World Journal of Microbiology and Biotechnology

, Volume 30, Issue 3, pp 855–863

Control of Alicyclobacillus acidoterrestris in fruit juices by a newly discovered bacteriocin

Authors

  • Jinjin Pei
    • Department of Food ScienceNorthwest A&F University
    • Department of Food ScienceNorthwest A&F University
  • Yahong Yuan
    • Department of Food ScienceNorthwest A&F University
Original Paper

DOI: 10.1007/s11274-013-1491-1

Cite this article as:
Pei, J., Yue, T. & Yuan, Y. World J Microbiol Biotechnol (2014) 30: 855. doi:10.1007/s11274-013-1491-1
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Abstract

Alicyclobacillus acidoterrestris is one of the most spoilage-causing bacteria in fruit juices. Control of A. acidoterrestris in fruit juices by bificin C6165 (Pei et al. in J Appl Microbiol 114(5):1273–1284, 2013), a bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, was described in this study. Activity spectrum of bificin C6165 was investigated and sixteen strains of A. acidoterrestris were sensitive to bificin C6165 in diluted Apple Juices. In the commercial fruit juices, vegetative cells of A. acidoterrestris were inactivated by bificin C6165 at 40 μg/ml. The inhibitory effect of bificin C6165 was better at lower pH (pH 3.5) and at a higher temperature of 45 °C. Furthermore, electron microscopy examination of the vegetative cells treated with bacteriocin revealed substantial cell damage and bacterial lysis. The result suggested that primary mode of action of bificin C6165 was most probably due to pore formation. Although no significantly activity of bificin C6165 was observed against the endospores of A. acidoterrestris in commercial apple juice, the addition of bacteriocin contributed to the reduction of the thermal resistance of A. acidoterrestris spores. Additionally, encapsulation of bificin C6165 with Ca-alginate gel was investigated. Encapsulation of bificin C6165 provided a promising method to control A. acidoterrestris in food juice industry.

Keywords

AlicyclobacillusBacteriocinBificin C6165BiopreservationFruit juice

Introduction

Spoilage incidents attributed to Alicyclobacillus species, especially Alicyclobacillus acidoterrestris, were reported in various fruit juices all over the world (Bevilacqua et al. 2012), fruit juice blends (Goto et al. 2008; Silva and Gibbs 2001), carbonated fruit juice drinks (Eiroa et al. 1999), fruit pulps (Gouws et al. 2005), lemonade and isotonic water (Yamazaki et al. 2000), iced tea (Groenewald et al. 2009) and even canned diced tomatoes (Alpas et al. 2003). Microorganisms of the genus Alicyclobacillus have a broad pH range of the growth between 2.0 and 7.0 and are able to grow in a temperature range of 20–70 °C. Alicyclobacillus spores have been shown to resist acidic environments and high temperatures applied in hot fill processes used for fruits and vegetable juices, rendering Alicyclobacillus as a potential cause of spoilage. Alicyclobacillus have been detected in a wide range of fruit juices and products as well as processing facilities, where they enter most probably on fruit surfaces contaminated from soil during production and harvesting (Silva and Gibbs 2001; Bevilacqua et al. 2012).

Bacteriocins of Lactic acid bacteria are defined as ribosomal synthesized proteins or protein complexes usually antagonistic to genetically closely related organisms. The use of bacteriocins for food preservation has been a matter of extensive work in recent years (Ansari et al. 2012; Cosentino et al. 2012; Santagati et al. 2012), but application of bacteriocins in preservation of fruit juices has seldom been studied (Grande et al. 2005). Model membrane studies with nisin have shown that lipid II acts as a docking station (Wiedemann et al. 2001). After binding, nisin inserts itself into the cell membrane to form short-lived pores which disturb the integrity of the cytoplasmic membrane and causes the efflux of ions and other cell components (Sablon et al. 2000). Bificin C6165 is a newly discovered bacteriocin-like substance produced by Bifidobacterium animalis subsp. animalis CICC 6165 bacteria discovered by our lab (Pei et al. 2013). The primary study on characterization of bificin C6165 in laboratory medium (Pei et al. 2013) suggested that this bacteriocin may be used for biopreservation. In this study, we described the antimicrobial activity of bificin C6165 against vegetative cells and endospores of A. acidoterrestris in fruit juices.

