Alicyclobacillus—Still Current Issues in the Beverage Industry

ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY

4

Barbara Sokołowska, Marzena Połaska, Agnieszka Dekowska Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology, Department of Fruit and Vegetable Product Technology, Warsaw, Poland

4.1 Introduction The presence of Alicyclobacillus, a thermoacidophilic and spore-forming bacteria, in acidic fruit juices poses a serious problem for the beverage and juice industry. Several species of Alicyclobacillus, especially A. acidoterrestris, have been identified as spoilage organisms in commercially pasteurized fruit juices and beverage. Spoilage manifests itself as the formation of off-odors caused by compounds such as guaiacol and halophenols (Orr et al., 2000; Jensen and Whitfield, 2003; Gocmen et al., 2005; Witthuhn et al., 2012, 2013) and a “cheese-like” off-aroma identified as 3-methylbutyric acid and 2-methylbutyric acid (Danyluk et al., 2011; Sokołowska et al., 2013b). The economic impact of such incidents can be very high. Alicyclobacillus spp. can be difficult to control in fruit juice products as their spores survive juice pasteurization temperatures and may subsequently germinate and grow after processing if conditions are suitable. A. acidoterrestris strains show the ability to germinate and grow at a pH range of from 2.0 to 6.0 at a temperature of 20–55°C. Two main factors preventing beverages from spoilage with most of the other bacteria, thermal treatment, and low pH values are not sufficient to eliminate Alicyclobacillus spp. (Splittstoesser et  al., 1994; Komitopoulou et al., 1999; Silva et al., 1999; Bahçeci and Acar, 2007; Sokołowska et al., 2008; Maldonado et al., 2008; Huertas et al., 2014). Due to the number of spoilage episodes and incidences, A. acidoterrestris is recognized as being the most important species and suggested as target of pasteurization in fruit juices and concentrates (Silva et al., 2000; Vieira et al., 2002). Safety Issues in Beverage Production. https://doi.org/10.1016/B978-0-12-816679-6.00004-8 © 2020 Elsevier Inc. All rights reserved.

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106  Chapter 4  ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY

During the last four decades since the first species of Alicyclobacillus was isolated from spoilage apple juice in 1982, 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 (Tianli et al., 2014). One of the unconventional methods used to inactivate Alicyclobacillus spores and vegetative cells is high hydrostatic pressure (HHP) pasteurization. Several reports have indicated the usefulness of this treatment as a method for controlling A. acidoterrestris (Vercammen et al., 2012; Silva et al., 2012; Skąpska et al., 2012; Sokołowska et al., 2013a). In order to enhance the bactericidal effect of HHP, it is combined with other factors such as high temperature or antimicrobial agents, mainly nisin and lysozyme, well-known natural food preservatives (Sokołowska et al., 2012). The wide spectrum of chemical, physical, and combined methods developed in the last decade against A. acidoterrestris was reported. For example, treatment by high-pressure carbon dioxide (Bae et  al., 2009; Porębska et al., 2016, 2017), ozon (Torlak, 2014a), and new disinfectant (Osopale et al., 2017). Also such methods as high-pressure homogenization (Roig-Sagués et al., 2015), microwaves (Giuliani et al., 2009), ohmic heating (Kim et  al., 2017b), UV-C light (Baysal et  al., 2013; Tremarin et  al., 2017), and application of neutral electrolyzed water (Torlak, 2014b) were used. Several researches have considered application of essential oils or their active constituents as natural anti-alicyclobacilli preservation agents (Bevilacqua et al., 2010; Maldonado et al., 2013; Huertas et  al., 2014). Apart from essential oils, plant extracts (from lemon, neroli, bicitro, grape seed, and rosemary) were tested for their antimicrobial potential as well (Bevilacqua et al., 2013; Molva and Baysal, 2015; Piskernik et al., 2016). The recent study (Dos Anjos et al., 2016) showed that the enzymes papain and bromelain have an antibacterial effect on A. Acidoterrestris alone and when combined with nisin. Alicyclobacillus have been isolated from orchard soils, fruits, the production environment and many final products—juices, juice concentrates, and beverage all over the world (Cerny et al., 1984; Baumgart et al., 1997; Eguchi et al., 2001; Parish and Goodrich, 2005; Witthuhn et al., 2006; Goto et al., 2008; Groenewald et al., 2008; Wang et al., 2010; Steyn et al., 2011; Zhang et al., 2013; Danyluk et al., 2011; Durak et al., 2010; McKnight et al., 2010; Oteiza et al., 2011; Sokołowska et al., 2016). The spores of Alicyclobacillus spp. survive for long periods in fruit concentrates and similar environments; however, more dilute environments are required for growth. However, the presence of these bacteria in juice does not always result in product spoilage. The behavior (growth, survival or inactivation) of Alicyclobacillus species is greatly affected by juice type (Splittstoesser et  al., 1994; Goto, 2007; Sokołowska et al., 2016)

Chapter 4  ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY   107

The International Federation of Fruit Juice Producers (IFU) undertook to develop internationally acceptable methods for the detection of Alicyclobacillus spp. and has subsequently published as general method for detection of these organisms (Massaguer et al., 2002). This method is widely used in Europe. The filtration method, in comparison to the enrichment method, increased the likelihood of detecting Alicyclobacillus contamination of fruit juice concentrates containing inhibitory compounds (McNamara et  al., 2011; Sokołowska and Niezgoda, 2017). Various methods have been developed for the differentiation and identification of Alicyclobacillus. The traditional methods used in the detection/enumeration of microorganisms involve their growth on an appropriate medium as well as visual detection. These methods are slow and require from several days to 2 weeks to obtain results. The juice industry needs rapid methods to appreciate microbiological quality and control the process. During the development and application of new methods, the most important criteria are limited analysis time and sensitivity.

4.2  Characteristics of Alicyclobacillus 4.2.1 History Most fruit juices and beverages exhibit acid pH, which is a natural control measure against spoilage. There are several species of bacteria capable of spoilage of acid food products. Some of the above mentioned species belong to the genus Alicyclobacillus. The first instance of isolation of thermoacidophilic bacteria from hot springs in the Tohoku district in Japan was reported by Uchino and Doi (1967). Endospores of these bacteria were shaped differently, and bacteria exhibited the ability to grow in more acidic and aerobic conditions than the better known at that time two thermophlic species Bacillus coagulans and Bacillus stearothermophilus. On the basis of apparent differences, and based on the morphological and cultural characteristics, the isolated bacteria were identified as a new strain of Bacillus coagulans. Similar thermoacidophilic species were isolated in 1971 from thermal acidic environments in the United States (Yellowstone National Park and Volcano National Park in Hawaii) by Darland and Brock (1971). Due to the different pH optimum, and different DNA base composition than the previously isolated by Uchino and Doi B. coagulans, a new Bacillus acidocaldarius species was created (Darland and Brock, 1971). An important feature of the newly discovered species was the composition of the lipids membrane containing ω-cyclohexane fatty acids as the major components

108  Chapter 4  ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY

of cell membrane (Darland and Brock, 1971). Hippchen et al., in 1981 isolated from soils some species very similar to B. acidocaldarius also containing similar properties of fatty acids in the cell membrane. The new species named Bacillus cycloheptanicus was discovered in 1983 by Poralla and König (1983). This strain possessed primarily ω-cycloheptane fatty acids in its cell membrane (Poralla and König, 1983). The first case of spoilage of pasteurized apple juice on a large scale, caused bacteria closely related to B. acidocaldarius, took place in Germany in 1984 (Cerny et al., 1984). This bacterium was classified as a new species, Bacillus acidoterrestris. Based on the analysis of 16 S rRNA sequences and due to the different composition of the cell membrane the new genus Alicyclobacillus was introduced into the bacterial nomenclature in 1992 (Wisotzkey et  al., 1992). Then researchers renamed Bacillus acidocaldarius, B. acidoterrestris, and Bacillus cycloheptanicus as Alicyclobacillus acidocaldarius, A. acidoterrestris, and Alicyclobacillus cycloheptanicus. The name Alicyclobacillus comes from the presence of specific ω-alicyclic fatty acids (ω-cyclohexane or ω-cycloheptane fatty acids) in their cell membrane (Wisotzkey et  al., 1992). Further studies of Goto and coworkers have shown that relatively few species such as: Alicyclobacillus pomorum, Alicyclobacillus aeris, Alicyclobacillus ferrooxydans, Alicyclobacillus macrosporangiidus, Alicyclobacillus contaminans do not contain ω-alicyclic fatty acids in the cell membrane. However phylogenetic analysis of 16S rRNA sequence and gyrB gene sequences recommended to classify these species to genus Alicyclobacillus (Goto et al., 2003). Nowadays genus Alicyclobacillus comprises of 25 species: Alicyclobacillus acidocaldarius, Alicyclobacillus acidophilus, Alicyclobacillus acidoterrestrs, Alicyclobacillus aeris, Alicyclobacillus cellulosilyticus, Alicyclobacillus consociatus, Alicyclobacillus contaminans, Alicyclobacillus cycloheptanicus, Alicyclobacillus daucy, Alicyclobacillus disulfidooxidans, Alicyclobacillus fodiniaquatilis, Alicyclobacillus fastidiosus, Alicyclobacillus ferrooxydans,Alicyclobacillus herbarius, Alicyclobacillus hesperidum, Alicyclobacillus kakegawensis, Alicyclobacillus macrosporangiidus, Alicyclobacillus pohliae, Alicyclobacillus pomorum, Alicyclobacillus sacchari, Alicyclobacillus sendaiensis, Alicyclobacillus shizuokensis, Alicyclobacillus tengchongensis, Alicyclobacillus tolerans, Alicyclobacillus vulcanalis (Deinhard et al., 1987a,b; Wisotzkey et al., 1992; Albuquerque et al., 2000; Goto et al., 2003, 2007; Tsuruoka et al., 2003; Simbahan et  al., 2004; Karavaiko et  al., 2005; Jiang et  al., 2008; Imperio et al., 2008; Guo et al., 2009; Kim et al., 2014; Masataka et al., 2014; Nakano et  al., 2015; Zhang et  al., 2015). Some important characteristics of all species of the genus Alicyclobacillus are described in Table 4.1.

