Controlling the Production of Off-odor Guaiacol by. Alicyclobacillus acidoterrestris in Apple juice or a. Microbiological Medium.

Transcription

1 Controlling the Production of Off-odor Guaiacol by Alicyclobacillus acidoterrestris in Apple juice or a Microbiological Medium Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Xiaohuan Hu, B.S. Graduate Program in Food Science & Technology The Ohio State University 2016 Thesis Committee: Prof. Sheryl Barringer, Advisor Prof. Ahmed Yousef Asso. Prof. Monica Giusti

2 Copyright by Xiaohuan Hu 2016

3 Abstract The presence of the off-odor compound guaiacol in juices spoiled by Alicyclobacillus acidoterrestris, is one of the major causes for consumer complaints and product rejection. This study tested how temperature, ph, substrate (vanillin) and antimicrobial compounds (ɛ-polylysine and lauric arginate) effected bacterial growth and guaiacol production in yeast starch glucose broth (YSG) and apple juice with new analytical instrument Selected Ion Flow Tube Mass Spectrometery (SIFT-MS). Incubated samples were held at C and ph with 10mg/L vanillin. Minimum inhibitory concentrations (MICs) of ɛ-polylysine and lauric arginate were determined at 37 C and ph 3.7. Guaiacol concentration was determined in the headspace by selected ion flow tube mass spectrometry (SIFT-MS). Incubation temperature didn t influence the concentration of bacteria in the stationary phase or guaiacol produced, but did influence bacterial growth rate which determined the time until guaiacol production occurred. Bacterial growth was inhibited at 20 and 25 C in apple juice but not at 37 or 45 C either in YSG or apple juice. ph 6.7 completely inhibited bacteria and ph 2.7 delayed bacterial growth, thus influencing guaiacol production; but the bacterium grew well at ph 3.7 and 4.7. Guaiacol was produced when the bacterial population reached ~10 5 CFU/mL. MIC values for lauric arginate and ɛ-polylysine were 9.4 and 75ug/ml, respectively. Both of these compounds inhibited vegetative cell multiplication and ii

4 spore germination and no guaiacol was produced. However, Alicyclobacillus acidoterrestris OSYE in YSG solution was not inhibited by lauric arginate. The correlation of vanillin concentration ( mg/l) to guaiacol production was highly linear. iii

5 Acknowledgements I would first like to thank my father and my mother, who gave me this chance to study in America and who always give me a lot of encouragement to explore new things. I also would like to thank my advisor Dr. Sheryl Barringer. She gave me countless advice relating to my research and classes. I also learned a lot from the way that she deals with things. What is more, she is always so nice no matter what kinds of problems I brought to her. She also taught me how to write thesis and how to manage my project efficiently. I also want to thank Dr Yousef and En Huang, they came up with a great project for me, I learned a lot by working with this project for about two years. En also taught me all necessary lab skill of microbial research step by step and gave me various great suggestions toward the troubles that I met in my project. What is more, I would also like to thank my committee member Dr. Monica Giusti for her supports to my research project. I also appreciate all the help from Dr. Yousef s lab such as the help from Xu Yang and Yang Song; the help from my lab mates, such as Yafei Han and Hacer Akpolat who taught me a lot knowledge about SIFT-MS. It was a wonder experience by working with all of you. iv

6 Vita 2014.B.S. Food Science, Jilin University 2014 to present M.S. Food Science, The Ohio State University Fields of study Major Field: Food Science and Technology. v

7 Table of Contents Abstract... ii Acknowledgements... iv Vita... v List of Figures... x List of Tables... xii Practical Application... xiii Chapter 1-Introduction... 1 Chapter 2- literature review Apple juice Apple juice composition Method for microorganism control in apple juice Physical treatment Heat Treatment Antimicrobial compounds Irradiation Properties of Alicyclobacillus spp... 9 vi