Materials and methods

Bacterial strains and cultivation conditions

Twenty strains of A. acidoterrestris were used in this study. A. acidoterrestris DSM3922 was purchased from Deutsche Sammlung von Mikroorgansmen und Zellkulturen; AAT11, AAT92, AAT10, AAT93, AAT96, AAT14, AAT94, AAT13, AAT12 and AAT95 (Wang 2010) were provided by Sanjing food Biotechnology Corporation in Japan; YL-3, YL-5 (Wang 2010), BS-2, BS-5, LC-8, LC-3, LC-4, BS-1(Cai 2005) were isolated by our lab from apple plantations in Shaanxi province in China; CFD-1 was isolated by our lab from workplace in Haisen Fruit Juice Corporation (one of the biggest concentrated apple juice producer in China) (Hu 2007). All Strains were grown at 45 °C in the Alicyclobacillus.spp medium (AAM) described by Yamazaki et al. (2000).

The bacteriocin-producer strain B. animalis subsp. animalis CICC6165 was purchased from China Center of Industrial Culture Collection (CICC) and cultivated in MRS broth at 37 °C. Bifidobacterium animalis subsp. animalis CICC 6165 and A. acidoterrestris were routinely stored at 4 °C on MRS-agar and AAM agar, respectively. Strains were maintained as frozen stocks at −80 °C in 20 % glycerol for long-term preservation.

Production of bificin C6165

Partially purified bificin C6165 was obtained as described by Pei et al. (2013). The activity of bacteriocin was determined at each step by spot-on-lawn method (Ennahar et al. 2001). One liter of cells free supernatant (CFS) (adjusted to pH 6.0 with 4 M NaOH) of B. animalis subsp. animalis CICC 6165 was prepared as described by Pei et al. (2013). Ammonium sulfate was gently added to the CFS maintained at 4 °C to obtain 80 % saturation (about 561 g/l), and the mixture was stored overnight at 4 °C. After the centrifugation at 20,000×g for 1 h at 4 °C, the resulting pellet was re-suspended in 20 mM sodium phosphate buffer (pH 6.0, buffer A) (Fraction I). Fraction I was applied onto a SP-Sepharose Fast Flow (15 mm internal diameter, 100 mm length, Sigma-Aldrich, USA) cation-exchange column equilibrated with buffer A. After the column was washed with 300 ml buffer A, the bacteriocin was eluted with linear NaCl gradient (0–0.5 mol/l) at pH 6.0 (fraction II). The fractions contained antimicrobial substances were pooled. The partially purified bificin C6165 was then lyophilized and stored at 4 °C until used.

Preparation of spore suspensions

Cultures grown for 24 h at 40 °C in AAM broth were spread onto agar plate in Petri dishes and incubated at room temperature (25 °C) for 10 days. Sporulation was confirmed by microscopy following staining with malachite green. After reaching more than 90 % of sporulation, spores in each plate were collected and re-suspended in distilled water (2 ml per plate). The pool of spores collected from the different plates was centrifuged at 5,000×g for 15 min at 4 °C. The spores were washed twice with distilled water followed by centrifugation, and finally re-suspended in distilled water (106 spores/ml, as determined by plating counts). Then the spores were stored at −20 °C until use.

Effect of bificin C6165 on A. acidoterrestris in fruit juices

Diluted Apple Juice (DAJ) samples were prepared by diluting the Concentrated Apple Juice collected from Haisheng Juice Co., Ltd. (Shannxi Province, China) with distilled water to the soluble solid content of 11°Brix (Atago Type N1 refractometer, Atago Co., Tokyo, Japan). The DAJ was then subjected to autoclave at 115 °C for 15 min. Before it was inoculated with vegetative cells of A. acidoterrestris from an exponential-phase culture or with spore suspensions, the DAJ was pre-incubated for 1 h at 45 °C. Then, bificin C6165 was added to the DAJ at the desired final concentration. Juice mixtures were incubated at 45 °C for 10 days. The activity of bificin C6165 against the growth of indicator strains was determined by measuring the OD600 (Cheikhyoussef et al. 2009). Cell viability was determined by plating onto the AAM agar.

