thoroughly, and often disinfectants (e.g. ClO₂) are added to the washing water [4,16,18]. Special attention must be taken to ensure that treatment chemicals do not remain in the products. Although they are regarded as safe for consumers, new methods of elimination of Alicyclobacillus (spore and vegetative cells) are in demand. Nisin and ascorbic acid are the first natural substances used for this purposes [1,17]. The impact of herbaceous material on growth of this bacteria was not investigated yet.

The aim of the present study was to evaluate the effect of selected herbal extracts on the Alicyclobacillus acidoterrestris (A.a.) cultures. If the inhibitory activity would be shown the potential application in fruit juices production would be found. The juices supplementation with small concentrations of extracts or the usage of higher amounts of them for washing can improve the pro-health properties of juices and simultaneously protect against A.a.-induced juice spoilage.

MATERIAL AND METHODS

Chemicals
Diammonium salt of the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid (ABTS diammonium salt); 2,2'-diphenyl-1-picrylhydrazyl(DPPH); (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox); catechin hydrate; Folin-Ciocalteau phenol reagent; and a phosphate buffer saline (PBS): 0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4 at a temperature of 25°C. All the chemicals listed were purchased from the SIGMA-Aldrich Company. A 96% ethanol was obtained from the ChemPur Company, and other base chemicals typical for laboratories were from the POCh Company.

Material
Fifteen herbs were used in the investigation. Dried, homogenous samples of herbs were purchased in Herbapol S.A. in Cracow. The name of herbaceous material, common name of herb, its Latin name and part of plant analyzed are listed in Table 1.

Table 1. The composition of medium 402

Raw material	Common name of herb	Latin name of herb	Part of plant
Fructus Rosae	Dog rose	Rosa canina L.	fruit
Fructus Myrtilli	Lingonberry	Vaccinium vitis-idaea L.	fruit
Lupuli Strobilus	Hop	Humulus lupulus L.	strobile
Folium Menthae piperitae	Peppermint	Mentha × piperita  L.	leaf
Folium Urticae	Nettle	Urtica dioica L.	leaf
Folium Betulae	Small-leaved Lime	Tilia cordata Mill.	leaf
Flos Lavandulae	Lavender	Lavandula angustifolia Mill.	flower
Inflorescentia Helichrysi	Dwarf everlast	Helichrysum arenarium (L.) Moench	flower
Anthodium Chamomillae	Chamomile	Matricaria recutita L.	flower head
Radix Valerianae	Valerian	Valeriana officinalis L.	root
Archangelicae Radix	Angelica	Angelica archangelica L.,	root
Glycyrrhizae Radix	Liquorice	Glycyrrhiza gabra L.	root
Rhizoma Agropyri	Couch Grass	Elymus regens (L.) Gould	rhizome
Cortex Frangulae	Buckthorn	Frangula alnus Mill.	bark
Foenugraeci Semen	Fenugreek	Trigonella foenum-graecum L.	seed

Bacteria
Pure cultures of Alicyclobacillus acidoterrestris (DSM 2498) were provided by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). Bacteria were grown in special medium 402 (Table 2) at 47°C.

Table 2. The herbaceous material used for study

Solution A (adjust pH to 4)	CaCl2 × 2 H2O	0.25 g
	MgSO4 × 7 H2O	0.50 g
	(NH4)2SO4	0.20 g
	yeast extract	2.00 g
	glucose	5.00 g
	KH2PO4	3.00 g
	distilled water (liquid medium)	up to 1000 mL
	distilled water (solid medium)	up to 500 mL
Solution B	ZnSO4 × 7 H2O	0.10 g
	MnCl2 × 4 H2O	0.03 g
	H3BO3	0.30 g
	CoCl2 × 6 H2O	0.20 g
	CuCl2 × 2 H2O	0.01 g
	NiCl2 × 6 H2O	0.02 g
	Na2MoO4 × 2 H2O	0.03 g
	distilled water	up to 1000 mL
Solution C	agar	15 g
	distilled water	up to 500 mL

Each solution was sterilized separately. For liquid medium solution A (with 1000 mL distilled water) was combined with 1 mL of solution B. For solid medium there were combined: solution A (with 500 mL distilled water), 1 mL of solution B and solution C.