Table 4.1  Cultural, Morphological and Colony Characteristics of Alicyclobacillus spp Alicyclobacillus Species

pH Range (Optimum)

Temperature Range (°C) (optimum)

Alicyclobacillus acidocaldarius

2.00–6.00 (3.5–4.00)

45–71 (53–65)

Aerobic

+ to variable

Alicyclobacillus acidocaldarius subsp. rittmannii

2.50–5.00 (4.00)

45–70 (63)

Aerobic

Alicyclobacillus acidophilus

2.50–5.50 (3.00)

20–55 (50)

Alicyclobacillus acidoterrestris

2.50–5.80 (4.50–5.00)

Alicyclobacillus aeris

Oxygen Requirement

Cell Size (Length Width mm)

Size (Diameter mm)

Endospore Characteristics

Color

Shape

1.5– 3.0 × 0.5– 0.8

Oval or ellipsoidal,

Unpigmented, cream yellow

+

2.0– 4.0 × 0.5– 2.0

Central to terminal

Cream, opaque

Circular, flat, or convex, smooth, irregular margins Convex, circular, entire margins

Aerobic

+

0.9–1.1 ×  4.8–6.3

Creamy white, opaque

Round, smooth

1.1–3.8

20–70 (36–53)

Aerobic

+ to variable

2.9–4.3 ×  0.6–0.8

Ellipsoidal to oval, terminal to subterminal Oval, 1.5–1.8 0.9–1.0 mm, terminal, subterminal and central

Creamy white to yellowish, translucent to opaque

Round

3.0–5.0

Soil/apple juice

2.0–6.0 (3.5)

25–35 (30)

Aerobic

+ to variable

1.5–2.5 ×  0.4–0.66

nr

Creamy white

Circular

0.5–1.0

Alicyclobacillus cellulosilyticus

3.5–6.5 (4.8)

40.0–67.5 (55)

Aerobic

–

2.0–6.0 ×  0.5–0.86

nr

White

nr

0.5–1.0

Alicyclobacillus consociatus

5.5–10.5 (6.5)

15–45 (30)

Aerobic

+

Terminal

Beige

Circular, convex

nr

Alicyclobacillus contaminans

3.50–5.50 (4.00–4.50)

35–60 (50–55)

Aerobic

+ to variable

2.0– 5.0 × 0.8– 1.0 4.0– 5.0 × 0.8– 0.9

Solfataric copper mine drainage Steamed Japanese cedar chips from a lumbermill Blood sample

Ellipsoidal, subterminal

Non-pigmented (creamy white), opaque

Circular, entire, umbonate

3.0–5.0

Gram Stain

Source

References

1.0–2.0

Thermal acid waters

0.8–1.0

Geothermal soil of Mount Rittmann, Antarctica Acidic beverage

Uchino and Doi (1967), Darland and Brock (1971), Wisotzkey et al. (1992) Nicolaus et al. (1998)

Soil from crop fields in Fuji city

Matsubara et al. (2002) Hippchen et al. (1981), Deinhard et al. (1987a), Wisotzkey et al. (1992) Walls and Chuyate (1998) Guo et al. (2009)

Masataka et al. (2014), Kusube et al. (2014)

Glaeser et al. (2013) Goto et al. (2007)

Continued

Table 4.1  Cultural, Morphological and Colony Characteristics of Alicyclobacillus spp—cont’d Alicyclobacillus Species

pH Range (Optimum)

Temperature Range (°C) (Optimum)

Alicyclobacillus cycloheptanicus

3.00–5.50 (3.50–4.50)

Alicyclobacills dauci

Cell Size (LENGTH width mm)

Oxygen Requirement

Gram Stain

40–53 (48)

Aerobic

+

2.5–4.5 ×  0.35–0.55

Oval, subterminal

3.0–6.0 (4.0)

20–50 (40)

Aerobic

+ to variable

2–5 × 0.5– 0.7

nr

Alicyclobacillus disulffidooxidans Alicyclobacillus fadiniaquatilis

0.50–6.00 (1.50–2.50) 2.5–5.5 (3.5)

4–40 (35) 20–45 (40)

Aerobic

+ to variable + to variable

Alicyclobacillus fastidiosus

2.50–5.00 (4.00–4.50)

20–55 (40–45)

Aerobic

+ to variable

0.9–3.6 0.3–0.5 1.5– 5.0 × 0.5– 0.8 4.0–5.0 ×  0.9–1.0

Alicyclobacillus ferrooxydans

2.00–6.00 (3.00)

17–40 (28)

Aerobic

+

1.0–1.5 0.4–0.6

nr

Alicyclobacillus herbarius Alicyclobacillus hesperidum

3.50–6.00 (4.50–5.00) 3.50–4.00

35–65 55–60 35–60 (50–53)

Aerobic

+

nr

Oval, subterminal

Aerobic

+

2.1–3.9 0.5–0.7

Terminal

Alicyclobacillus kakegawensis

4.00–4.50

40–60 (50–55)

Aerobic

+ to variable

4.0–5.0 0.6–0.7

Oval, subterminal

Aerobic

Endospore Characteristics

Size (Diameter mm)

Source

Reference

Color

Shape

Creamy white, opaque White, opalescent

Round, small, smooth Circular, opaque

nr

Soil

Poralla and König (1983), Wisotzkey et al. (1992)

1.0–1.5

Nakano et al. (2015)

Oval, subterminal or terminal nr

nr

nr

nr

Light yellow

Circular and flat

1–2

Spoiled mixed vegetable and fruit juice product Waste water sludge Acid mine water

Ellipsoidal, subterminal

Nonpigmented (creamy white), opaque Not pigmented

Circular, entire, flat

3.0–4.0

Apple juice

Goto et al. (2007)

Pinpoint, circular, entire Circular

0.3–0.5

Solfataric soil

Jiang et al. (2008)

2.0–3.0

Herbal tea

Goto et al. (2002a,b)

nr

1.0–2.0

Albuquerque et al. (2000)

Circular, entire, flat

2.0–3.0

Solfataric soils of São Miguel, Azores Soil from crop fields in Kakegawa

Not pigmented Not pigmented

Nonpigmented (creamy white)

Dufresne et al. (1996), Karavaiko et al. (2005) Zhang et al. (2015)

Goto et al. (2007)

Alicyclobacillus macrosporangiidus Alicyclobacillus pohliae

3.50–6.00 (4.00–4.50)

35–60 (50–55)

Aerobic

+ to variable

5.0–6.0 0.7–0.8

Oval, terminal

4.50–7.50 (5.50)

42–60 (55)

Aerobic, facultatively anaerobic

+

1.5–2.5 0.4–0.6

Alicyclobacillus pomorum Alicyclobacillus sacchari

3.00–6.00 (4.00–4.50) 2.50–5.50 (4.00–4.50)

30–60 (45–50) 30–55 (45–50)

Aerobic

+ to variable + to variable

2.0–4.0 0.8–1.0 4.0–5.0 0.6–0.7

Alicyclobacillus sendaiensis Alicyclobacillus shizuokensis

2.50–6.50 (5.50) 3.50–6.00 (4.00–4.50)

40–65 (55) 35–60 (45–50)

Aerobic

–

Aerobic

Alicyclobacillus tengchongensis

2.0–6.0 (3.2)

30–50 (45)

Alicyclobacillus tolerans Alicyclobacillus vulcanalis

1.50–5.00 (2.50–2.70) 2.00–6.00 (4.00)

<20–55 (37–42) 35–65 (55)

NR, not reported.