Commercial orange juice (Hui Yuan, China; pH 3.58), apple juice (Rongshi, China; pH 4.06), peach juice (Hui Yuan; pH 4.21), and grapefruit juice (Hui Yuan, China; pH 3.62) were purchased in a local supermarket.

Effect of temperature, pH value and long time preservation on the activity of bificin C6165 in DAJ

The activity of bificin C6165 in DAJ which pH value was adjusted to 3.5 and 4.5, respectively, was tested in this study. Different incubation temperatures (25 and 45 °C) were carried out as well. The effect of extended storage at low refrigerated temperature on bificin C6165 stability in DAJ was evaluated by placing partially purified bificin C6165 in DAJ containing A. acidoterrestris at 4 °C for 100 days. The activity of bificin C6165 in DAJ was determined by spot-on-lawn method (Ennahar et al. 2001)). The minimum concentration of bificin C6165 that inhibited the growth of A. acidoterrestris was recorded as MIC.

Scanning electron microscope

Exponential-phase cells of A. acidoterrestris DSM 3922 strain (ca. 108 CFU/ml) in DAJ were treated with bificin C6165 (0.1 mg/ml) and incubated at 45 °C. After 24 h of incubation, samples (1 ml) were collected by centrifugation (8,000×g for 20 min) and then subjected to the preparation procedure according to the manual instruction for scanning electron microscopy examination and Field Emission Gun Scanning Electron Microscopy examination at the Technical Services of the Northwest A&F University.

Thermal treatment

DAJ (10 ml) containing the spores of A. acidoterrestris (ca.105 spores/ml) and desirable concentrations of bificin C6165 were heated at water bath. After the temperature of samples reached to 90 °C and was maintained for desirable time, the samples were cooled down in ice-water as quickly as possible. The surviving populations of A. acidoterrestris were determined by plating on AAM agar and then incubated at 45 °C for 2 days. Dt value (heat resistance) was defined as the minutes of heat treatment at temperature for survivors with 1 log dropping.

Encapsulation of bificin C6165

Sodium alginate was dissolved in hot distilled water (60 ml) with constant stirring. After cooling to room temperature, partially purified bificin C6165 solution was added under stirring condition to form a uniform mixture and the final volume was made to 100 ml. This mixture was added dropwise to 1,000 ml of desired concentration of CaCl2. The sample was kept overnight at 4 °C. The volume of rest lipid (V) was recorded. Protein concentrations (C) were determined by using Pierce BCA Protein Assay Reagent. Encapsulation efficiency (EE) = (G-CV)/G × 100 %. G was the amount of bificin C6165. The beads were washed with distilled water and stored at 4 °C prior to use. Random selective gel beads (thirty) were closely arranged in line and checked with vernier caliper (Di). The diameter of gel bead was defined as D = ΣDi/30.

Effect of concentration of CaCl2 and sodium alginate on encapsulation of bificin C6165 was investigated. CaCl2 was added at 1.0, 1.5 or 2.0 %, respectively and sodium alginate was added at 1.0, 1.5 or 2.0 %, respectively. Rapid encapsulation formation rate, completed shape and high encapsulation efficiency were the parameters for optimal encapsulation.

Apple juice quality evaluation

The DAJ samples before and after treatments with bacteriocin were analyzed for total sugar, titratable acidity, color value, and clarity. Total sugar of apple juices was determined by direct titrimetric method with Fehling`s reagent (SB/T 10203-1994). Titratable acidity was measured by the titrimetric method (SB/T 10203-1994). The determination of color value and clarity of apple juices was carried out by a Shimadzu Spectrophotometer (UV2550, Shimadzu Scientific Instruments). Transmittance values at 440 and 625 nm were used, respectively, to measure color value and clarity (GB/T, 18963).

Statistical analysis

All experiments were performed in triplicate, and the data are presented as mean ± standard deviation (SD). Data was subjected to One-way analysis of variance by using the Statistical Analysis System. Statistical significance was considered to exist at p < 0.05.