Herbal extracts preparation
Extracts were prepared by boiling 2.000 g of particular herb in 100 mL of distilled water for 10 min. The whole mixture was filtered and the supernatants obtained were collected into twisted test-probes. Samples were stored in cold (+4°C) until analysis.

Assessment of the antioxidant activity
The antioxidant activity was assayed on the basis of a protocol represented by Re et al. [8] with some modifications incorporated. The ABTS radical was generated during a chemical reaction between the 7 mM aqueous solution of diammonium salt of the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid and the 2.45 mM potassium persulfate. The solution was kept at a room temperature in darkness throughout the night, in order to complete the reaction and to stabilize the ABTS cation-radical. Prior to analysis the radical solution was diluted with PBS (pH 7.4) in such a way that allowed for obtaining the final absorbance of A = 0.70 ± 0.02 (ABTS_0.7) measured at 734 nm (spectrophotometer BECKMAN DU 650). 100-µL aliquots of the properly diluted extract or of Trolox solutions (their concentration ranging from 0 to 100 mg/L) were added to 1 mL of ABTS_0.7, and the absorbance was measured 6 min after mixing. The antioxidant capacity of extracts under study was calculated using a standard curve drawn up for solutions of the synthetic vitamin E (Trolox) and expressed in mg of Trolox/g of dried weight. All determinations were performed in 5 replications.

Total polyphenol content assay
Total polyphenols content was assayed by Folin-Ciocalteu method on the basis of a protocol represented by Swain and Hillis [15]. An amount of 45 mL of redistilled water, 0.25 mL of Folin-Ciocalteu reagent (water dissolved 1:1 v/v) and 0.5 mL of 7% Na₂CO₃ were added to the 5 mL of herb extract. The mixture was left out for 30 min in dark. Then the absorbance was measured on a spectrophotometer (BECKMAN DU 650) at 760 nm. The obtained results of total polyphenols content were expressed as mg of catechin/g of dry weight based on the standard curve drawn up for catechin methanol solutions (their concentration ranging from 0.1 to 15 mg/L). All determinations were performed in 5 replications.

Effect of herbs on Alicyclobacillus acidoterrestris growth
To assess the influence of herbal extracts on A. acidoterrestris (A.a.) culture 1, 2 or 3 mL of particular extract was added to tubes containing medium 402 (the final volume was 10 mL) and then inoculated with bacterial cells (1x10⁶). The number of cells was determined by turbidimetric evaluation (in NTU, Eutech Instruments Turbidimeter TN-100) and calculated using a calibration curve based on the results from cell counting chamber. After 6, 12, 18, 36, 42, and 60 h the bacterial growth was determined by turbidimetric evaluation (in NTU), and the amount of bacterial cells in 1 mL was calculated using the standard curve drawn up for the A.a. cultures in 402 medium. The results were then expressed as % of control samples (A.a. cultures in 402 medium without herbal extracts). In order to exclude the impact of herbal extract on turbidimetry value the measurement was also done for the medium with herbal extract without bacteria inoculation. All determinations were performed in triplicate.

Statistical analysis
The results were shown as an arithmetic mean (± standard deviation). A single-factor Analysis of Variance test (ANOVA) with a post hoc Tukey test was applied to perform a statistical analysis. A Kołmogorov-Smirnov test was applied to examine the normality of distribution. All statistical calculations were performed using GraphPad InStat version 3.01 for Windows (GraphPad Software, San Diego, California, USA).