8 2.3 Reasons for survival of Alicyclobacillus spp in juice Methods for the control of A. acidoterrestris Off-flavor of juice Flavor properties of guaiacol Formation pathway of guaiacol in drink The influence of substrates on guaiacol production The sensory threshold of guaiacol The influence factors on guaiacol production The concentration of Alicyclobacillus spp Temperature of storage Heat shock Other off-flavor compounds Chemical properties of 2, 6-dibromophenol and 2, 6-dichlorophenols The synthetic pathway of 2, 6-dibromophenol (2, 6-DBP) and 2, 6- dichlorophenols (2, 6-DCP) Detection method of guaiacol Instrumental analysis of guaiacol a. Peroxidase enzyme colorimetric assay b. High performance liquid chromatography with UV-diode array detection (HPLC-DAD) vii

9 c. Headspace gas chromatography-mass spectrometry (HS GC-MS) Sensory analysis Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) Principle of selected ion flow tube mass spectrometry (SIFT-MS) Chemical characters of H3O +, NO +, and O Chapter 3 - Materials and Methods Yeast Starch and Glucose (YSG) solution YSG (Yeast, Starch and Glucose) broth preparation YSG agar medium base Sporulation agar medium Apple juice, 2-ethoxyphenol and vanillin Peptone water Internal standard curve for YSG broth and apple juice samples Selected Ion Flow Tube Mass Spectrometry Detection of Guaiacol Culturing vegetative Alicycobacillus acidoterrestris Culturing Alicycobacillus acidoterrestris spores The relationships of guaiacol and bacterial counts over time at different temperatures in YSG broth or juice samples The relationship of guaiacol and bacterial counts over time at different ph in YSG broth viii

10 3.13 Antimicrobial compounds Limitation of vanillin for guaiacol production Statistical Analysis Chapter 4- Results and Discussion The influence of temperature on bacterial growth and guaiacol production The influence of ph on guaiacol production and bacterial growth Effect of antimicrobial compounds Limitation of substrate (vanillin) on guaiacol production Chapter 5-Conclusions References Appendix: Tables and Figures ix

11 List of Figures Figure 1.The process of forming vanillin acid from ferulic acid Figure 2.The process of forming guaiacol from vanillic acid Figure 3.The process of ferulic acid degradation through the producing of 4-vinylguaiacol Figure 4.The pathway of guaiacol production by A. acidoterrestris FB Figure 5. Bacterial counts influenced by temperature in YSG solution (above) and apple juice (below) a measurements with the same letter within the same sample have no significant difference (p 0.05) Figure 6.Guaiacol production influenced by temperature in YSG solution (above) and apple juice (below) a measurements with the same letter within the same sample have no significant difference (p 0.05) Figure 7.The influence of ph on guaiacol production and bacterial growth in YSG a measurements with the same letter within the same sample have no significant difference (p 0.05) Figure 8.The influence of antimicrobial compounds on vegetative cells (above) and spores (below) of Alicycobacillus acidoterrestris OSYE in YSG x

12 Figure 9.Relationship between the amount of vanillin (mg/l) and guaiacol concentration in the headspace (ppb) Figure 10.The influence of antimicrobial compounds on vegetative cell of Alicycobacillus acidoterrestris ATCC in YSG Figure 11.The influence of antimicrobial compounds on spores of Alicycobacillus acidoterrestris ATCC in YSG Figure 12.The influence of antimicrobial compounds on vegetative cell of Alicycobacillus acidoterrestris ATCC in juice Figure 13.The influence of antimicrobial compounds on spore of Alicycobacillus acidoterrestris ATCC in juice Figure 14.The influence of antimicrobial compounds on vegetative cell of Alicycobacillus acidoterrestris OSYE in YSG Figure 15.The influence of antimicrobial compounds on spore of Alicycobacillus acidoterrestris OSYE in YSG Figure 16.The influence of antimicrobial compounds on vegetative cell of Alicycobacillus acidoterrestris OSYE in Juice Figure 17.The influence of antimicrobial compounds on spore of Alicycobacillus acidoterrestris OSYE in Juice xi