Results

Effect of bificin C6165 on A. acidoterrestris in DAJ

Activity spectrum of bificin C6165 against all twenty indicator strains was determined by measuring the growth of indicator strains (optical density at 600 nm) in DAJ containing 0.1 mg/ml bificin C6165. Alicyclobacillus acidoterrestris DSM 3922 as well as the fifteen strains of A. acidoterrestris in DAJ was sensitive to bificin C6165. Strain LC 4, YL-5, AAT96 and BS-2 were not inhibited by bificin C6165 (Table 1). According to previously study, bificin C6165 had wider inhibitory spectrum in AAM broth. In AAM broth, bificin C6165 was active against all 20 strains of A.acidoterrestris.
Table 1

Antimicrobial spectrum of bificin C6165 in DAJ

Indicator strains

DSM 3992

AAT 10

AAT 11

AAT12

AAT 13

Activity of Bificin C6165 in DAJ

+

+

+

+

+

Indicator strains

AAT 92

AAT 93

AAT 94

AAT 95

AAT 96

Activity of Bificin C6165 in DAJ

+

+

+

+

Indicator strains

YL 3

YL 5

BS 1

BS 2

BS 5

Activity of Bificin C6165 in DAJ

+

+

+

Indicator strains

AAT14

LC 3

LC 4

LC 8

CFD 1

Activity of Bificin C6165 in DAJ

+

+

+

+

Vegetative cells from A. acidoterrestris DSM 3922 and A. acidoterrestris CFD1 were incubated in AAM at 45 °C with bificin C6165 at concentrations of 0, 10, 20, 40, 80 and 160 μg/ml. For bacteriocin concentration of 20 μg/ml tested, viable cell counts of A. Acidoterrestris decreased rapidly within the first 3 days of incubation (Fig. 1). However, the remaining viable cells were able to multiply during the following incubation period (up to 10 days). For bacteriocin concentration at 40 μg/ml or higher, no viable cells were detected after 24 h (Fig. 1).
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Fig. 1

Effect of bificin C6165 on vegetative cells of A. acidoterrestris DSM 3922 (a) and A. acidoterrestris CFD-1 (b) in DAJ. Controls (filled star). Cultures added bificin C6165 at a concentration of 10 μg/ml (square), 20 μg/ml (filled triangle) and 40 μg/ml (triangle). Data represent the average of three assays ± SD

Effect of bificin C6165 on A. acidoterrestris DSM 3922 in commercial fruit juices

For fruit juices inoculated with vegetative cells without bacteriocin, the concentrations of viable cells increased to a variable degree within the 10 days of incubation. Highest counts were obtained from apple juice, followed by orange, peach and grape fruit juice (Fig. 2). In fruit juices added bificin C6165 (40 μg/ml), no viable cells were detected after 24 h.
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Fig. 2

Effect of bificin C6165 (40 μg/ml) on vegetative cells of A. acidoterrestris DSM 3922 in commercial apple juice (a), orange juice (b), peach juice (c) and grape fruit juice (d). Symbols: square (without bifiicn C6165) and filled square (with bificin C6165). Data represent the average of three assays ± SD

In fruit juices inoculated with endospores, addition of bificin C6165 (80 μg/ml) reduced the concentrations of viable counts below the detection limits within 24 h in grape, peach and orange fruit juices. However, cell viability was not significantly reduced in commercial apple juice (Fig. 3).
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Fig. 3

Effect of bificin C6165 on endospore of A. acidoterrestris DSM 3922 inoculated in commercial fruit juices. Symbols: triangle apple; filled square grape; square orange; filled diamond peach. Data represent the average of three assays ± SD

Effect of pH and temperature on activity of bificin C6165 in DAJ

The ability for bificin C6165 to inhibit A. acidoterrestris was increased at lower pH (Table 2). The sensitivity of A. acidoterrestris to bificin c6165 varied among the strains. At pH 3.5 and 4.5, MIC values for the vegetative cells among the strains were 20–40 and 40–80 respectively. Among the tested strains, A. acidoterrestris AAT 11 and DSM 3922 were the most resistant to bificin C6165, requiring 40-80 mg/ml for the inhibition at pH 3.5 and 4.5, respectively. It is considered that the use of bificin C6165 is effective in preventing A. acidoterrestris in acidic drinks.
Table 2

Factors affecting the antimicrobial activity of bificin C6165 agaisnt A. acidoterrestris in DAJ