Round, terminal

Non-pigmented, (creamy white), opaque Cream colored

Circular, entire, convex Entire, convex

Oval, subterminal

Not pigmented

Circular

3.0–4.0

Ellipsoidal, subterminal

Non-pigmented (creamy, white)

3.0–5.0

Round or ellipsoidal, terminal Oval, subterminal

Soil, Japan

1.0–2.0

Aerobic

+

nr

nr

White and semi-transparent Nonpigmented (creamy, white) opaque nr

1.0

+ to variable

2.0–3.0 0.8 4.0–5.0 0.7–0.8

Circular, entire, umbonate Circular, convex Circular, entire, convex nr

Soil from crop fields in Fujieda city Geothermal soil of Mount Melbourne, Antarctica Mixed fruit juice Liquid sugar

Aerobic

+

nr

0.3–0.5

+

Oval, terminal or subterminal Terminal

nr

Aerobic

3.0–6.0 0.9–1.0 1.5–2.5 0.4–0.7

Semitransparent to white

Convex

1.0

Soil from crop fields in Shizuoka city Soil of a hot spring at Tengchong in China Oxidizable lead zinc ores Geothermal pool, Coso hot springs, California

Aerobic

2.0–4.0

1.5–2.0

nr

Goto et al. (2007)

Imperio et al. (2008)

Goto et al. (2003) Goto et al. (2007)

Tsuruoka et al. (2003) Goto et al. (2007)

Kim et al. (2014)

Karavaiko et al. (2005) Simbahan et al. (2004)

112  Chapter 4  ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY

4.2.2  General Characteristics of Alicyclobacillus Alicyclobacillus species consists of a group of rod shaped, Grampositive with a tendency to be Gram- variable in old cultures, nonpathogenic, strictly areobic, spore forming, and soil-borne bacteria (Goto et al., 2003, 2008). Alicyclobacillus spores can be terminal, subterminal, or central with or without swollen sporangium. There are two species A. sendaiensis and A. cellulosilyticus which are dying gram-negative (Tsuruoka et  al., 2003; Steyn et  al., 2011; Masataka et al., 2014). Most species are strictly aerobic, however A. pholiae is sometimes facultatively anaerobic. Bacteria belonging to the Alicyclobacillus genus show the ability to grow at high temperatures 20–70°C, with an optimum range from 35°C to 65°C (Walker and Philips, 2008; Smit et al., 2011). Only one species A. disulfidooxidans exhibits the ability to germinate at an extremely low temperature 4°C, and the other two exceptions: A. ferrooxydans and A. tolerans, are able to grow at temperature below 20°C (Karavaiko et al., 2005; Jiang et al., 2008; Tianli et al., 2014). Highly acidic environments with pH range 2.0–6.0, with optimum pH 3.0–5.0 are also very important for Alicyclobacillus growth (Tianli et al., 2014; Tyfa et al., 2015). There are only two species: A. tolerans and A. disulfidooxidans, which can survive in the very low 1.5–2.0 pH range, and only two species: A. contaminas and A. pohliae, which can grow at a high pH 10.5 and 7.5, respectively (Dufresne et  al., 1996; Karavaiko et al., 2005; Goto et al., 2007; Imperio et al., 2008). Most of Alicyclobacillus strains tolerate less than 5% NaCl (Tianli et al., 2014), the exceptions are A. macrosporangiidus and A. shizuokensis which show a higher tolerance for salt, and can grow at 7% NaCl (Goto et al., 2007). In 1984 Kannenberg et  al., carried out research on the role of ω-­ cyclic fatty acids in heat and acid resistance. The conclusions of their research indicate that the presence of ω-cyclohexane fatty acids reduced permeability, increased packing of the lipids in the membrane core, and led to the stabilization of the membrane structure and protection against high temperatures and high acid conditions (Kannenberg et al., 1984; Chang and Kang, 2004; Ciuffreda et al., 2015). No ω-cyclic fatty acids were detected for seven species, including A. consociatus, A. contaminans A. ferrooxydans, A. macrosporangiidus, A. pohliae, and A. pomorum, A. aeris (Goto et al., 2003, 2007; Guo et al., 2009; Jiang et al., 2008).

4.2.2.1  Off-Flavor Production Another noteworthy feature of the Alicyclobacillus species is the ability to produce guaiacol and some halophenoles which cause spoilage of fruit products (Jensen, 1999). The data about aromatic

Chapter 4  ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY   113

compounds produced by A. acidoterrestris in spoilage fruit juice are summarized in Table 4.2. Spoilage is difficult to detect visually, because there are no changes in the appearance of the product, no gas is produced, and sometimes only light sediment may appear (Gocmen et al., 2005). The only evidence of the presence of Alicyclobacillus in the given product is phenolic, antiseptic off-flavor identified as guaiacol (2-methoxyphenol) or halophenoles (2,6-dibromophenol and 2,6-dichlorophenol) (Bahçeci et al., 2005; Cai et al., 2015). Guaiacol is formed in non-oxidative decarboxylation of vanilic acid catalyzed by the vanilic acid decarboxylase enzyme system (Bahçeci et al., 2005; Osopale et al., 2017). The conversion of vanillic acid to guaiacol is more rapid than that of vanillin (Molva and Baysal, 2016). Formation of guaiacol by Alicyclobacillus depends on several factors: the strain and species of Alicyclobacillus, the concentration and presence of vegetative cell or spores, temperature, oxygen, and nutrients availability (Yamazaki et  al., 1997a; Sokołowska et al., 2013b; Molva and Baysal, 2016). According to Bahçeci et al. (2005) a concentration of 104 cfu/mL of vegetative cells of Alicyclobacillus is necessary for producing sensory detectable amounts of guaiacol. Bahçeci et  al. (2005) inoculated apple juice with 103 cfu/mL of A. acidoterrestris strain, and observed that guaiacol production started after 30 h, when the bacterial concentration of 104 cfu/mL had been reached. However, Alicyclobacillus inoculation at the level of 105 cfu/mL resulted in the immediate production of guaiacol. The highest concentration of guaiacol production was reported after 75 h of incubation at a temperature of 46°C, and for the same inoculum stored at 25°C production of guaiacol was not observed (Bahçeci et al., 2005). Spoilage potential is also limited by the amount of oxygen that is available in the growth medium, which is necessary for Alicyclobacillus growth (Siegmund and Pollinger-Zierler, 2007). Most commonly isolated from the pasteurized beverages spoilage species is A. acidoterrestris. Aerobic bacilli of A. acidoterrestris are 2.9 do 4.3 μm long, with terminal or subterminal endospores. Growth of A. acidoterrestris is possible at a temperature between 20°C and 60°C, with the temperature optimum in the range of 40–53°C. Growth of A. acidoterrestris is observed in pH 2.5–6.0 with optimum pH 3.5–4.5. Potential for fruit juices spoilage has been attributed also to several other species such as A. acidiphillus, A. daucy, A. herbarius, A. cycloheptanicus, A. pomorum, A. contaminans, and some strains of A. hesperidum (Wisotzkey et al., 1992; Albuquerque et al., 2000; Goto et al., 2003, 2007; Nakano et al., 2015).

4.2.2.2  Heat Resistance of Alicyclobacillus Spores A serious and still unsolved problem in the food industry created by Alicyclobacillus species is their ability to produce heat resistant endospores, surviving the typical pasteurization process (90–95°C

Table 4.2  Aromatic Compounds Produced by Alicyclobacillus acidoterrestris in Spoilage Fruit Juice Kind of Juice Orange juice

Apple juice

Fruit drink Ice tea Spoilage mix fruit juice

Incubation [°C/ Days]

Population [cfu/ mL]

Odor-Active Compound—Amount

25°C/6 days 35°C/6 days 44°C/6 days 25°C/10 days 35°C/6 days 44°C/6 days 35°C/3 days 44°C/3 days –

6.0 × 105 1.0 × 106 6.0 × 106 2.0 × 107 3.0 × 106 2.0 × 107 1.0 × 105 1.0 × 106 4.1 × 105

Guaiacol—14,1 μg/L Guaiacol—13.3 μg/L Guaiacol—1.2 μg/L Guaiacol—11.6 μg/L Guaiacol—17.3 μg/L Guaiacol—33.7 μg/L Guaiacol—32.3 μg/L Guaiacol—100.8 μg/L 2,6-dibrmophenol—5 ng/L

–

3.0 × 101 8.0 × 101 3.0 × 101 8.0 × 101 1.0 × 105

2,6-dibrmophenol 2–4 ng/L 2,6-dichlorophenol 16–20 ng/L 2,6-dibrmophenol—20 ng/L 2,6dichlorophenol—20 ng/L Guaiacol—8.1 μg/L Guaiacol—11.4 μg/L nd Guaiacol—0.68 μg/L Guaiacol—1.08 μg/L Guaiacol—3.98 μg/L Guaiacol—4.05 μg/L Guaiacol—4.87 μg/L Guaiacol—0.68 μg/L Guaiacol—1.36 μg/L Guaiacol—2.40 μg/L Guaiacol—2.98 μg/L Guaiacol—4.09 μg/L Guaiacol—5.76 μg/L 2,6-dibrmophenol— 0.46 μg/L 2,6-dibrmophenol— 0.86 μg/L 2,6-dibrmophenol— 0.84 μg/L 2,6-dibrmophenol— 1.05 μg/L

– Mix fruit juice

1 day

Apple juice

21°C/8 days 37°C/8 days 21.5°C/5 days 21.5°C/10 days 21.5°C/15 days 21.5°C/20 days 21.5°C/25 days 21.5°C/30 days 30°C/5 days 30°C/10 days 30°C/15 days 30°C/20 days 30°C/25 days 30°C/30 days 21.5°C/25 days 21.5°C/30 days 30°C/25 days 30°C/30 days

Apple juice

ND, not detected.

