Culture dependent and independent genomic identification of Alicyclobacillus species in contaminated commercial fruit juices

Highlights

• Fruit juices were examined for presence of Alicyclobacillus.

• shc, 16S rRNA gene sequencing and RAPD PCR identified Alicyclobacillus spp.

• Guaiacol production detected among Alicyclobacillus acidoterrestris isolates.

• PCR-DGGE differentiated guaiacol-producing A. acidoterrestris.

Abstract

Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming, bacteria species which occasionally cause spoilage of heat-processed fruit juices by producing guaiacol taint. In this study, Alicyclobacillus contamination of commercial fruit juices in West Africa was investigated using culture-dependent and -independent approaches. Firstly, a total of 225 fruit juice products from Ghana (n = 39) and Nigeria (n = 186) were enriched with yeast–starch–glucose (YSG) broth (pH 3.7) following heat shock at 80 °C for 10 min. Alicyclobacillus was detected in 11.6% (26) of samples. Isolates were identified to the genus taxonomic level by genus-specific PCR which targeted the squalene-hopene-cyclase (shc) gene followed by analysis of the almost-complete 16S ribosomal RNA (rRNA) gene sequences that identified 16 Alicyclobacillus acidoterrestris, 7 Alicyclobacillus acidocaldarius and 3 Alicyclobacillus genomic species 1 (Alicyclobacillus sp. 1). Whole-genome fingerprinting using PCR-RAPD primers Ba-10, F-61 and F-64 grouped the 16 A. acidoterrestris isolates into two genetic clusters. Furthermore, high performance liquid chromatographic (HPLC) analyses revealed the activity of vanillic-acid decarboxylase (vdc) in all A. acidoterrestris isolates due to guaiacol production from vanillic-acid. Lastly, species-specific PCR-DGGE targeting the 16S rRNA gene clearly discriminated between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius group. Information provided by this study is fundamental to the development of effective strategies for the improvement of quality and shelf-life of processed tropical fruit juices in W. Africa.

Introduction

Food industries generally employ different physical, chemical or a combination of both methods in the control of spoilage and pathogenic microorganisms in food products. Industrial food processing uses pasteurization to guarantee food quality and safety by destroying vegetative cells without much lethal effects on endospores previously assumed to be of very limited safety consequence in acidified foods such as fruit juice. The emergence of Alicyclobacillus species as spoilage microorganisms that can survive pasteurization has posed serious challenge to the quality issues in the fruit juice industry.

Alicyclobacillus species are thermo-acidophilic, endospore-forming bacteria with distinct ω-alicyclic fatty acids in their cell membranes (Wisotzkey et al., 1992). Due to the heat and acid tolerance of their endospores, Alicylobacillus spp. are capable of evading fruit juice pasteurization and subsequently germinating in the acidic fruit juices (Chang and Kang, 2004, Walker and Phillips, 2008, Durak et al., 2010). In fact pasteurization is believed to stimulate germination of Alicyclobacillus endospores (Gouws et al., 2005, Groenewald et al., 2008). Alicyclobacillus induced spoilage of fruit juices is usually devoid of spoilage attributes such as turbidity, heavy sediments, gas production or package swelling (Brown, 1995, Walls and Chuyate, 1998, Danyluk et al., 2011). Usually, spoilage is evidenced by phenolic or medicinal off flavours caused by guaiacol or the halophenols (Pettipher et al., 1997, Splittstoesser et al., 1998, Walls and Chuyate, 1998, Jensen and Whitfield, 2003, Chang and Kang, 2004, Bevilacqua et al., 2008). Guaiacol is a product of the non-oxidative decarboxylation of vanillic acid and the vanillic-acid decarboxylase (vdc) complex involved in this reaction has been identified in Alicyclobacillus and other taint producing microorganisms (Crawford and Olson, 1978, Chow et al., 1999, Álvarez-RodrÍgue et al., 2003, Niwa and Kawamoto, 2003, Witthuhn et al., 2012).

Several studies have reported the occurrence of Alicyclobacillus spp. in fruit juices and other acidic food products, ingredients and processing environments, indicating that the problem of Alicyclobacillus contamination is widespread (Splittstoesser et al., 1994, Yamazaki et al., 1996, Eiora et al., 1999, Jensen, 2000, Goto et al., 2008, Groenewald et al., 2009, Zhang et al., 2013). Many reports have also identified Alicyclobacillus acidoterrestris as the predominant spoilage Alicyclobacillus species due to its high occurrence in spoiled products, fruit juice processing environments, and its ability to produce taints in fruit juices (Goto et al., 2002, Matsubara et al., 2002, Chen et al., 2006, Walker and Phillips, 2008, Groenewald et al., 2009, Durak et al., 2010, Wang et al., 2010, Danyluk et al., 2011). Consequently, A. acidoterrestris is recognised as the target species for quality control in the fruit juice industry (Yamazaki et al., 1997, Goto et al., 2008). Other Alicyclobacillus relevant to the fruit juice industry include Alicyclobacillus acidiphilus, Alicyclobacillus herbarius, Alicyclobacillus pomorum and Alicyclobacillus genomic species 2 (Matsubara et al., 2002, Goto et al., 2002, Goto et al., 2003, Chen et al., 2006), however these species are rarely encountered (Niwa, 2004, Goto et al., 2008).

Although Alicyclobacillus contamination of fruit juices and related materials have been widely studied and reported in many parts of the world, there is dearth of information about genetic diversity of Alicyclobacillus in West Africa and their spoilage potential during food processing. Previous studies have reported variations in the phenotypic characteristics and sensitivity of A. acidoterrestris strains to chemical treatments. For example, Goto et al. (2008) reported that the guaiacol production varied among strains of A. acidoterrestris isolated in Japan while Yamazaki et al. (2000) reported that the MIC of nisin against A. acidoterrestris varied among strains isolated from different geographical sources, highlighting the importance of considering diverse strains during the design of control strategies against A. acidoterrestris isolates.

With the rapid growth of small and large scalefruit juice industry in W. Africa, it is important that product quality and shelf-life are consistent in order to boost consumer confidence and prevent economic loss to manufacturers and retailers. As Alicyclobacillus spp. may impact negatively on the shelf-life of processed fruit juices, this study was conducted to identify the predominant Alicyclobacillus species in locally processed fruit juices in W. Africa, aimed at understanding of diverse strains of Alicyclobacillus as to facilitate development of strategy that can aid shelf-life improvement and mitigation of economic losses to fruit juice manufacturers in W. Africa.