Strain

MIC (minimum inhibitory concentration) mg/ml

Temperature

pH

25 °C

45 °C

3.5

4.5

DSM

80

40

40

80

AAT 11

80

40

40

80

CFU 1

40

20

20

40

BS 1

80

40

40

80

YL 3

40

20

20

40

All data represent an average of three repeats. The values recorded in each experiment did not vary b more than 5 %. Single data points are, therefore, presented in the figures without SD bars

Sensitivity of A. acidoterrestris to bificin C6165 was determined in DAJ at 25 °C, representing ambient storage, and at 45 °C, representing the optimal growth temperature for the organism. At the lower temperature, viability of A. acidoterrestris cells was inhibited by lower concentration of bificin C6165. At 45 °C, the organism was less-sensitive to bificin C6165 as we can see in Table 2. Unfortunately, bificin C6165 lost most of its activity in DAJ at 4 °C for 100 days (date not shown).

Ultra-structural changes of vegetative cells of A. acidoterrestris DSM3922 in DAJ treated with bificin C6165

Electron microscopy examination of vegetative cells of A. acidoterrestris DSM3922 treated with bificin C6165 revealed severe structural changes compared to untreated control cells (Fig. 4). Short after bacteriocin addition, cell wall damage and loss of cytoplasmic content were observed (data not shown). Cell disorganization was more pronounced after 24 h of incubation, yielding abundant cell debris (Fig. 4b). The bacteriocin-treated sample (24 h) also contained small membrane vesicles, some of which seemed to protrude from the cells (Fig. 5).
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Fig. 4

Electron microscopy examination of A. acidoterrestris DSM3922 vegetative cells treated with bificin C6165. Cell suspensions in fruit were examined at time zero without bacteriocin (a) or at 24 h with bificin C6165 (b)

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Fig. 5

Field emission gun scanning electron microscopy examination of A. acidoterrestris DSM3922 vegetative cells treated with bificin C6165 for 24 h

Effect of bacteriocin concentration on thermal death kinetics of A. acidoterrestris

The D90 °C values obtained from A. acidoterrestris DSM3922 and CFD1 spores are shown in Fig. 6. The heat resistance of the bacterial spores declined gradually as bificin C6165 concentration increased. The D90 °C values of CFD1 were 20.8, 19.1, 18.5, 16.5, 14 and 11.9 min with addition of 0 (control), 10, 20, 40, 80 and 160 μg/ml bificin C6165, respectively and the D90 °C values of DSM3922 were 24.5, 22, 21.6, 19, 18 and 16.3 min with addition of 0 (control), 5, 10, 20, 40, 80 and 160 μg/ml bificin C6165, respectively.
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Fig. 6

Influence of bificin C6165 on the thermal resistance of A. acidoterrestris DSM 3922 (square with line) and A. acidoterrestris CFD1 (filled square) spores at 90 °C in DAJ. Data represent the average of three assays ± SD

Encapsulation of bificin C6165

When concentration of CaCl2 was lower than 1.5 %, microcapsule was much less of hardness and roundness. However, the Encapsulation efficiency (EE) decreased as the concentration of CaCl2 increased (Table 3). So the optimal concentration of CaCl2 was determined as 1.5 %. When the concentration of sodium alginate was lower than 1 %, granule roundness wasn’t completed; when the concentration of sodium alginate was higher than 1.5 %, round and completed microcapsule could be obtained (Table 3). Additionally, the EE of microcapsule increased simultaneously with the increase of concentration of sodium alginate. According to these results, high concentration (2 %) of sodium alginate was preferred. Diameter of the microcapsule was 1.3 mm, and encapsulation efficiency (EE) of bificin C6165 was 86.28 %.
Table 3

Effects of calcium chloride concentration and sodium alginate concentration on the morphology and encapsulation efficiency of microcapsules

Concentration of CaCl2 (%)

Concentration of sodium alginate (%)

Shape of encapsulation

EE (encapsulation efficiency)