7.5 × 102 1.8 × 102 –

References Pettipher et al. (1997)

Baumgart et al. (1997) Jensen and Whitfield (2003)

Orr et al. (2000) Siegmund and Pollinger-Zierler (2007)

Chapter 4  ALICYCLOBACILLUS—STILL CURRENT ISSUES IN THE BEVERAGE INDUSTRY   115

for 30–60 s) (Cerny et  al., 1984). The thermal resistance of bacterial spores is influenced by several environmental factors like pH or soluble solids content of the medium (Yamazaki et al., 1997a; Silva et al., 1999, 2015). Numerous studies can be found that report thermal inactivation parameters of Alicyclobacillus, that is, the D value (time at a determined temperature required to cause one-log cycle decrease in the population of a target bacterium) and the z value (temperature increase required to result in one-log cycle decrease of D value) (Table  4.3). D- and z-values of Alicyclobacillus are affected by the particular conditions or study characteristics (protocols, kind of fruit juice, Brix, pH, temperature, culture medium, inactivation method, bacterial strain, etc.) under which they were obtained. Therefore, variability in D- and z-values among primary studies is expected to occur, even among studies investigating the same type of fruit beverage. The heat resistance of spores is also influenced by dipicolinic acid (DPA) content, presence of heat stable proteins, and degree of mineralization. Yamazaki et al. (1997a) observed that under low pH conditions, spores of A. acidoterrestris have stronger binding characteristics of Ca2+ and Mn2+ than that of other tested Bacillus species. Remineralizing of spores with divalent cations, such as manganese or calcium, can increase heat resistance of the demineralized spores.

4.3  Occurrence of Alicyclobacillus in the Soil and Production Environment Most of the known species of Alicyclobacillus were isolated first from samples of soil. Soil contamination of fruits and the production line surface is probably the main reason of Alicyclobacillus presence in the final commercial fruit products (Chang and Kang, 2004). Parish and Goodrich (2005) have confirmed that soil was a primary source of Alicyclobacillus spores. They observed that contamination rate was lower at the facility which did not processed oranges picked up from the ground. The area of Alicyclobacillus occurrence is wide. The presence of these bacteria has been reported in South Africa, Japan, China, Germany, Poland, United States, Argentina, Australia, and even Antarctica. Alicyclobacillus is widespread in orchards, and has been isolated from single-strength and concentrated fruit juices at vari­ ous stages of production (Cerny et  al., 1984; Albuquerque et  al., 2000; Goto et  al., 2003; Simbahan et  al., 2004; Gouws et  al., 2005; Imperio et  al., 2008; Sokołowska et  al., 2010, 2013b; Sokołowska, 2014; Steyn et  al., 2011; Masataka et  al., 2014; Tayefe et  al., 2014).

Table 4.3  Heat Resistance of Alicyclobacillus acidoterrestris Spores in Various Fruit Juices Kind of Juice

pH

Soluble Solid Content [%]

Apple juice

3.5

11.4

Grape juice

3.3

15.8

Orange drink Apple ice tea Vitamin and fruity nectar Orange juice

4.1 3.5 3.5 –

5.3 4.8 6.1 –

Cupuaçu extract

3.6

11.3

Orange juice

3.5

11.7

Blackcurrant juice concentrate

2.5

Apple juice

3.5

26.1 58.5 –

Orange juice

3.9

–

Grapefruit juice

3.4

–

Apple juice

3.68

12.2

Apple nectar

2.97

14.0

Apple nectar with ascorbic acid—250 mg/dm3

2.95

14.0

T [°C]

D [min]

z [°C]

References

85 95 85 95 95 95 95 85 90 95 85 97 85 91 91 91 80 95 80 95 80 90 95 90 96 100 90 96 100 90 96 100

56 2.8 57 2.4 5.3 5.2 5.1 60.8–94.5 10.0–20.6 2.5–8.7 17.5 0.57 65.6 11.9 3.84 24.1 41.2 2.30 54.3 3.59 37.9 5.95 1.85 11.1 2.1 0.7 14.4 3.3 1.2 14.1 3.1 1.0

7.7

Splitstoesser et al. (1994)

7.2 9.5 10.8 9.6 7.2–11.3

Baumgart et al. (1997)

9.0

Silva et al. (1999)

Eiroa et al. (1999)

7.8 – 12.2

Komitopolou et al. (1999)

12.9 11.6

8.5

9.2

8.8

Bahceci and Acar (2007)

Mango pulp

4.0

–

Lemon juice concentrate

2.28–4.0 2.28–4.0

50.0 (clarified) 68.0 (nonclarified)

Apple juice

3.40

11.2

Apple juice concentrate

3.12

70.0

Orange juice concentrate

3.68

64

Passion fruit juice Tangerine juice

– 3.50

–

Tomato juice

4.4

12.0

Orange juice

–

11.0

Orange juice concentrate

3.57

66.5

80 85 90 95 82 86 92 95 82 86 92 95 90 95 90 95 92 95 98 102 95 90 95 100 105 85 90 95 80 87 95 99 80 87 95 99

40.0 25.0 11.7 8.33 17.36–33.66 18.06–68.95 7.60–23.19 6.2–12.63 15.50–50.50 14.54–95.15 8.81–85.29 8.55–23.33 20.0–47.8 3.4–15.1 15.8–161.3 5.3–29.8 25.6 12.9 6.16 2.01 1.7 15.0 6.20 2.10 0.63 40.65 9.47 1.5 16.25 12.5 10.8 1.38 18.4 13.4 10.6 1.67

21.27

De Carvalho et al. (2008)

–

Maldonado et al. (2008)

5.4–12.8

Sokołowska et al. (2008)

4.9–14.2 9.1

Peña et al. (2009)

7.6 10.8

McKnight et al. (2010) López et al. (2011)

7.0

Bevilacqua and Corbo (2011)

13.6

Alberice et al. (2012)

14.9

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The main species identified in Japanese orchards were A. acidoterrestris and A. contaminans, and slightly less common was A. acidocaldarius. Results of the study of Goto et  al. (2006, 2007, 2008) suggest that most of the previously known Alicyclobacillus species (except for A. disulfidooxidans, A. tolerans, and A. vulcanalis and recently discovered in China A. aeris, A. fadiniaquatilis, and A. tengchongensis) can be found in Japan. Similar results were obtained by Groenewald et  al. (2008) in samples of soil, in the factory environment and in the fruit concentrate from an accredited fruit processing facility in the Western Cape region of South Africa. Researchers isolated two species: A. acidoterrestris and A. acidocaldarius from soil and A. acidoterrestris from water which was used in the facility to wash fruits prior to the pulping (Groenewald et al., 2008). Alicyclobacillus species were detected in many fruits juices and beverages; however, the area of occurrence of this bacterium is much wider. Genus of Alicyclobacillus also has been isolated from organic compost, manure, water, tea, flowers, and even from flavorings. (Groenewald et al., 2008; Zhang et al., 2013; Oteiza et al., 2014). Oteiza et al. (2014) has isolated six strains of A. acidoterrestris from apple and pear flavorings and five of these strains were able to produce guaiacol. Survey conducted by the European Fruit Juice Association in 2005 presents the enormous scale of spoilage incidents in the fruit industry. Of the surveyed producers, 45% were affected by spoilage problem caused by thermoacidophilic, endospore forming Alicyclobacillus, in the 3 years preceding the survey and 33% of them experienced spoilage caused by presence of the Alicyclobacillus in the product more than three times. The species most frequently isolated from the production environment were A. acidoterrestris, and A. acidocaldarius (Groenewald et al., 2008; Sokołowska et al., 2016). In 2002–2015 Sokołowska et al., investigated 1164 samples of apple juice concentrate and 146 samples of dark berry juice concentrates, including 48 samples of cherry, 24 of strawberry, 8 of raspberry, 38 of blackcurrant, and 28 of chokeberry juice for the presence of Alicyclobacillus sp. In apple juice concentrates the level of Alicyclobacillus contamination ranged between 27.3% to 86.8%, depending on the year; A. acidoterrestris consisted of 12.7% to 100% of the isolated species. The samples of dark berry juice concentrates were also heavily contaminated. The percent of Alicyclobacillus positive samples ranged from 57.1% (chokeberry juice concentrates) to 63.1% (blackcurrant juice concentrates). A. acidoterrestris ranged from 4.2% in blackcurrant juices, up to 40.0% in raspberry juices, of all the Alicyclobacillus strains isolated from these juices. All tested samples were delivered by a limited number of manufactures, mainly due to suspected problem with microbiological contamination, and results are not representative of the quality of the juices produced across Poland (Sokołowska et al., 2016).

