Alicyclobacillus spoilage and isolation--a review

Until recently, acidic products such as fruit juice and fruit based products were generally thought to be susceptible to spoilage by yeasts, mycelia fungi and lactic acid bacteria, as the low pH of these products acts as natural control measures against spoilage by most bacteria. Alicyclobacillus seem to be prevalent in fruit based products as they survive the acidic fruit juice environment, even when they are exposed to pasteurisation temperatures during production. In this review the historical background of the discovery of these bacteria is summarised. The bacterial characteristics and the reported spoilage incidences caused by members of this genus are discussed. As the isolation methods for these bacteria are controversial, this review includes a discussion of the various media that have been reported in the literature for the use in the isolation and enumeration of members of the genus Alicyclobacillus.     

Alicyclobacillus acidoterrestris: an increasing threat to the fruit juice industry?

The quality of food can be defined in various ways with processors and consumers considering flavour, odour and appearance as well as extended shelf life among the most important of its attributes. Spoilage of food by bacterial contamination may occur at any point during the processing, altering any one, or all, of these characteristics, rendering the product unusable. Alicyclobacillus spp. and Alicyclobacillus acidoterrestris, in particular, are emerging food spoilage organisms in the fruit juice and fruit juice products industry. Spores of the latter are able to survive heat treatments presently used in the industry and their elimination from products may be used as a measure of the effectiveness of any processing protocol to remove potential spoilage. This paper reviews the history, methods of detection, both traditional and rapid and the protocols that may be effective in controlling the growth of the organism and hence spoilage in the finished product.     

Isolation,identification and typification of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius strains from orchard soil and the fruit processing environment in South Africa

Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius are thermo-acidophilic, non-pathogenic, spore-forming bacteria that can survive the typical heat processing of fruit juices and concentrates. Bacterial endospores then germinate, grow and cause spoilage of acid food products. Species of Alicyclobacillus were isolated from orchard soil and a fruit concentrate production factory in South Africa. Preliminary identification of the isolates was based on morphological, biochemical and physiological properties. Identification at species level was done by PCR amplification using genus-specific primers and 16S ribosomal RNA (rRNA) gene sequencing. The majority of isolates belonged to the species A. acidoterrestris, but A. acidocaldarius was also isolated and identified. As far as we could determine, this is the first report of the isolation of A. acidoterrestris from wash water and soil outside a fruit processing plant, as well as the isolation of A. acidocaldarius from vinigar flies. The genotypic relatedness between strains of A. acidoterrestris and between strains of A. acidocaldarius was determined by RAPD-PCR. Sixteen isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Three isolateT:/PGN/ELSEVIER/YFMIC/web/00001155/s identified as A. acidocaldarius gave three unique banding patterns.     

Identification and haplotype distribution of Alicyclobacillus spp. from different juices and beverages

Alicyclobacillus spp. is an important thermoacidophilic, spore-forming spoilage bacterium that is a major concern for beverage and juice industries. In order to develop effective control strategies and adequately address the prevalence of contamination sources, it is necessary to characterize Alicyclobacillus' ecology in fruit, juice and beverage production and processing environments.Alicyclobacillus spp. isolates were collected from juice, beverage, ingredients, and environmental samples over a period of ten years. A total of 141 isolates were characterized as Alicyclobacillus spp. by 16S rRNA analysis and the most frequently isolated species was found to be Alicyclobacillus acidoterrestris (45%), A. acidocaldarius subsp. acidocaldarius (30%), and A. acidocaldarius (11%).The majority of thermotolerant sporeformers isolated from apple juices and concentrates was found to be A. acidoterrestris (24 out of 36 total apple isolates); while A. acidoterrestris was most frequently associated with citrus, citrus concentrates, and their associated environments, isolated by University of Florida (UF) (15 out of total 28 UF citrus isolates). However, A. acidocaldarius and subsp. acidocaldarius were frequently isolated by Cornell University (CU) (29 out of 35 CU citrus isolates), from citrus juices made from concentrate. Four major haplotypes of Alicyclobacillus spp. were identified based on the 16S rRNA gene sequencing from the 141 isolates tested. The Allelic Types (ATs) matched the phylogenetic analysis grouping of the different Alicyclobacillus spp. based on the isolation source.Our results suggest a predisposition for certain ATs of Alicyclobacillus spp. depending on juice or ingredient isolation source.     