13 List of Tables Table 1. Vitamin Content of Fresh Apples per 100 Gram of Tissue... 5 Table 2.Amino Acid Content (%) of Fresh Apples and Apple Products... 5 Table 3. Mean, Standard Deviation (SO), Coefficient of Variation (CV), minimum and maximum for apple juice mineral content... 6 Table 4. Difference between the initial (0h) and final (48h for YSG and 72h for juice) bacterial counts and guaiacol concentration Table 5.Difference between the initial (0h) and final bacterial counts (48h for YSG or 72h for juice) among different conditions Table 6. Difference between the initial (0h) and final (48h for YSG and 72h for juice) guaiacol concentration among different conditions Table 7.Volatile Compounds Measured in Headspace Scan Volatile Compound Table 8.The influence of storage time in water bath on volatilization xii

14 Practical Application Alicyclobacillus acidoterrestris growth and guaiacol production were inhibited by storage at 20 or 25 C, ph 6.7 and addition of ɛ-polylysine or lauric arginate. Guaiacol production can be decreased, but not prevented, by decreasing the substrate (vanillin) concentration in food. Therefore, storage apple juice at low temperatures (20 or 25 C) and adding ɛ- polylysine or lauric arginate into apple juice products can be used in practical processing of juice to prevent both vegetative cell growth and spore generation as well as prevent guaiacol production. In ideal condition, adjusting juice to high ph (6.7) or reduce vanillin concentration in it can help inhibit bacterial growth and guaiacol production. xiii

15 Chapter 1-Introduction Alicyclobacillus acidoterrestris is a thermophilic, acidophilic, spore-forming microorganism (Chang and Kang 2004). Because of its thermophilic properties, this bacterium can survive in a large temperature range, from 20 to 70 C (Chang and Kang 2004) C is the optimal temperature range (Chang and Kang 2004). It can survive commercial pasteurization of juice in the form of spores and spoil fresh or processed fruit juice products by producing an off-odor when its count reaches 105CFU/mL (Chang and Kang 2004). Alicyclobacillus acidoterrestris can produce guaiacol at 25 or 45 C, but bacterial grow more slowly at 25 than 45 C (Witthuhn and others 2013). The bacteria can also grow in a wide range of ph, from (Chang and Kang 2004). Guaiacol is a metabolite, which is the main factor that leads to the smoky or medicinal off-flavor in juice associated with Alicyclobacillus. spp. Guaiacol is produced by a biological conversion reaction from ferulic acid to vanillic acid and then decarboxylation to guaiacol (Chang and Kang 2004). Vanillin, vanillic acid and ferulic acid are three important precursors of guaiacol, but only vanillin or vanillic acid can be decomposed by Alicyclobacillus. spp directly without participation of other bacteria (Witthuhn and others 2012). Higher concentration of substrates (vanillin or vanillic acid) tended to produce higher final amounts of guaiacol by 1

16 Alicyclobacillus acidoterrestris in Bacillus acidoterrestris (BAT) broth (Witthuhn and others 2012). Lauric arginate (LAE) is a new and natural cationic surface-active molecule which has a large range of antimicrobial activity (Rinrada and others 2014). Lauric arginate is a surfactant because of its polar cationic head group which is formed from L- arginine, and nonpolar tail which comes from lauric acid, so it can attach to the oilwater interface or biofilm (Rodríguez and others 2004; Loeffler and others, 2014). It is mostly used in meat products and has not been used in juice against Alicyclobacillus. spp. Lauric arginate is generally recognized as safe (GRAS) by the US. Food and Drug Administration (FDA) (Food and Drug Administration 2005). ε-polylysine was reported to have antimicrobial activity against yeasts, fungi, grampositive and gram-negative bacteria, and bacteriophages (Chang and others 2010). It is a GRAS additive and was approved by the FDA in 2004 (Food and Drug Administration, 2004). ε-polylysine can inhibit bacterial growth, mainly depending on electrostatic adsorption to the bacterial cell surface and its cationic properties which can destroy the outer membrane and finally lead to abnormal cytoplasmic distribution (Shima and others 1984; Yoshida and Nagasawa 2003; Chang and others 2010). Both ε-polylysine (300mg/L) and lauric arginate (200mg/L) have been reported to effectively reduce the amount of Salmonella on inoculated chicken carcasses (Benli and others 2011) Alicyclobacillus acidoterrestris ATCC had been reported to produce the off-odor guaiacol in juice (Gocmen and others 2005). Alicyclobacillus acidoterrestris OSYE is an industrial strain isolated from commercial juice which it is considered to be the most resistant strain found commercially. The objective of this study was to test how different temperature and ph affects bacterial growth and guaiacol production by 2