RESULTS AND DISCUSSION

The supplementation of foodstuffs with herbal extracts can be benefit due to inhibition of pathogen microorganisms growth and reduction of financial losses connected with spoilage of food. Moreover, herbs can improve the sensory, nutritional and pro-healthy value of food [2]. Antioxidants are compounds that exert strong activity not against free radicals only, but also against many microorganisms. Most efficient natural antioxidants are vitamins A and C, tocopherols, carotenoids and polyphenols, secondary metabolites of plants. Among the factors that influence the antioxidant activity are: quality of plant raw material, its variety, maturity stage, place of origin, climatic and soil conditions, time of harvesting and storage condition [3,6,7,12].

In the study antioxidant activity was determined by ABTS assay, and obtained results are presented in Table 3. Very big differences were shown between particular herbs. The herb with the highest antioxidant activity were leaves of lime (337.4 ± 23.3 mg Trolox/g d.m.) and it was above 300-times more that the lowest value, obtained for dog rose fruit and couch grass rhizome (1.7 ± 0.3 and 1.2 ± 0.5, respectively). Also flowers of  dwarf everlast had high antioxidant potential (243.0 ± 0.8).

Table 3. Antioxidant  activity  (mg of Trolox/1 g d.m.) of extracts prepared form herbaceous material determined by ABTS assay; in the table mean ± SD was shown (n = 5)

Herbal extract	Antioxidant activity (mean ± SD)
Dog rose fruit	1.7 ± 0.3 a
Chamomile flower head	156.4 ± 4.3 b
Dwarf everlast flower	243.9 ± 0.8 d
Hop strobile	150.4 ± 33.4 b
Liquorice root	142.7 ± 0.8 b
Lavender flower	95.7 ± 17.3 c
Lime leaf	337.4 ± 23.3 e
Peppermint leaf	132.7 ± 22.9 b
Couch grass rhizome	1.2 ± 0.5 a
Nettle leaf	69.0 ± 21.2 c
Fenugreek seed	17.2 ± 13.8 a
Angelica root	7.8 ± 4.8 a
Lingonberry fruit	64.6 ± 34.5 c
Valerian root	7.5 ± 2.4 a
Buckthorn bark	82.6 ± 8.7 c

Red color was used for extracts of the highest antioxidant activity. Blue color was used for extracts of the lowest potential. a,b,c,d,e – the same letters denote the lack of statistically significant differences between means, at p < 0.05.

Table 4. Total polyphenols content (mg of catechin/1 g d.m.) of herbal extracts determined by Folin-Ciocalteu method; in the table mean ± SD was shown (n = 5)

Herbal extract	Total polyphenols content (mean ± SD)
Dog rose fruit	0.5 ± 0.0 a
Chamomile flower head	8.6 ± 0.3 c
Dwarf everlast flower	12.5 ± 0.5 d
Hop strobile	8.5 ± 0.2 a,b
Liquorice root	4.6 ± 0.4  a,b
Lavender flower	6.8 ± 0.3  a,b
Lime leaf	17.8 ± 0.2  b
Peppermint leaf	10.5 ± 0.8  a,b
Couch grass rhizome	2.1 ± 0.0 a
Nettle leaf	8.0 ± 0.9  a,b
Fenugreek seed	4.6 ± 1.1  a,b
Angelica root	2.0 ± 0.0 a
Lingonberry fruit	4.4 ± 0.2  a,b
Valerian root	4.2 ± 0.9 a
Buckthorn bark	6.6 ± 0.1  a,b

Red color was used for extracts of the highest polyphenols concentration. Blue color was used for extracts of the lowest level of total polyphenols. a,b,c,d – the same letters denote the lack of statistically significant differences between means, at p < 0.05.

Folin-Ciocalteu method was used to evaluate total polyphenols content in extracts of particular herbs (Table 4). The highest polyphenols content was demonstrated in extracts of lime leaves and dwarf everlast flowers (17.8 and 12.5 mg catechin/g d.m., respectively), which correlated with their antioxidant activity. Very low level of polyphenols was noted in extracts of dog rose (0.5), couch grass rhizome (2.1) and angelica root (2.0). Such low values for dog rose, were caused by the form of the herbs. It is very difficult to extract bioactive compounds from the whole fruits, without their breaking up. Moreover, water is no the best extrahent, when the fruit is covered by a layer of wax.