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In 1996–1997 the Brazilian Association for Citrus Exporters (ABECitrus) also selected three orange processing factories in Sao Paulo State to check them for Alicyclobacillus sp occurrence. Scientists collected the raw material of fruit juice (harvested fruits, fruits from the ground, and fruit before and after storage in silos), soil from orange farms around factories, and sprinkler water in orchards. Both in the soil samples, in the fruit surface, and in the water, species of Alicyclobacillus were present (de Cássia Martins Salomão et al., 2014). The highest level 104–105 cfu/g of Alicyclobacillus contamination was recorded in tested soil samples. Much lower amounts were found on the surface of fruits and in the water. The level of contamination of fruits depends on the season. In the dry season spores of Alicyclobacillus from the ground are transferred to fruit surfaces by wind. In the rainy season spores adhered to fruit surface are washed away by rain (de Cássia Martins Salomão et al., 2014). Chen et al. (2006) also isolated Alicyclobacillus strains from a concentrated apple juice-processing environment. Alicyclobacillus was present in wash water, distilled water, and apple juice concentrate.

4.4  The Ability of A. acidoterrestris to Spoil of Different Juices and Beverages A. acidoterrestris has been isolated from many juices and concentrates including apple, orange, pear, cherry, grapefruit, mango, tomato, white grape, aloe vera, pineapple, lemon, passion fruit, coconut cream, blueberry, pomegranate, strawberry, blackcurrant, chokeberry, raspberry, watermelon, tomato, and banana (Baumgart and Menje, 2000; Eguchi et  al., 2001; Witthuhn et  al., 2006; Sokołowska, 2009; McKnight et  al., 2010; Danyluk et  al., 2011; Oteiza et  al., 2011; Sokołowska et al., 2016) and kiwi (Zhang et al., 2013). These bacteria were also isolated from various types of acidic beverages (Yamazaki et  al., 1996b; Sokołowska, 2009), including ice tea (Baumgart et  al., 1997; Duong and Jensen, 2000) and also from beverage ingredients such as sugar (Durak et al., 2010). However, the presence of these bacteria in juice does not always result in product spoilage. The behavior (growth, survival or inactivation) of Alicyclobacillus species is greatly affected by juice type. Splittstoesser et  al. (1994) have investigated the growth of heat-activated spores of two A. acidoterrestris strains, in different types of fruit juices. The inoculated strains grew very well in apple, tomato, and white grape juices. Very strong growth inhibition was observed in red grape juice, prune juice, cranberry cocktail, and mixed fruit juices: apple/grape/cherry, apple/raspberry/grape, apple/red grape. A similar study was carried out by Walls and Chuyate

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(2000). The growth of A. acidoterrestris was observed in apple, orange, pear, white grape, tomato, and grapefruit, but not in pineapple and apple-cranberry juices. Also Goto (2007), tested the growth behavior of several strains of A. acidoterrestris in a variety of fruit juices, and concluded that the behavior of the strains depended on the type of juice and also on the source of isolation of the strains. A recent study showed the ability of A. acidoterrestris to grow in mango and pineapple juice (Danyluk et al., 2011). Juice concentrates are unlikely to be spoiled by Alicyclobacillus due to their high soluble solid contents that prevent the germination and outgrowth of spores (Walls and Chuyate, 2000; Sinigaglia et  al., 2003; Sokołowska and Łaniewska-Trockenheim, 2008; Peña and Massaguer, 2006; Peña et  al., 2011; Oteiza et  al., 2015) and moreover, natural compounds present in some of them may decrease spore count. When a concentrate is diluted to produce ­single strength juice or used to produce juice-based beverages, the spores may find in such product a favorable environment for germination and growth that under certain conditions, can lead to product deterioration.

Fig. 4.1  Changes in the populations of eight A. acidoterrestris strains in apple juice (11.2 °Bx, pH 4.2) during the incubation at 45°C. (Source: Sokołowska, B., Niezgoda, J., Dekowska, A., Porębska, I., Nasiłowska, J., Waldon-Wiewióra, E., Kowalska, M., 2016. Incidence of Alicyclobacillus spp. in polish apple and dark berry juice concentrates and the ability of isolated A. acidoterrestris strains to spoilage of these juices. Post. Nauki. Technol. Przem. Rol.-Spoż. 71 (1), 5–20.)

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The changes in eight wild strains of A. acidoterrestris populations over a period of 28 days of incubation at 45°C in apple, chokeberry, raspberry, strawberry, cherry, and blackcurrant single strength juices were described by Sokołowska et al. (2016). The growth of A. acidoterrestris was observed only in apple juice (Fig. 4.1). After two days of incubation at 45°C, the population of all strains achieved levels of >7 log cfu/mL. The growth curves of all strains are similar to the growth described in orange juice (Gocmen et al., 2005; Goto, 2007), mango and pineapple juices (Danyluk et al., 2011), and kiwi juice (Zhang et al., 2013). No A. acidoterrestris growth was observed in dark berry juices during 28 days incubation at 45°C. The survival curves obtained in chokeberry and raspberry juices (Figs.  4.2 and 4.3) are similar. The population of A. acidoterrestris in these juices was reduced by 1.67– 3.63 log and by 1.45–3.15 log, respectively, after 28 days, ­depending

Fig. 4.2  Changes in the populations of eight A. acidoterrestris strains in chokeberry juice (15.0 °Bx, pH 3.56) during the incubation at 45°C. (Source: Sokołowska, B., Niezgoda, J., Dekowska, A., Porębska, I., Nasiłowska, J., Waldon-Wiewióra, E., Kowalska, M., 2016. Incidence of Alicyclobacillus spp. in polish apple and dark berry juice concentrates and the ability of isolated A. acidoterrestris strains to spoilage of these juices. Post. Nauki. Technol. Przem. Rol.-Spoż. 71 (1), 5–20.)

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Fig. 4.3  Changes in the populations of eight A. acidoterrestris strains in raspberry juice (7.0 °Bx, pH 3.17) during the incubation at 45°C. (Source: Sokołowska, B., Niezgoda, J., Dekowska, A., Porębska, I., Nasiłowska, J., Waldon-Wiewióra, E., Kowalska, M., 2016. Incidence of Alicyclobacillus spp. in polish apple and dark berry juice concentrates and the ability of isolated A. acidoterrestris strains to spoilage of these juices. Post. Nauki. Technol. Przem. Rol.-Spoż. 71 (1), 5–20.)

on the strain. At the end of incubation time, in strawberry juice reduction achieved was 1.04–4.34  log, depending on the strain (Fig.  4.4). Significant differences in the population of individual strains were found in this juice. The reductions were significantly higher (P < .05), in all data points, for two strains: TO-29/4/02 and TO-57/1/04. However, the behavior of the strains did not depend on the source of isolation of the strains. Similar reduction was observed (Silva et al., 2000) in cupuaçu pulp during 1 month storage in aerobic and anaerobic conditions. In cherry and blackcurrant juices, the population of A. acidoterrestris decreased systematically during 28 days of incubation, reaching reduction even up to 5.0 log (Figs. 4.5 and 4.6). Significant differences in the survival population of individual strains in these juices, after 21 and 28 days of incubation, were observed. In cherry juice (Fig. 4.5), after 21 days of incubation reduction was significantly lower (P < .05) for

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Fig. 4.4  Changes in the populations of eight A. acidoterrestris strains in strawberry juice (7.0 °Bx, pH 3.64) during the incubation at 45°C. (Source: Sokołowska, B., Niezgoda, J., Dekowska, A., Porębska, I., Nasiłowska, J., Waldon-Wiewióra, E., Kowalska, M., 2016. Incidence of Alicyclobacillus spp. in polish apple and dark berry juice concentrates and the ability of isolated A. acidoterrestris strains to spoilage of these juices. Post. Nauki. Technol. Przem. Rol.-Spoż. 71 (1), 5–20.)

TO-169/06 and 216/11 strains, and reached 1.54 and 1.81 log, respectively. After 28 days of incubation, only for one strain: 216/11, the significantly lower (P < .05) reduction was observed, and it was 2.30 log. The reduction were significantly lower (P < .05), and reached 2.5– 3.2 log, for TO-117/02, TO-169/06 and 216/11 strains, in blackcurrant juice after 28 days of incubation (Fig. 4.6). The behavior of the strains did not depend on the source of isolation of the strains. Berry fruits are rich sources of bioactive compounds, such as phenolics and organic acids, which have antimicrobial activities against many microorganisms, including human pathogens. The antimicrobial activities of blueberry and blackberry against foodborne pathogens, including Listeria monocytogenes, Salmonella Typhimurium, Campylobacter jejuni, and Escherichia coli O157:H7 (Biswas et  al., 2012; Yang et  al., 2014) were demonstrated. The antimicrobial activity of eight Nordic berries (bilberry, lingonberry, cranberry, red

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Fig. 4.5  Changes in the populations of eight A. acidoterrestris strains in cherry juice (13.5 °Bx, pH 3.51) during the incubation at 45°C. (Source: Sokołowska, B., Niezgoda, J., Dekowska, A., Porębska, I., Nasiłowska, J., Waldon-Wiewióra, E., Kowalska, M., 2016. Incidence of Alicyclobacillus spp. in polish apple and dark berry juice concentrates and the ability of isolated A. acidoterrestris strains to spoilage of these juices. Post. Nauki. Technol. Przem. Rol.-Spoż. 71 (1), 5–20.)

raspberry, strawberry, cloudberry, blackcurrant, and sea-buckthorn berry) and berry phenolics were reported by Puupponen-Pimiä et al. (2005b). In this report, it was found that phenolic compounds, especially ellagitannins of berries, were strong inhibitory compounds against foodborne pathogens (Staphylococcus aureus and Salmonella typhimurium). Several mechanisms in the growth inhibition of bacteria were involved, such as destabilization of the cytoplasmic membrane, permeabilization of the plasma membrane, inhibition of extracellular microbial enzymes, direct actions on microbial metabolism, and deprivation of the substrates required for microbial growth (PuupponenPimiä et al., 2005a). There are no data in the literature regarding the mechanism of the antimicrobial activities of these compounds against spores; however, inhibition of spore germination or inhibition of the outgrowth of germinated spores is possible.