17 Alicyclobacillus acidoterrestris ATCC and OSYE during storage. The minimum inhibition concentrations (MICs) were determined for lauric arginate, and ε- polylysine and they were tested for their ability to inhibit the growth of vegetative cells and spore germination in YSG broth and apple juice. The relationship between vanillin concentration and guaiacol production was also tested with Selected Ion Flow Tube Mass Spectrometry (SIFT-MS). 3

18 Chapter 2- literature review 2.1. Apple juice Apple juice composition Apple juice is a high acid product (ph<4.6), it is treated with pasteurization process. The contents of apple juice are mainly fiber, vitamin, pectin, amino acid, mineral, organic acid. In the peel of apple, there are 0.7~0.8% higher of fiber content than oranges, bananas or grapefruits (Downing 1995). Ascorbic acid is the highest vitamin compound in apple which is about 5mg/100g (Downing 1995; Gebhardt and others 1982). Other vitamins in apple with and without peels can been seen from the table 1. Apple with skin usually have higher content of ascorbic acid than without skin. For apple grew in America, the pectin content is constant during season until apples become soft, and then the total pectin will be decrease. Amino acid is a minor compound in apple. Protein in fresh apples with peel is about 0.19%. Aspartic and glutamic acid are the predominant amino acid followed by lysine and leucine in fresh apple and apple products (Table 2). The average mineral content in apple juice is about 0.207% among apple from different areas because of the various of soil content (Mattick and Moyer 1983). Potassium contents are the main mineral compounds followed by phosphorus and calcium (Table 3). Organic acid is one of important contents in apple. Temperature condition and the length of growing season will 4

19 influence the organic acid content. Malic acid is the primary organic acid in fruit, but citric acid, oxalic acid as well as lactic acid are also presented. The contents of acid in fruit will influence flavor or eating quality (Downing 1995). Table 1. Vitamin Content of Fresh Apples per 100 Gram of Tissue (Gebhardt and others 1982) RE*= Retinol equivalent Table 2.Amino Acid Content (%) of Fresh Apples and Apple Products (Gebhardt and others 1982) 5

20 Table 3. Mean, Standard Deviation (SO), Coefficient of Variation (CV), minimum and maximum for apple juice mineral content (Mattick and Moyer 1983) Microbiology in apple juice Apple juice is a low ph product, so it can inhibit the growth of a large variety of bacteria. Only molds, yeast, lactic acid and acetic acid bacteria or some spore-forming and acidophilic bacteria such as Alicyclobacillus spp can survive in juice. Most of the microorganism will usually produce off-flavors, turbidity, alcohol or gas in products such as yeast, mold, acetic acid bacteria or Alicyclobacillus spp (Downing 1995). But the survive of some bacterial toxin such as salmonellosis or patulin in juice after pasteurization process also can lead to outbreaks in juice. For example, contaminated commercial apple cider had leaded to outbreak of salmonellosis in New Jersey (Downing 1995) which was thought that was because of the contamination of Salmonellae presented in manure. And patulin which is a fungal toxin and carcinogenic chemical compounds contamination of apple juice is usually because of using unsound raw apple or samples are not processed under actual peocessing conditions. There are two main sources that microorganism can present in apple juice- raw apples and equipment (Downing 1995). Therefore, the quality of final juice products 6