Discrimination of <Emphasis Type="Italic">Alicyclobacillus</Emphasis> Strains by Lipase and Esterase Fingerprints

Alicyclobacillus spp. have recently become a major issue in the fruit juice industry due to its implication in the spoilage of pasteurized juices/beverages. In this study, lipase and esterase fingerprints of 37 Alicyclobacillus strains and 14 Bacillus strains in apple juice were collected using a five-substrate cocktail including four C8-esters and one C4-diester. Characterization of each strain could be finished in a single assay followed by high-performance liquid chromatography (HPLC) analysis for product separation and quantification. The obtained lipase and esterase fingerprints were highly species-dependent. Differences between the relative peak areas of each product for different species were significant. Coupled to chemometric techniques including principal component analysis (PCA) and stepwise linear discriminant analysis (SLDA), the lipase and esterase profiles led to 100 % of correct species identification for Alicyclobacillus and Bacillus strains. This approach is promising, reliable, and with good repeatability and could be used as an alternative tool to discriminate Alicyclobacillus spp. in fruit juices.     

Spoilage by Alicyclobacillus Bacteria in Juice and Beverage Products: Chemical,Physical, and Combined Control Methods

Alicyclobacillus is a genus of spoilage bacteria causing contamination of juices and other beverage products that cannot easily be contaminated by other microbes because of their high acid contents. During the last 4 decades since the first species of Alicyclobacillus was isolated in 1967, Alicyclobacillus has become a major concern to the global juice and beverage industries, and many promising methods have been developed and applied to control them. After introducing the history and general characteristics of Alicyclobacillus, as well as their heat resistance and spoilage, this review focuses on the control methods against Alicyclobacillus, including chemical and physical methods and combined methods. All these control methods show inhibitory or killing effects against Alicyclobacillus to some extent and, moreover, some of them have been put to use in the juice and beverage industries for decades and shown to be quite effective, although further developments can be achieved and new methods are constantly being established and investigated. Although it is difficult to compare the effects with one another among these methods because of the different experimental conditions in different reports, some of them, such as the treatments of nisin and high hydrostatic pressure, are well studied and proved to have extensive application prospect. The inhibitory factors, test strains and media, and especially the detailed experimental conditions and main results of these control methods are summarized here. The limitations of some methods mainly relating to the changes of products’ sensory qualities are also presented.     

Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.     

Fluid dairy product quality and safety: looking to the future

The fiercely competitive nature of the US beverage industry will drive the fluid milk sector of the dairy industry to improve product quality and shelf life to enable dairy beverages to compete with innovative new introductions as well as with currently popular shelf-stable products. The recent substantial growth in the volume of flavored milk sales specifically suggests that attention is needed to improve these products. Further, increasing public awareness and regulatory attention directed toward food safety issues highlight the need for the dairy industry to proactively address and eliminate emerging food safety issues that may negatively impact the image of dairy products. Shelf life and sensory profiles of high temperature short time pasteurized fluid milk products are presented, illustrating the need for greater attention to controlling contaminating microorganisms in processed fluid milk products. Bacterial spoilage patterns of flavored versus unflavored milks are compared, and suggestions are presented for extending flavored product shelf lives. Strategies currently applied to extend shelf life are reviewed. Food safety issues facing the dairy industry are presented within the context of an overview of foodborne illnesses in the United States. The pressing need to determine thermal resistance characteristics of Mycobacterium paratuberculosis is described.     

中国果蔬饮料生产与市场

中国果蔬饮料业与整个饮料业几乎是同时发展，但发展缓慢，一直处于对市场的不断摸索阶段。经过近二十年的曲折发展，开始成熟，在苹果，橙，桃，杏，山楂，芒果，菠萝、西番莲、胡萝卜、黑加仑、草莓、树莓等果蔬饮料的加工上形成了一定的基础或优势，制定了部分产品检测标准，2001年全国果蔬饮料产量为146万吨，比上年增长了49．87％，成为增幅最大加工，全方位提高质量管理，进一步提高出口产品质量，拓宽市场，是中国果蔬饮料业需迫切解决的问题和发展方向。     

Bioactive phytochemicals, nutritional value, and functional properties of cape gooseberry (Physalis peruviana): An overview

Exotic fruits play an important role in nutrition as an excellent base for low calorie and dietetic products. Highly valued for its unique flavor, texture and color, recent research has shown cape gooseberry (Physalis peruviana) to be high in many beneficial compounds. The diversity of functional applications to which P. peruviana can be put gives this fruit great importance. The food industry has used cape gooseberry in different products including beverages, yogurts and jams. With the rapidly growing popularity of this unique fruit it is important to have a comprehensive reference for its nutritional benefits. This review provides a valuable source for current knowledge on bioactive phytochemicals and further development of P. peruviana for functional foods as well as nutraceutical and pharmaceutical industries.     