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Fig. 4.6  Changes in the populations of eight A. acidoterrestris strains in blackcurrant juice (11.6 °Bx, pH 2.85) during the incubation at 45°C. (Source: Sokołowska, B., Niezgoda, J., Dekowska, A., Porębska, I., Nasiłowska, J., Waldon-Wiewióra, E., Kowalska, M., 2016. Incidence of Alicyclobacillus spp. in polish apple and dark berry juice concentrates and the ability of isolated A. acidoterrestris strains to spoilage of these juices. Post. Nauki. Technol. Przem. Rol.-Spoż. 71 (1), 5–20.)

4.5  The Reliability of the Results Obtained According to IFU No 12 Detection Method The IFU undertook to develop internationally acceptable methods for the detection of Alicyclobacillus spp. in concentrates and juices (Massaguer et  al., 2002) and has subsequently published as general method for detection of these organisms. After revision in March 2007 “Method on the detection of taint producing Alicyclobacillus in fruit juices” is based on the use of K-agar in combination with BAT agar or YSG agar. Samples that can be filtered should be analyzed by the “Membrane Filtration Method”. Samples that are not easily membrane filtered are tested by “Spread Plating Method” or by performing the “Enrichment Method”. Differentiation is based on the inability of Alicyclobacillus to grow on standard agar at neutral pH, growth and spore production at 45°C on media at pH 3.7 (K-agar, BAT/YSG) and growth at 65°C on YSG. Taint producing

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strains of Alicyclobacillus are unlikely to grow at 65°C. The peroxidase test may be used to confirm the ability of an isolate to produce guaiacol from vanillic acid. This IFU No 12 detection method is widely used in Europe. The presence of inhibitory compounds in fruit juice concentrates can lead to false-negative test results if a high level of these compounds are present in the medium (Oteiza et al., 2015; Sokołowska et  al., 2016; Sokołowska and Niezgoda, 2017). The reliability of the results obtained according to IFU No 12 method was conducted. The comparison filtration and enrichment method was carried out (Sokołowska and Niezgoda, 2017). About 500 samples of apple juice concentrates in which the presence of Alicyclobacillus spp. was detected by filtration method and were examined using the enrichment method (Table 4.4). The detection of Alicyclobacillus spp. was achieved only in 65 out of 181 samples (32.4%) when, according to IFU No 12 method, distilled water (1:10) was used as a diluent. When apple juice concentrates was diluted using BAT broth (1:10) detection was achieved in 160 out of 315 samples (50.8%). When BAT broth was used as the diluent, the recovery was higher, but in some cases the growth was restrained, indicating the presence of inhibitory compounds in apple juice concentrates. Among the samples of dark berry juice concentrates (blackcurrant, raspberry, strawberry, cherry, and chokeberry) contamination was detected only using the filtration method, the enrichment method gave no positive results (Table  4.4) when distilled water or BAT broth (1:10) were used as a diluent. However, when dark berry juice concentrate was diluted using BAT broth (1:50) the detection of Alicyclobacillus spp. was higher and achieved 57.1%. In some cases the growth was restrained by inhibitory compounds. These results are consistent with the studies (McNamara et al., 2011), which showed that the filtration method gives more cases of detection of Alicyclobacillus in fruit juice concentrates containing inhibitory compounds, than the spread plate method. It was also found that natural antimicrobials present in lemon juice concentrate can inhibit germination and outgrowth of Alicyclobacillus spores if a dilution of 1:9 is used during detection procedures. Thus, a modification in the dilution proportion used in the detection procedures from 1:9 to 1:19 is suggested mainly for juice concentrates in which natural compounds presenting antimicrobial activity may inhibit the germination and outgrowth of Alicyclobacillus spp. These findings have relevance for the juice industry and may impact the success of screening and quality control programs as well as the costs resulting from spoilage episodes (Oteiza et al., 2015). The relationship between juice concentration and A. acidoterrestris growth characteristic was investigated by the Members of the Japan Fruit Juice Association (JFJA) Working Group (Goto, 2007).

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Table 4.4  Frequency of the Detection of Alicyclobacillus spp. in Concentrated Fruit Juices Depending on the Method and Type of Diluent Number of Positive Samples for Alicyclobacillus spp.

Type of Juice Apple juice concentrates 67–73°Bx Dark berry (blackcurrant, raspberry, strawberry, cherry, chokeberry), juice concentrates 63–68°Bx

Type of Diluent for Enrichment Method (Dilution Ratio)

Using the Enrichment Method

Using the Membrane Filtration Method

Distilled water (1:10) BAT broth (1:10) Distilled water (1:10) BAT broth (1:10) BAT broth (1:50)

65 160 0 0 8

181 315 33 12 14

(Source: Sokołowska, B., Niezgoda, J., 2017. Optimized methodology for detection of Alicyclobacillus spp. in fruit juice concentrates. Fruit Process. 3, 86–89.)

A strong correlation with the type of juice was observed. Single strength orange juice usually showed good growth promotion, but there was a tendency toward complete inhibition of growth when the juice concentration was over 50%–60% (Niwa, 2005). For juices with a high polyphenol content, such as red grape juice, the detection of Alicyclobacillus was difficult even when the juice concentration was in excess of 10%–20%. The dilution ratio recommended in the JFJA unified methodology for the detection of thermoacidophilic bacteria, was 20–50 times for red grape juice, 20 times for white grape and grapefruit juice, and 10 times for apple, orange, carrot, lemon, and peach juice (Goto, 2007). Moreover in the enrichment method, the JFJA suggested YSG broth (yeast extract, starch, and glucose) as a diluent. Whereas using the IFU No. 12 method, for testing concentrates, the dilution ratio is 10 times, and sterile distilled/demineralized water is acceptable as providing an environment similar to that of a final juice product. Only for products such as sugar solutions and water, additional nitrogen or carbon sources may be required to support bacterial growth. BAT broth or YSG broth may provide these nutrients, but they also provide additional minerals that are not generally present in most juice products. Currently used, the IFU and JFJA methods for Alicyclobacillus detection, may lead to different results.

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4.6  Differentiation and Identification of Alicyclobacillus Species During recent years, the increasing awareness of the threat posed by Alicyclobacillus in the fruit and vegetable industry has created the need to develop new methods and applications enabling fast and accurate identification and differentiation of these bacteria. The classical IFU method, most commonly used by the industry, enables only to differentiate between spoilage and non-spoilage Alicyclobacillus species. The analysis takes about 15 days. The techniques used for Alicyclobacillus identification and differentiation include plating methods, genetic methods, immunodetection methods, taint smell detection, instrumental methods, and chemical methods. During the development and application of new methods, the most important criteria are limited analysis time and sensitivity.

4.6.1  Plating Methods A wide range of different media have been used for Alicyclobacillus isolation and identification, among them three are recommended by the IFU method No. 12 (2004/2007): B. acidoterrestris/B. acidocaldarius medium (BAT/BAM) (Deinhard et al., 1987a,b), K agar (Walls and Chuyate, 1998), and YSG (Goto et al., 2002a,b). Also, acidified Orange Serum Agar (OSA) and Potato Dextrose Agar (PDA) were proven to give high Alicyclobacillus recovery (Witthuhn et  al., 2007, 2011). Despite this, new media are developed and described. The erythritol medium (BAM medium supplemented with erythritol and bromophenol blue) is sometimes used as a supplementary test for the IFU method no 12 to indentify A. acidoterrestris, because this species, unlike other Alicyclobacillus, is able to assimilate the sugar with acid production (Baumgart et al., 1997). A. acidoterrestris presence is indicated by two parameters: the growth is observed at 46°C, but not at 60°C, and the color of the colonies with surrounding medium changes to yellow. In comparison, A. acidocaldarius grows both at 46°C and 60°C, and forms blue colonies without the medium color change. The SK medium is an altered version of K medium, suited for best Alicyclobacillus spp. recovery (Chang and Kang, 2005). The research had been carried out, optimizing the pH, and concentration of acid, Tween80, and divalent cations. As a result, the novel SK medium was described, showing significantly higher recovery of Alicyclobacillus spp. than PDA and K agar. Recently, a selective-differential SK2 medium for Alicyclobacillus has been described. The medium is based on SK agar, supplemented with Chrome Azurol S (CAS) and 70 ppm vanillic acid.