The impact of different concentrations of herbal extracts on Alicyclobacillus acidoterrestris growth is presented in figures 1–15. After 6 h of incubation, in all analyzed samples the bacteria count was higher than in control (from 103% for buckthorn bark up to 17 443% for lime leaves), and this results was independent on concentration and kind of extract used. It should be emphasized that the turbidimetry of medium did not change after extract addition (medium without A.a., data not shown). After longer incubation in samples exposed to angelica root (Fig. 1), liquorice root (Fig. 2), dwarf everlast flower (Fig. 3), nettle leaf (Fig. 4), and dog rose fruit (Fig. 5) extracts the bacteria number decreased below the control level. The others extracts caused the stimulation of bacteria growth. We suppose that it was caused by the valuable nutrient and bioactive compounds present in the herbal extract. Only after their depletion the impact of other components, as those of antibacterial activity, could be visible.

Fig. 1. The effect of different concentrations of angelica root extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

1, 2 or 3 mL of extract was added to culture medium inoculated with 1 x 106 bacterial cells (final volume of the culture was 10 mL). The amount of AA cells was deteremined after 6, 12, 18, 36, 42, and 60 h of incubation at 47°C by turbidimetric evaluation, and the amount of bacterial cells in 1 mL was calculated and expressed as % of control samples (AA cultures in medium 402 without herbal extracts).

Fig. 2. The effect of different concentrations of liquorice root extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 3. The effect of different concentrations of dwarf everlast flower extracts on AA growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 4. The effect of different concentrations of nettle leaves extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 5. The effect of different concentrations of dog rose fruit extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

The most efficient in Alicyclobacillus acidoterrestris inhibition was nettle leaf extract (Fig. 4). The A.a. cells number after 60 h of culturing with 1, 2, and 3 mL of the extract was 33.6%, 12.3%, and 6.7% of control, respectively. The potent inhibitor were also extracts from dog rose fruit (16.4%, 19.4% and 17.0%), angelica root (45.6%, 52.2% and 54.7%) and liquorice root (50.2%, 49.4% and 37.1%, respectively for 1, 2 and 3 mL of extract in comparison to control culture). Also extract prepared from flowers of dwarf everlast had exerted inhibitory effect (Fig. 3). Although, the values below control level had been reached only after more that 40 h of incubation, the results were very promising: 19.2% (for 1 mL of extract), 24.8% (2 mL) and 38.1% (3 mL) of control.

Addition of valerian root extract (Fig. 6) caused rapid fall of cells concentration after 12 h of culture (to 20% of control) but subsequently the bacteria number increased, and reach after 60 h from 114% (3 mL) to 284% (1 mL of extract). Similar dynamics were observed for cultures with buckthorn bark extracts (Fig. 7); the lowest bacteria number was at 12 h (~9% of control) and then slowly increased. Only in samples with 3 mL of this extract the amount of A.a. cells was lower than in control without extract.

Fig. 6. The effect of different concentrations of valerian root extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 7. The effect of different concentrations of buckthorn bark extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

We showed that in the case of buckthorn bark the higher was concentration of extract added the lower was the bacteria count in particular culture points. The same tendency was observed for extracts of nettle leaves (Fig. 4), peppermint leaves (Fig. 8), and couch grass rhizome (Fig. 9). However the amount of bacteria in the samples incubated with peppermint and couch grass were always higher than in control medium without herbs.

Fig. 8. The effect of different concentrations of peppermint leaves extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 9. The effect of different concentrations of couch grass rhizome extracts on Alicyclobacillus acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Interesting results were obtained for hop strobile extract (Fig. 10); after initial increase, A.a. cell number decreased slowly during next hours of the experiment. The higher dose of extract the higher final bacterial cell number. In samples with 1 and 2 mL of extract, after 60 h of cultivation, the amount of bacterial cells was 39% and 88% of control, respectively. In case of cultures exposed for 3 mL of hop strobile extract, the significant growth of A. acidoterrestris was detected during whole experiment, with final value 2-times higher than in control.