Mitigation of Alicyclobacillus spp. spores on food contact surfaces with aqueous chlorine dioxide and hypochlorite

The prevalence of Alicyclobacillus spp. and other spore-forming spoilage organisms in food handling and processing environments presents a sanitation challenge to manufacturers of products such as juices and beverages. The objectives of this study were to determine the efficacy of chlorine dioxide and sodium hypochlorite in killing Alicyclobacillus spores in situ and to evaluate the efficacy of various chlorine dioxide and hypochlorite sanitizing regimes on Alicyclobacillus spp. spores on stainless steel, wood, and rubber conveyor material. Five or two log CFU/ml spore concentrations were left in aqueous solution or inoculated onto stainless steel, rubber, or wood coupons and challenged with sanitizer for varied time intervals. After treatment, the coupons were placed in sterile sample bags, massaged with neutralizing buffer, and enumerated on Ali agar. Surfaces were also examined before and after treatment by scanning electron microscopy to confirm destruction or removal of the spores. For both five and two log CFU/ml spore concentrations, treatments of 50 and 100 ppm of chlorine dioxide and 1000 and 2000 ppm of hypochlorite, respectively, were the most effective. Of the range of chlorine dioxide concentrations and contact time regimes evaluated for all surfaces, the most effective concentration/time regime applied was 100 ppm for 10 min. Reductions ranged from 0 to 4.5 log CFU/coupon. Chlorine dioxide was least effective when applied to wood. Hypochlorite was not efficient at eliminating Alicyclobacillus spores from any of the food contact surfaces at any time and concentration combinations tested. Chlorine dioxide is an alternative treatment to kill spores of Alicyclobacillus spp. in the processing environment.     

现制饮料中腐败微生物及其检测与防控技术研究进展

我国现制饮料种类丰富,产业发展快速且前景广阔。然而,近年来现制饮料中由腐败微生物引起的食品安全问题频发,受到了人们的高度重视。因此,本文归纳并总结了现制饮料中存在的主要腐败微生物种类、污染源及其产生的不良影响。介绍了传统检测法和快速检测法在腐败微生物检测中的应用,其中近红外光谱技术和高光谱成像技术等快速检测法具有样本量少、检测结果快、识别准确率较高等优点。阐述了物理杀菌法和食品防腐剂法等在腐败微生物防控中的应用,具有高效抗菌性、成本低、对现制饮料的感官品质和活性成分影响较小等优点。现制饮料行业的可持续发展离不开对腐败微生物的有效防控。本文为现制饮料腐败微生物控制和产业的可持续发展提供了理论依据和技术支撑。     

Prevalence, concentration, spoilage, and mitigation of Alicyclobacillus spp. in tropical and subtropical fruit juice concentrates

The presence of Alicyclobacillus in fruit juices and concentrates poses a serious problem for the juice industry. This study was undertaken to determine the (i) prevalence, concentration, and species of Alicyclobacillus in tropical and subtropical concentrates; (ii) efficacy of aqueous chlorine dioxide in reducing Alicyclobacillus spp. spores on tropical and subtropical fruit surfaces; and (iii) fate of and off-flavor production by Alicyclobacillus acidoterrestris in mango and pineapple juices. One hundred and eighty tropical and subtropical juice concentrates were screened for the presence and concentration of Alicyclobacillus spp. If found, the species of Alicyclobacillus was determined by 16S rDNA sequencing and analysis with NCI BLAST. Of these samples, 6.1% were positive for Alicyclobacillus, and nine A. acidoterrestris strains and two Alicyclobacillus acidocaldarius strains were identified. A five-strain cocktail of Alicyclobacillus spp. was inoculated onto the surface of fruits (grapefruit, guava, limes, mangoes, oranges and pineapple), which were then washed with 0, 50, or 100 ppm aqueous chlorine dioxide. Significant reductions due to chlorine dioxide were only seen on citrus fruits. A five-strain cocktail of A. acidoterrestris was inoculated into mango and pineapple juices. Microbial populations were enumerated over a 16-day period. Aroma compounds in the juice were analyzed by GC-olfactometry (GC-O) and confirmed using GC-MS. GC-O of mango juice identified previously reported medicinal/antiseptic compounds. GC-O of pineapple juice revealed an unexpected “cheese” off-aroma associated with 2-methylbutyric acid and 3-methylbutyric acid.     

Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: results of a 14-year survey

The purpose of this study was to determine the incidence of Alicyclobacillus spp. in fruit/vegetable juices (concentrated pulps and clarified and non-clarified juices) marketed in Argentina between 1996 and 2009.The presence of Alicyclobacillus was determined in a total of 8556 samples of fruit and vegetable juices (apple, pear, grape, peach, blend of juices, tangerine, pineapple, orange, mango, plum, guava, apricot, lemon, banana, kiwi, carrot, strawberry, grapefruit, and beetroot) collected in seven Argentinean provinces. Multiple factor analysis (MFA) was carried out on a data matrix that contained the percentage of positive samples, type of juice, raw material and production year.Except for kiwi and orange, Alicyclobacillus was found in juices from all the evaluated raw materials. The highest percentage of positive samples was found for beetroot, strawberry, banana, peach, mango, carrot and plum juices. The percentage of positive samples for these juices ranged from 100% to 24%.Furthermore, the application of multivariate techniques provided an insight on the relationship between the incidence of Alicyclobacillus and production variables. This approach enabled the identification of the most relevant variables that increased the percentage of positive samples among the juices, which could help in developing strategies to avoid the incidence of this bacterium.By means of hierarchical cluster analysis seven groups (clusters) of juices which showed different percentages of positive samples for Alicyclobacillus spp. were identified. This analysis showed that pineapple, peach, strawberry, mango and beetroot juices had higher rates of positivity for Alicyclobacillus than the rest of the evaluated juices. MFA analysis also showed that some clear relationships could be highlighted between the percentage of samples positive for Alicyclobacillus and five types of fruit juices (strawberry, beetroot, grapefruit, pineapple and mango). It was observed that a large proportion of juices produced in 2000, 2005 and 2008 were located in clusters with higher incidence of Alicyclobacillus spp., whereas a larger proportion of clarified concentrate juice and concentrate pulp samples showed higher probability of incidence of Alicyclobacillus in these products. Data presented in this study brings a contribution to the ecology of Alicyclobacillus in fruit/vegetable juices marketed in Argentina. This information would be useful to enhance the microbiological stability of fruit juices regarding the presence of Alicyclobacillus spp.     

Assessment of the response of indigenous microflora and inoculated Bacillus licheniformis endospores in reconstituted skim milk to microwave and conventional heating systems by flow cytometry

Heat treatment is one of the most widely used processing technologies in the dairy industry. Its primary purpose is to destroy microorganisms, both pathogenic and spoilage, to ensure the product is safe and has a reasonable shelf life. In this study microwave volumetric heating (MVH) was compared with a conventional tubular heat exchanger (THE), in terms of the effects of each at a range of temperatures (75°C, 85°C, 95°C, 105°C, 115°C, and 125°C) on indigenous microflora viability and the germination of inoculated Bacillus licheniformis endospores in reconstituted skim milk. To assess the heat treatment–related effects on microbial viability, classical agar-based tests were applied to obtain the counts of 4 various microbiological groups including total bacterial, thermophilic bacterial, mesophilic aerobic bacterial endospore, and thermophilic aerobic bacterial endospore counts, and additional novel insights into cell permeability and spore germination profiles post-heat treatment were obtained using real-time flow cytometry (FC) methods. No significant differences in the plate counts of the indigenous microorganisms tested, the plate counts of the inoculated B. licheniformis, or the relative percentage of germinating endospores were observed between MVH- and THE-treated samples, at equal temperatures in the range specified above, indicating that both methods inactivated inoculated endospores to a similar degree (up to 70% as measured by FC and 5 log reduction as measured by plate counting for some treatments of inoculated endospores). Furthermore, increased cell permeability of indigenous microflora was observed by FC after MVH compared with THE treatment of uninoculated skim milk, which was reflected in lower total bacterial count at a treatment temperature of 105°C. This work demonstrates the utility of FC as a rapid method for assessing cell viability and spore inactivation for postthermal processing in dairy products and overall provides evidence that MVH is at least as effective at eliminating native microflora and inoculated B. licheniformis endospores as THE.     

Discussion between alternative processing and preservation technologies and their application in beverages: A review

The alternative technologies discussed in this review are able to eliminate pathogenic bacteria while retaining bioactive compounds such as anthocyanins, phenols, pigments, and vitamins, improve the extraction processes of fruit or vegetable juices. The use of nonthermal processing technologies in beverages might provide a higher quality value to the final product and consumers tended to prefer more natural products with minimum preservative substances. This review is analyze and discuss the advantages and disadvantages of alternative technologies applied in beverages, evaluating the parameters that can optimize beverage processing such as time, temperature, pressure, frequency, tension, among others. Additionally, finding solutions to beverage processing issues will be considered analyzing the results obtained with other applied methods, and an overview of new strategies such as microfiltration or the combination of two alternative technologies to improve the quality of the final product will also be evaluated.