21 relying on the quality of raw apples and the sanitation of equipment. Microbial flora of apples varies with the soundness of apple and seasons. The epiderm and core of sound apple can inhibit the growth of bacteria, but for unsound apples, there ae high microbial counts in the flesh (Marshall and Walkley 1951). What is more, handpicked apples also have lower bacterial counts than mechanically harvested apples during storage (Davenport 1980) Inadequately cleaned equipment can provide pools of residual fruit and juice which usually bring the fasted growth strains of bacteria to apple products. Therefore, routine daily cleaning of equipment is pretty important to keep the initial counts of bacteria at a low level and prevent potential spoilage Method for microorganism control in apple juice Methods used for preventing bacterial growth in juice varies with the different nature of final products. If juice is processed with soundness raw apple and go through with adequate sanitation, it can simplify the preservation procedures (Downing 1995). The most commonly used preservation method are physical treatments, heat treatment, irradiation and chemical antimicrobial compounds Physical treatment Centrifugation apple juice at the speed of 9,000*g can reduce the population of microbial by more than about 99.8% (Kosikowski and Moreno 1970). Filtration of juice with diatomaceous earth can help reducing microbial counts (Swanson and others 1985), but ultrafiltration can remove molds, yeast and bacteria (Heatherbell and others 1977). 7

22 Heat Treatment Heat treatment is the most commonly used method for removing bacteria from apple product. For shelf-stable apple juice, usually samples are processed with heat treatment at 71 C for 15-20min, 88 C for 1 or 2 min, or >121 C with treat time less than 30 sec. The effective of heat treatment depending on the initial number of microorganism (Downing 1995). However, heat treatment is not effective on inhibiting heat resistance or spore-forming organisms such as Alicyclobacillus spp Antimicrobial compounds Sorbic acid, sodium and potassium sorbate have been used in apple juice for a long time. Research showed that sodium benzoate is more effective on controlling the growth of yeasts and model than sorbic acid or sodium sorbate. But benzoate can arouse off-flavor, so sorbate is wildly used antimicrobial compound in juice (Downing 1995). Sulfur Dioxide can reduce the bacterial population to a undetectable levels with the concentration of 150 ppm (Downing 1995, Warth 1985). Only fermenting type of yeast can survive in this condition, but its lag time will be extended (Warth 1985). Other preservatives such as pimaricin which can inactivate Saccharomyces spp in apple juice within twenty-five days with at 5ppm (Baerwald 1976); dichlorofluoromethane or acetaldehyde also are used in apple juice for inhibiting yeasts (Cousin and others 1997, Barkai-Colan and Aharoni 1976). 8

23 Irradiation Different kinds of irradiation have been used in inactivation of bacteria in apple juice (Downing 1995). Ultraviolet irradiation is used in pasteurization of apple juice can achieve 99% reduction of microbial counts without any turbidity or off-flavor production (Harrington and Hills 1968). Gamma-irradiation is used in freezeconcentrated apple juice which can extend its shelf life to 10 months without any other off-odor (Downing 1995, Kiss and Farkas 1968). 2.2 Properties of Alicyclobacillus spp Alicyclobacillus spp. is a gram-positive, rod-shaped, spore-forming, thermophilic, and acidophilic bacteria (Chang and Kang 2004). They can live in a large temperature range from 20 to 70 C. The optimal range is C. They also can grow in a wide range of ph, from (Chang and Kang 2004). The most special character of Alicyclobacillus. spp is the membrane component of ω-alicyclic fatty acids, which can provide them with ability of surviving in high temperature and acid environment (Albuquerque and others 2000). Alicyclobacillus spp is a major microorganism that can cause the spoilage of food. However, it has no pathogenicity which was tested by directly injecting spores into mice or indirectly adding spores to juice and then feeding spores to guinea pigs (Walls and Chuyate 2000). No symptoms of disease were produced. However, Alicyclobacillus spp is the main concern of fruit juice companies, because it can survive in commercial pasteurized production and produce off-flavor guaiacol (Splittstoesser and others 1994; Chang and Kang 2004). The appearance of this kind 9