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The vanillic acid in this concentration inhibited the growth of most of non-guaiacol producing Alicyclobacillus strains. Additionally, colonies of the guaiacol-producing strains appeared as dark purple or royal blue, while colonies of one non-guaiacol producing strain able to grow on the medium, appeared as yellow to pale white (Chang et al., 2013).

4.6.2  Membrane Filtration Method Membrane filtration was applied in Alicyclobacillus detection as it enables to examine large volumes of the sample; however, not all types of samples can be filtered. The filters with the sample passed through them are incubated on the surface of agar media. IFU method no 12 recommends the use of 0.45 μm pore size membranes. It is also recommended to test the filter for bacteria recovery prior to the introduction of it into conventional use, because of very divergent recovery counts, depending on the filter manufacturer (Splittstoesser et al., 1994; Albuquerque et al., 2000; Pettipher, 2000; Lee et al., 2007).

4.6.3  Microscopic Method The DEFT method (Direct Epifluorescent Filter Technique) was applied in Alicyclobacillus detection by Pettipher et  al. (1997). The method employs membrane filtration followed by acridine orange staining and epifluorescence microscopic observations. Recently, the EZ-Fluo system, a rapid fluorescent staining-based technology for fast, quantitative detection of microbial contaminants in filterable matrices was developed. This system uses industry standard membrane filtration techniques and fluorescent-based technology to detect viable and culturable microorganisms, i.a. Alicyclobacillus sp. (Sokołowska and Niezgoda, 2015).

4.6.4  Genetic Methods PCR (Polymerase Chain Reaction) based methods are widely used in taxonomy, and were successfully applied for Alicyclobacillus identification and differentiation. RAPD-PCR (Random Amplification of Polymorphic DNA) employs short, 8–12 nucleotide primers, with many possible binding sites across the bacterial genome, which means that amplified segments of DNA are random. Obtained DNA fragments are separated in agarose gel, creating a specific pattern. The use of RAPD-PCR in order to differentiate the Alicyclobacillus species has been described in several p ­ apers (Yamazaki et  al., 1997b; Groenewald et  al., 2008; McKnight et  al., 2010; Zhang et  al., 2013; Bevilacqua et  al., 2015; Franceschini et  al., 2015; Osopale et al., 2017).

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Yamazaki et al. (1997b) proposed three primers that allow distinguishing between A. acidoterrestris, A. acidocaldarius, and other related bacteria. The primers were used by Groenewald et al. (2008) to characterize Alicyclobacillus species isolated from orchard soil and the fruit processing environment, by McKnight et al. (2010) to identify Alicyclobacillus in pasteurized exotic fruit juices, by Bevilacqua et al. (2015) to characterize thermoacidophilic strains from soil and a spoilage incident, by Franceschini et al. (2015) in study on tomato products spoilage by Alicyclobacillus spp., and by Osopale et al. (2017) as a part of analysis of Alicyclobacillus isolated from contaminated commercial fruit juices. Zhang et al. (2013) used freshly described primers in their study on Alicyclobacillus contamination in the production line of kiwi products. RAPD-PCR is a rapid method, the analysis takes several hours, however, it is sensitive to changes in reaction conditions, which can result in low reproducibility. One attempt of using ERIC-based PCR, a variant of Rep-PCR, was described by Felix-Valenzuela et al. (2015) for A. acidocaldarius differentiation. Rep-PCR (Repetitive Element PCR Fingerprinting) is based on the use of primers binding to naturally occurring interspersed repetitive elements in bacterial genome ERIC (Enterobacterial Repetitive Intergenic Consensus) which are repetitive sequences common to enteric bacteria and vibrios, but ERIC-PCR have been also successfully employed in the differentiation of other bacteria. ERIC primers are longer than primers usually used in RAPD, giving more reproducible fingerprints. The patterns obtained for A. acidocaldarius by ERICPCR showed high diversity, but clusters formed by ERIC-PCR and 16S rRNA sequence analysis were different. Restriction fragment length polymorphism (RFLP) method targets a specific DNA fragment, carried by all microorganisms tested. The fragment is amplified by PCR and cut by restriction endonuclease. The differences in sequence of this fragment in studied samples are reflected in the different arrangement of restriction sites. After the restriction enzyme treatment, the fragments are separated by gel electrophoresis and create a unique fingerprint. Like in RAPDPCR, relatedness of the tested microorganisms is established based on similarity of the patterns. 16S rDNA RFLP analysis has been described in the characterization of thermoacidophilic bacteria from the apple juice processing environment (Chen et  al., 2006). Comparing to RAPD-PCR, this method takes more time, since it includes the restriction cut stage, but is less sensitive to changes in reaction conditions. Another way to determine microbial taxonomic relations is the analysis of the sequence polymorphism of specific genes. Based on known sequences, sequence-specific detection methods are d ­eveloped.

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Those methods employ real-time PCR, reverse-transcription PCR, LAMP-PCR, and fluorescence hybridization. The most popular markers in bacterial taxonomic studies are the 16S rDNA gene and the ITS (Internal Transcribed Spacer) region of rDNA operon. Universally distributed among bacteria, the 16S rDNA gene includes highly conserved, as well as variable, species-­specific sites. Based on the conserved regions, universal primers were designed, while the species-specific regions are the target for species-­ specific sequence based detection methods. The noncoding ITS region sequence shows larger diversity than 16S rDNA gene and is a powerful tool in the differentiation of strains of the same species. 16S rDNA sequence analysis was one of the parameters the Alicyclobacillus genus creation was based on (Wisotzkey et al., 1992), and along with the housekeeping gyrB gene sequence analysis, is an important criterium in the new Alicyclobacillus species propositions (Albuquerque et al., 2000; Goto et al., 2003, 2007; Jiang et al., 2008; Guo et al., 2009; Glaeser et al., 2013; Kim et al., 2014; Kusube et al., 2014; Nakano et al., 2015; Zhang et al., 2015). 16S rDNA sequence analysis was used in the identification of A. ­acidoterrestris isolated from acidic beverages (Yamazaki et  al., 1996b), to establish the occurrence and distribution of thermoa­ cidophilic bacteria isolated from orchard soils in Japan (Goto et  al., 2008), to compare the thermoacidophilic bacteria isolated from various acidic environments in Japan (Goto et  al., 2002b), to examine the haplotype distribution of 141 Alicyclobacillus strains isolated from several sources (Durak et al., 2010), to determine the impact of the hypoxic environment of bottled juices on the growth of A. acidoterrestris (Kinouchi et al., 2014), in a study on heterogeneity of A. acidoterrestris isolated from soil and a spoilage incident (Bevilacqua et al., 2015), in differentiation of A. acidocaldarius strains (Felix-Valenzuela et al., 2015), and to investigate if enrichment requirements for A. acidoterrestris detection by real-time PCR is influenced by the fruit juice and puree characteristics (Shayanfar et al., 2015). The immunomagnetic separation (IMS) method, based on capturing the analyzed cells by magnetized beads, coated with appropriate antibodies, was incorporated into 16S rDNA PCR Alicyclobacillus detection, and was proven to enhance its sensitivity (Wang et al., 2013a). Later, the IMS method was also mixed with two 16S rDNA targeting real-time PCR systems. Both the TaqMan system and SYBR Green I system showed improved sensitivity of detection of Alicyclobacillus in fruit juices, when combined with IMS (Wang et al., 2014; Cai et al., 2015). The shc gene-based procedure of Alicyclobacillus identification has been described. Shc is a single copy gene coding squalenehopene cyclase, the key enzyme involved in hopanoid synthesis. Hopanoids are pentacyclic triterpenoid lipids improving plasma

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membrane ­stability of numerous bacteria. The gene sequence is highly heterogenic at the species level. Primers and probe targeting shc gene have been designed to detect Alicyclobacillus using real-time PCR. The method does not allow differentiating detected bacteria and requires further verification. It has not been proved that the method would allow detecting all Alicyclobacillus species (Luo et al., 2004). A 275 bp fragment of shc gene was amplified in order to initial screening of isolates in a study on fruit juice products from Ghana and Nigeria (Osopale et al., 2017). The primers used in this study had been previously described in the paper on UV-C inactivation of microorganisms, where they were used for differentiation of Alicyclobacillus from other tested genera (Huch et al., 2010). During the last few years, the development of next-generation sequencing techniques has resulted in a rapid fall of the difficulty and cost of sequencing, providing huge amounts of sequence data in a relatively short space of time. The number of complete genome sequences deposited in internet databases is rising rapidly. These techniques are also widely used in metagenome analyses, enabling us to collect data about whole microbial communities, including unculturable bacteria. So far, in the GenBank database there are two fully sequenced genomes deposited (both of A. acidocaldarius), and 19 are partially sequenced (including, among others, A. acidoterrestris, A. hesperidum, A. herbarius and A. acidiphilus). The classical and next-generation sequencing techniques were employed in studies on metagenomes isolated from various environments, determining Alicyclobacillus species presence and significance in the bacterial communities. Healthy and diseased konjac rhizosphere soils analysis placed Alicyclobacillus among 14 species significantly affected by the duration of continuous cropping and plant status (Wu et al., 2017). Alicyclobacillus have been found in microbial communities in pyrrhotite mine tailings in Morocco (Bruneel et  al., 2017), in a creek contaminated by acid mine drainage (Sun et al., 2016), in the Chinoike Jigoku hot spring in Japan (Masaki et al., 2016), in uranium-contaminated soil in China, where Alicyclobacillus has been established as one of the three dominant genera in radioactive soil (Yan et al., 2016).The Alicyclobacillus species was a part of the microbiomes of lung and its extracellular vesicles in nonsmokers, healthy smokers, and COPD (chronic obstructive pulmonary disease) patients, most commonly found in all tested groups (Kim et al., 2017a). Németh et  al. (2014) have incorporated Denaturing Gradient Gel Electrophoresis (DGGE) in analysis of the microbiome of deep subsurface geothermal well waters in Hungary. DDGE targeting the 16S rDNA gene was also used to discriminate between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius isolated from contaminated commercial fruit juices (Osopale et al., 2017).