Practical applications

The applications of alternative technologies mentioned in this article, are a feasible option for the preservation of beverages in the area of industry. The capacity of each of them has been demonstrated, in addition to the use of microfiltration and ultrafiltration, but the combination between them sounds very encouraging to achieve the quality and safety of beverages, with an extended shelf life and greater protection of its main components. This provides a wide range of research opportunities for any type of beverage.     

Alicyclobacillus acidoterrestris in pasteurized exotic Brazilian fruit juices: Isolation,genotypic characterization and heat resistance

In this study, the population of Alicyclobacillus spp. was estimated in pasteurized exotic Brazilian fruit juices using the most probable number (MPN) technique followed by biochemical tests. Pasteurized passion fruit (n = 57) and pineapple (n = 50) juices were taken directly from Brazilian manufacturers. While Alicyclobacillus spp. was isolated from passion fruit juice, the microorganism was not found in any pineapple juice samples. A higher incidence of Alicyclobacillus was observed in samples taken in June and July (dry months in Brazil) in comparison to the other months (March, April, May and August), and the highest Alicyclobacillus counts were recovered from these samples(>23 MNP/100 mL). Sixteen (n = 16) Alicyclobacillus strains were typed using the randomly amplified polymorphic DNA method (RAPD-PCR). RAPD-PCR revealed great genetic similarity between the passion fruit juice strains and Alicyclobacillus acidoterrestris DSM 2498. The heat resistance of three isolates was determined, and the mean D_95°  (1.7 min) and z (7.6 °C) values in the passion fruit juice were not significantly different (p > 0.05) from those obtained for the DSM 2498 strain (D_95°  = 1.5 min and z = 7.1 °C). This is the first report on the isolation of A. acidoterrestris from exotic fruit juices such as passion fruit juice. It is worth pointing out the importance of applying good agricultural practices in the field and applying controls for the fruit selection and washing steps, as well as controlling the time/temperature conditions for pasteurization so as to reduce the incidence and chances of A. acidoterrestris spoilage in these juices.     

Clostridial spore germination versus bacilli: genome mining and current insights

Bacilli and clostridia share the characteristic of forming metabolically inactive endospores. Spores are highly resistant to adverse environmental conditions including heat, and their ubiquitous presence in nature makes them inevitable contaminants of foods and food ingredients. Spores can germinate under favourable conditions, and the following outgrowth can lead to food spoilage and foodborne illness. Germination of spores has been best studied in Bacillus species, but the process of spore germination is less well understood in anaerobic clostridia. This paper describes a genome mining approach focusing on the genes related to spore germination of clostridia. To this end, 12 representative sequenced Bacillus genomes and 24 Clostridium genomes were analyzed for the distribution of known and putative germination-related genes and their homologues. Overall, the number of ger operons encoding germinant receptors is lower in clostridia than in bacilli, and some Clostridium species are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli. The in silico germination model constructed for clostridia was linked to recently obtained experimental data for selected germination determinants, mainly in Clostridium perfringens. Similarities and differences between germination mechanisms of bacilli and clostridia will be discussed.     

Influence of high hydrostatic pressure processing on physicochemical characteristics of a fermented pomegranate (Punica granatum L.) beverage

The effect of high hydrostatic pressure at 500 MPa/10 min (HHP1), 550 MPa/10 min (HHP2) and 600 MPa/5 min (HHP3) on the microbiological, physicochemical, antioxidant and sensory characteristics of a fermented pomegranate (FP) beverage, stored for 42 days (4 ± 1 °C), was evaluated. The FP beverage was also pasteurized at 63 °C/30 min (VAT) and 72 °C/15 s (HTST). The high hydrostatic pressure (HHP) and VAT pasteurized beverages did not show microbial growth (<10 CFU/mL) throughout 42 days of storage. The physicochemical characteristics were not affected (p > 0.05) by HHP or pasteurization. Color of the samples showed significant differences (p ≤ 0.05) in all HHP processed and pasteurized beverages. Antioxidant activity, total phenolic compounds, flavonoids and anthocyanins increased slightly after HHP processing. Antioxidants decreased throughout the storage in all treatments. Both HHP processed and pasteurized beverages were well accepted by average consumers when evaluated using a 9-points hedonic scale.Industrial relevanceThe high hydrostatic pressure (HHP) improves the microbiological, antioxidant and sensorial stability of fermented pomegranate beverages during storage. The HHP is more common for processing fruit juices than for fermented beverages; therefore, it can be expanded to the fermented beverages industry, which could modify the today usual thermal processing methods and, or the addition of preservatives, that are not natural, for delivering high quality and healthier pomegranate fermented beverages to consumers.