Fig. 10. The effect of different concentrations of hop strobile extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 11. The effect of different concentrations of small-leaved lime leaves extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

The same tendency (the higher extract concentration the higher amount of A.a. cells) was demonstrated in cultures exposed to extracts of lime (Fig. 11), lingonberry (Fig. 12), dwarf everlast (Fig. 3), chamomile (Fig. 13), and fenugreek (Fig. 14). The extract of small-leaved lime caused tremendous stimulation of A.a. growth (Fig. 11). After 6 h of incubation the amount of bacterial cells reached from 5000% (for 1 mL of extract) up to 17443% (for 3 mL). Although, the cell number decreased with time, at the end of the experiment it was still much higher than in control samples.

Fig. 12. The effect of different concentrations of lingonberry fruit extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 13. The effect of different concentrations of chamomile flower head extracts on Alicyclobacillus acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 14. The effect of different concentrations of fenugreek seeds extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SD

Fig. 15. The effect of different concentrations of lavender flower extracts on A. acidoterrestris (AA) growth in medium 402. Points in graphs represent the arithmentic mean of 3 replicates ± SDD

Among the examined extracts of herbaceous material one can find such of bactericidal or bacteriostatic activity against A. acidoterrestris. Unfortunately, contrary to our expectations, the majority of the herbs stimulated the A.a. growth. It was probably caused by the fact that extracts are abundant in valuable nutrient and bioactive compounds that stimulated bacterial growth. Only after their depletion the impact of other components, as those of antibacterial activity, could be visible. In the presented study, the correlation between antioxidant potential of examined herbs and their inhibitory effect on A.a. growth was not shown.

The most efficient in inhibition of Alicyclobacillus acidoterrestris growth was extract of dog rose, characterized by low antioxidant activity. Culture exposition on 10% extract of dog rose fruit or dwarf everlast flower enabled the reduction of A.a. cells number by about 80% (when compared with control without herbal extract) after 60 h of culturing. In the same concentration extracts from nettle leaves diminished the growth by 70%, while roots of liquorice and angelica by 50%.

Although the extracts concentrations applied in the experiments were too high to become competitive (economic considerations) with chemical disinfectants, in the many of cases the inhibitory effect was bigger for lower doses of herbs. It suggest that the further research on the impact herbal extracts (especially in concentration <1%) on A. acidoterrestris growth is needed Moreover the mixtures of different herbs can be used in the future. Matławska and Bylka [5] shown, that herbal preparation produced form 40 mg of hop strobile and 60 mg of valerian root had the same activity as 400 mg valerian root. This phenomenon is called synergism, and we will make use of it in further experiments.

It should be also highlighted, that in the study the hot water extracts were used. We realize that the high temperature used during boiling can destroy some bioactive compounds present in the raw material. However, on the other hand, it allows extracting bigger amounts of valuable substances. The usage of organic solvents would be probably the best, but the extracts obtained from analyzed herbal plants are destined for food technology. Therefore, the selection of adequate solvent and extraction method should focus first of all on avoiding the possible toxicity, and then on getting the high efficiency of process and good extracts quality. The further research with different extraction methods should be performed.

CONCLUSIONS

Some herbaceous materials exert inhibitory effect against Alicyclobacillus acidoterrestris. The studies on the possibility of their usage during production of apple juice in order to diminish the amount of this bacterium should be performed. This supplementation could be the natural way of preventing the spoilage of fruit juices. The highest inhibitory activity among examined herbs had: rod rose fruit, nettle leaf, dwarf everlast flower, and roots of angelica and liquorice. No correlation between antioxidant potential of examined herbs and their inhibitory effect on A.a. growth was shown.

REFERENCES

1. Bahceci K.S., Acar J., 2007. Modeling the combined effects of pH, temperature and ascorbic acid concentration on the heat resistance of Alicyclobacillus acidoterrestris. Int. J. Food Microbiol. 120 (3), 266–273.