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The growing amount of known sequences isolated from microorganisms, enables developing sequence-specific detection methods. As mentioned above, 16S rDNA gene and the ITS region are the main bacterial markers, and their extensive use in bacterial taxonomy led to the formation of large public domain databases. Based on the 16S rDNA gene sequence, A. acidoterrestris specific primers have been designed (Yamazaki et  al., 1996a) to detect A. acidoterrestris using reverse-transcription PCR, as well as a set of primers and probe to detect A. acidoterrestris using real-time PCR (Connor et al., 2005). Reverse transcription PCR combined with microchip capillary electrophoresis were applied for A. acidoterrrestris detection in orange juice. Besides tracking of the bacterial growth, the method was used to ­verify cell diminution and can be applied in the monitoring of i­ nhibitor susceptibility (Funes-Huacca et al., 2004). Several commercial PCR-based Alicyclobacillus detection systems are available on the market (e.g., Veriflow, Biotecon, R-Biopharm). Real-time PCR targeting the 16S rDNA gene was conjuncted with aptamer employing (SELEX, systematic evolution of ligands by exponential enrichment) method. Aptamers are relatively short, single-stranded DNA, RNA molecules, or peptides, which are able to bind to a specific target molecule. DNA aptamers selected for Alicyclobacillus spp. spores were used in enrichment of the spores from orange juice by magnetic separation, followed by quantification of the isolated DNA by real-time PCR (Hünniger et al., 2015a,b). The aptamer technique was also implemented in lateral flow assay applications (Fischer et al., 2017). The authors examined the use of aptamers on a wide spectrum of target classes, from small sized (metabolites) to large sized (whole cell/spore). Alicyclobacillus spores in buffered orange juice were employed as the large-sized class example, and limit of detection was determined at >8 CFU/mL. A method of detection of A. acidoterrestris using loop-mediated isothermal amplification (LAMP) has been described (Chen et  al., 2011). The LAMP method employs four primers and the reaction proceeds at a constant temperature. Products of the reaction can be detected photometrically or by turbidity measurement. A. acidoterrestris detection is based on ITS region amplification. The LAMP method is highly specific and does not require expensive equipment, which makes it an attractive alternative to the classic PCR methods. A biochip for rapid detection of spore-forming bacteria in foods, including Alicyclobacillus, has been designed. The detection is based on species and genera specific 16S rDNA gene sequences, combined with genes encoding metabolic functions, toxins, or structural genes. Fluorescently labeled probes indicate the presence or absence of targeted bacteria during real-time PCR (Postollec et al., 2010, 2012).

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Another rDNA-based Alicyclobacillus detection method employs fluorescently labeled probe hybridization (Pieper et  al., 2006). The method combines FISH (fluorescent in situ hybridization) and flow cytometry. The fluorescently labeled probes hybridize with complementary DNA sequences inside living cells and then the labeled bacteria can be detected by the cytometer. Fluorescently labeled probes, designed specifically for Alicyclobacillus, have been used in detection of these bacteria in foods. Details concerning the probes were not described, since the test is available commercially. The test differentiates between Alicyclobacillus sp., glowing green, and A. acidoterrestris, glowing both green and red (Thelen et al., 2003).

4.6.5  Immunodetection Methods The enzyme-linked immunosorbent assay (ELISA) method, using a specific polyclonal anti-Alicyclobacillus antibody, was applied to detect Alicyclobacillus in apple juice (Wang et al., 2012, 2013b). The technique was later developed by adding IMS to the procedure, employing specific anti-Alicyclobacillus antibodies, immobilized on surface of magnetic nanoparticles (Wang et al., 2013c), which shortened the total analysis time and improved the procedure’s sensitivity limit from 105 to 103 CFU/mL (Wang et al., 2013c). Mast et  al. (2016) described the use of sandwich ELISA employing polyclonal rabbit antibodies in the detection of A. acidoterrestris spores. The spores with bound antibodies were visualized by immunofluorescence. The method was tested with several types of juices and detection limits were determined at 2.1 × 103–3.8 × 104 spores/mL.

4.6.6  Spectroscopic Methods Fourier transform infrared spectroscopy (FTIR) and Fourier transform near-infrared (FT-NIR) spectroscopy are widely used in bacterial classification and identification. These vibrational spectroscopic methods are based on the measurement of the vibration of chemical bonds, excited by infrared or near infrared radiation. Applied to bacterial cells, the methods enable to scan overall cells compositions. The spectra obtained are reproducible and specific for different bacteria. The FTIR method has been successfully used to discriminate between Bacillus and Alicyclobacillus species (Al-Holy et al., 2015), between different A. acidoterrestris strains (Lin et al., 2006), and between E. coli O157:H7 and Alicyclobacillus strains in inoculated apple juice (Al-Qadiri et al., 2006). FT-NIR was tested in order to establish if it enables distinguishing between different Alicyclobacillus species. The researchers concluded

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that differentiation at the species level was possible with a combination of FT-NIR and chemometrics (Wang et al., 2015). Grasso et  al. (2009) described a method for detection and differentiation of Alicyclobacillus species based on hydrophobic grid membranes and attenuated total reflectance infared microspecroscopy (ATR-IR).

4.6.7  Impedance Measurement Fernández et al. (2017) have described the detection of A. acidoterrestris in contaminated apple juice by electrical impedance. Both direct and indirect impedance measurement techniques were used. The study shows that a direct impedance method was not valid for the determination of A. acidoterrestris, because the high salt content of the BAT medium used in the study prevented the detection of small changes in conductance, produced by the microorganisms. On the other hand, the indirect technique, measuring changes in the medium conductivity due to the CO2 formation, showed a high accuracy of detection. The CO2 was produced by the bacteria during the guaiacol formation.

4.6.8  Guaiacol Detection Method Manifestation of spoilage Alicyclobacillus in fruit and juice products can be exposed indirectly, by the detection of guaiacol. This can be done using sensory, chemical, or analytical methods. The sensory method is based on the collaboration of trained sensory panels. Several studies aimed at establishing the best estimate threshold (BET) of guaiacol have yielded quite divergent results: from 2.00 μg/L for taste in apple, orange, and noncarbonated fruit juice (Pettipher et al., 1997) and 2.23 μg/L for aroma in apple juice (Orr et al., 2000), to 0.48 μg/L in water and 0.91 μg/L in apple juice for aroma, and 0.17 μg/L in water and 0.24 μg/L in apple juice for taste (Eisele and Semon, 2005). The differences in the results indicate that proper training and individual sensitivity of panelists are crucial for this method. Guaiacol can be also detected chemically, using the reaction of its oxidation by peroxidase, in the presence of H2O2. As a result, brown colored 3,3′-dimethoxy-4,4′-biphenoquinolone is formed. Change of color of the reaction mix can be measured spectrophotometrically, but it is also visible to the naked eye. This reaction is the basis of guaiacol detection kits (Niwa and Kawamoto, 2003; Niwa and Kuriyama, 2003; Anonymous, 2006). High-performance liquid chromatography (HPLC) and gas chromatography (GC) have been applied for guaiacol detection. Formation

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of this component by A. acidoterrestris, measured by HPLC in inoculated apple juice supplemented with vanillin, showed approximate values between experimental and fitted data (Bahçeci et  al., 2005; Bahçeci and Acar, 2007). GC combined with solid and liquid-phase extraction methods has been described for the detection of guaiacol, 2,6-dibromophenol, and 2,6-dichlorophenol. Pettipher et al. (1997) used liquid-liquid extraction method (LLE) followed by gas chromatography-mass spectrometry (GC-MS) identification for inoculated orange and apple juice, and a noncarbonated fruit juice-containing drink. Gocmen et  al. (2005) used solid-phase microextraction (SPME) and LLE combined with gas chromatography-olfactometry (GC-O) and GC-MS in the detection of off-aroma compounds, finding SPME more suitable, because it was less time consuming and did not degrade the column. The GC-O analysis identified three components whose odor was described as medicinal/antiseptic. Zieler et al. (2004) described SPME extraction followed by GC-MS detection of Alicyclobacillus borne taint odors in apple juice. The analysis parameters were optimized, the method was fully validated, and limits of quantification and detection of guaiacol and 2,6-dibromophenol was established.

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