1.0

1.0

Unshapely

N

1.5

Granularity completed, but not round

85.76 ± 1.53

2.0

Granularity completed, but not round

87.52 ± 1.21

1.5

1.0

Granularity completed, almost round

80.45 ± 2.62

1.5

Granularity completed, almost round

83.92 ± 2.28

2.0

Granularity completed, round

86.28 ± 1.39

2.0

1.0

Granularity completed, almost round

78.53 ± 2.33

1.5

Granularity completed, round

80.35 ± 1.57

2.0

Granularity completed, round

85.15 ± 1.64

Activity of encapsulated bificin C6165 in fruit juices

The final concentration of bificin C6165 applied to DAJ in this work corresponded to 10 μg/ml. This concentration was used to evaluate whether the encapsulated bificin C6165 presented better inhibitory effect against A. acidoterrestris than free bificin C6165 during storage. A bacteriocidal action caused by higher concentrations could impair the comparison between free and encapsulated bificin C6165 along the time, since no further bacterial growth would be observed. The initial population of A. acidoterrestris in the DAJ was 4 log CFU/g (Fig. 7). On day eight, A. acidoterrestris in the control DAJ (without added bacteriocins) reached the stationary phase of growth, presenting the counts of 8–8.5 log CFU/ml. In the first 4 days, the growth of A. acidoterrestris was inhibited in the DAJ containing both free bificin C6165 and bificin C6165 encapsulation. However, after 8 days, encapsulation presented significantly higher inhibitory effect than free bificin C6165 (Fig. 7). A. acidoterrestris population didn’t increase between 8 and 12 days when bificin C6165 encapsulation was applied. At day 14 the cell viability of A. acidoterrestris treated with encapsulation was 3.5 log CFU/ml lower than the control and approximately 2 log CFU/ml lower than free bificin C6165 applied.
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Fig. 7

Growth of A. acidoterrestris in DAJ containing free or encapsulated bificin C6165. Viable counts were monitored in fruit juice containing encapsulated bificin C6165 (diamond), free bificin C6165 (square) and without the addition of bificin C6165 (filled square). Control juice (without bificin C6165 and A. acidoterrestris DSM 3922) (filled circle). Data represent the average of three assays ± SD

Juice quality

The effect of bificin C6165 on the quality of DAJ was investigated by checking the total sugar, titratable acidity, color and clarity value of the DAJ after treatment with free (80 μg/ml) and encapsulated bificin C6165 (80 μg/ml), respectively. The results showed that there was no significant difference (p > 0.05) in the values of total sugar, titratable acidity between the treated juice with free or encapsulated bificin C6165 and the control sample, as we can see in Table 4. Additionally, color and clarity stayed the same when the DAJ was treated with encapsulation bificin C6165. However, color and clarity reduced when free bificin C6165 was added to the DAJ.
Table 4

Effects of bificin C6165 treatments on the quality attributes of DAJ

 

Total sugar (g/100 ml)

Titratable acidity (g/100 ml)

Claritya (%)

Colorb (%)

Control

10.91 ± 0.03

0.156 ± 0.005

99.58 ± 0.53

65.29 ± 0.82

Free bificin C6165

10.95 ± 0.07

0.151 ± 0.008

96.85 ± 0.72

57.13 ± 1.02

Encapsulation

10.94 ± 0.06

0.154 ± 0.007

99.74 ± 0.43

65.57 ± 0.93

Values are means + SD

aValues for clarity indicate transmittance at 625 nm

bValues for color indicate transmittance at 440 nm

Discussion

The globalization of the food market and modern food-processing technologies create new ecological niches for microbes. One example can be found in A. acidoterrestris, a species of endospore-forming bacteria frequently found in soil. Due to its capacity to grow in a broad pH range of 2.0–7.0, A. acidoterrestris can easily multiply in acidic fruit juices. Endospores of A. acidoterrestris are also capable of germinating and outgrowing under high acidic conditions in apple, orange and grapefruit juices with pH values of 3.42–3.90 (Komitopoulou et al. 1999). Vegetative cells of A. acidoterrestris are highly thermotolerant, requiring more intense heat treatments for inactivation than any other microbial cells that may be present in juices like, for example, lactic acid bacteria or yeasts. In addition, they also produce endospores of a strong heat resistance, with reported D values exceeding 60 min at 85 °C and close to 8 min at 97 °C (Eiroa et al. 1999). Since the hot fill processes applied in manufacture of most commercial juices usually hold the products for about 2 min at temperatures between 88 and 96 °C, endospores of A. acidoterrestris have become the potential spoilage concern. Fruit juices and fruit juice-containing drinks that are most susceptible to A. acidoterrestris are either fresh (not heat-treated) or pasteurised (but not UHT-treated) (Grande et al. 2005; Bevilacqua et al. 2012). Therefore, alternative methods to control spoilage caused by A. acidoterrestris are required, especially if less intense treatments are to be applied in order to satisfy consumers’ demands for better preserved and fresh-tasting juices and drinks.