2. Czapska A., Bałasińska B., Szczawiński J., 2006. Działanie przeciwbakteryjne i przeciwutleniające ekstraktów przypraw żywnościowych [Antibacterial and antioxidant activity of food seasoning extracts]. Med. Weter. 62(3), 152–153 [in Polish].

3. Kondo S., Truda K., Muto N., Ueda J., 2002. Antioxidative activity of apple skin or flesh extracts associated with fruit development on selected apple cultivars. Sci. Hortic. 96, 177–185.

4. Lee S., Gray P., Dougherty R., Kang D., 2004. The use of chlorine dioxide to control Alicyclobacillus acidoterrestris spores in aqueous suspension and on apples. Int. J. Food Microbiol. 92, 121–127.

5. Matławska I., Bylka W., 2006. Synergizm działania w lekach roślinnych [Synergism of action in plant medicaments]. Herba Polon. 52(3), 128–133 [in Polish].

6. McGhie T.K., Hunt M., Barnett L.E., 2005. Cultivar and growing region determine the antioxidant polyphenolic concentration and composition of apples grown in New Zealand. J. Agric. Food Chem. 53 (8), 3065–3070.

7. Podsędek A., Wilska-Jeszka J., Anders B., Markowski J., 2000. Compositional characterization of some apple varieties. Eur. Food Res. Technol. 210, 368–372.

8. Re R., Pellegrini N., Porteggente A., Pannala A., Yang M., Rice-Evans C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Res. 26 (9/10), 1231–1237.

9. Silva F., Gibbs P., 2001. Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes. Trends Food Sci. Technol. 12, 68–74.

10. Silva F., Gibbs P., 2004. Target selection in designing pasteurization processes for shelf-stable high-acid fruit products. Leatherhead Food Research Association, Surrey.

11. Silva F., Gibbs P., Vieira M., Silva C., 1999. Thermal inactivation of Alicyclobacillus acidoterrestris spores under different temperature, soluble solids and pH conditions for the design of fruit processes. Int. J. Food Microbiol. 51, 95–103.

12. Sluis van der A., Dekker M., de Jager A., Jongen W.M.F., 2001. Activity and concentration of polyphenolic antioxidants in apple: effect of cultivar, harvest year, and storage conditions. J. Agric. Food Chem. 49, 3606–3613.

13. Sokołowska B., Jakubowski A., Przestrzelska A., 2004. How to prevent juices spoilage; Alicyclobacillus acidoterrestris – quality hazard of polish fruit juices [Jak zapobiegać zepsuciu soków; Alicyclobacillus acidoterrestris – zagrożenie jakości polskich soków owocowych].  Przem. Ferm. Owoc.-Warz. 2, 24–25 [in Polish].

14. Sokołowska B., Niezgoda J., Bytońska M., Łaniewska-Trokenheim Ł., 2008. The heat resistance of Alicyclobacillus acidoterrestris spores [Ciepłooporność przetrwalników Alicyclobacillus acidoterrestris]. Przem. Ferm. Owoc.-Warz. 12, 211–217 [in Polish].

15. Swain T., Hillis W.E., 1959. The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10, 63–68.

16. Wilson L., 2001. Microbial food contamination. CRC Press, Boca Raton.

17. Yamazaki K. , Murakami M., Kawai Y., Inoueand N., Matsuda T., 2000. Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks. Food Microbiol. 17, 315–320.

18. Yokota A., Fujii T., Goto K., 2007. Alicyclobacillus. Thermophilic Acidophilic Bacilli. Springer, Tokyo.

Accepted for print: 15.11.2009

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Aleksandra Duda-Chodak
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Phone: (+48 12) 662 47 92
email: aduda-chodak@ar.krakow.pl

Tomasz Tarko
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland
Phone: (+48 12) 662 47 92

Joanna Gajny
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland

Tadeusz Tuszyński
Department of Fermentation Technology
and Technical Microbiology,
Food Technology Institute,
Agricultural University in Cracow, Poland
Balicka 122, 30-149 Cracow, Poland

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