The antimicrobial spectrum and the physico-chemical properties of bificinC6165 suggest that this bacteriocin may be used for biopreservation. In this work, bificin C6165 was active to sixteen strains of Alicyclobacillus. In the study of enterocin AS-48 (Grande et al. 2005), four strains in fruit juices were sensitive and most of them showed a similar sensitivity to AS-48. The results shown in this work indicated that strains of Alicyclobacillus could be inhibited by bificin C6165 in both laboratory growth medium and commercial fruit juices of several types. In this study, the result suggested that A. acidoterrestris CFD1 was more sensitive to bificin C6165 than A. acidoterrestris DSM 3922. Importantly, higher concentration of bificin C6165 (40 μg/ml) was needed to inactivate A. acidoterrestris DSM 3922 in DAJ than in AAM broth (13 μg/ml) according to previously study by out lab (Pei et al. 2013). Bificin C6165 at concentration of 80 μg/ml was enough to inactivate endospores of A. acidoterrestris in commercial orange, grape and peach juices. However, this bacteriocin wasn’t active against endospres of A. acidoterrestris in apple juice. Nisin was reported to be inefficient against Alicyclobacillus in clear apple juice by Yamazaki et al. 2000. The same phenomenon was observed in our study. Niwa et al., (1991) and Yamazaki et al. 2000 reported that the difference in inhibitory effect of bacteriocins among the drinks may be attributed to the combination effect of bacteriocins and the polyphenols contained in acidic drinks, although the exact factors are not completely understood. Typically, fruit juices will be pasteurized at temperatures around 95 °C for 2 min our results and those reported previously by other authors demonstrated the ability of endospores to survive on such treatment (Peña et al. 2009). The work presented here indicated a strategy for control of A. acidoterrestris with the addition of bificin C6165. Inclusion of bificin C6165 in the juice prior to heat treatment would increase the organism’s heat sensitivity and would thus increase the lethality of pasteurization. Since bificin C6165 is relatively heat-stable at acidic pH value, it is very promising to apply in acidic drinks.

Some studies have shown that application of bacteriocins to fruit juices can be an additional hurdle to reduce the levels of A. acidoterrestris. However, direct application of bacteriocins may result in decrease or loss of antimicrobial activity due to problems related to interaction with food components (Aasen et al. 2003). Sant’Anna reported that the antimicrobial activity of BLS P34 was affected by sugar or amino acids components in MRA medium. The interaction of bacteriocins with food components may alter its bioavailability, thus the nature of food matrix influences the bacteriocin effectiveness. Nisin inactivation by some meat components, such as phospholipids and glutathione S-transferase has been reported (Cleveland et al. 2001). EDTA has significant effect on antimicrobial activity of BLS P34 (Sant’Anna et al. 2011). Encapsulation technology has been shown to protect bacteriocin from interfering food components, potentially enhancing their efficacy and stability. In this study, Ca-alginate gel was chosen for the encapsulation experiment as it is cheap, abundant and nontoxic. The encapsulation of bificin C6165 resulted in better inhibitory effect after 8 days, suggesting that the bificin C6165 were being released from the microcapsule after this time.

Unfortunately, addition of free bificin C6165 may damage the quality of DAJ. The color and clarity of DAJ with 80 μg/ml bificin C6165 was reduced to 57.13 and 96.85, respectively. According to the National Standard of People Republic of China, the color standard was ≧45 and the clarity standard was ≧95. Although the color and clarity was decreased after treatment with free bificin C6165, the quality of DAJ was acceptable.

Acknowledgments

This study has been supported by China State “12th Five-Year Plan” scientific and technological support scheme (2012BAD31B01); National Natural Science Foundation of China (31071550, 31171721); “948” project of the Ministry of Agriculture of china (2011-G8-3).

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© Springer Science+Business Media Dordrecht 2013