24 of bacteria would not make people sick but it will influence the flavor of productions and arouse consumer complaint. 2.3 Reasons for survival of Alicyclobacillus spp in juice There are two main ways for juice to be contaminated by microorganism. Fruit can contact with microorganism directly and then microorganisms can adhere to the peel of fruit, after that the bacteria can enter the processing of juice leading to juice contamination (Chang and Kang 2004). Other media, such as wind, rain, animal, etc., also can cause the accumulation of microorganisms on the surface of fruit (Chang and Kang 2004). Therefore, if the fruit is not washed well, Alicyclobacillus spp can enter the juice easily. Most spore-forming bacteria can be killed or controlled by low ph (ph<4.6). What is more, most juice industries attach great importance to other bacteria such as mold, yeast, and under heat and acidic conditions, most of the growth of heat reliable microorganism can be inhibited in juice (Change and Kang 2004). However, spore of Alicyclobacillus spp. can survive in pasteurization temperature and high acid environment. Thus, the processing of juice cannot control the survival or germination of Alicyclobacillus spp. It is a leading microorganism can cause deterioration of juice (Cerny and others 1984). In juice samples, only sugar when its content is higher than 18 Brix, or phenolic compounds can inhibit the growth of Alicyclobacillus spp (Chang and Kang 2004). Apple juice and tomato juice are also susceptible to this kind of bacteria, and normal 10

25 it cannot be detected until there are consumer complaints because of the off-odor of juice (Chang and Kang 2004). Lower temperature (T<20 C) can inhibit the growth or germination of spore, but most of the juice are shelf stable and stored at room temperature, thus it is not an effective way to inhibit the spoilage of Alicyclobacillus spp (Chang and Kang 2004). Therefore, with the growth in consumption of apple juice, more attention should be given to control or detect the Alicyclobacillus spp. 2.4 Methods for the control of A. acidoterrestris The optimization of heat treatment has been regarded as effective method to inactive A. acidoterrestris. However, high temperature is not such an effective method to inhibit spores of the bacteria, so during the storage period, spore of A. acidoterrestris can still germinate in product. Moreover, heat can destroy lots of nutritional compounds, so it is not ideal for inhibiting the growth of A. acidoterrestris in food (Chang and Kang 2004). High pressure processing is another effective method. A. acidoterrestris spores can be inactivated at high pressure (200 to 600Mpa) with gentle temperatures (45 to 65 C). Compared to traditional thermal processing (85~95 C), this method can lower process temperature which can provide a fresher and higher quality preserved food (Silva and others 2012). Excepting to these two main physical methods to control A. acidoterrestris, there are lots of chemical compounds that also can inhibit the growth of bacteria. Natural antimicrobial compounds or additives combine can reduce the amount of bacteria as 11

26 well as inhibit the germination of spores especially when samples are stored at high temperature or low ph conditions (Bevilacqua and others 2009). Additive of cinnamic aldehyde, and a moderate heat and low ph treatment is considered to be an effective method to control the number of bacteria (Bevilacqua and others 2009). In this method, cinnamaldehyde (40ppm-50ppm) works as inhibitor of the spore germination and thermal (80 to 86 C) and lower ph (3.5 to 5.5) treatment can reduce the original number of bacteria. Saponin extracts with a combination of heat-treatment can also inactivate A. acidoterrestris and produce less influence on the quality of products than high temperature treatment (Alberice and others 2012). Bovicin HC5 make contribution to the effective of heat treatment by promote the thermal sensitive of A. acidoterrestris (Carvalho and others 2008). When spores of A. acidoterrestris was treated by heat, D-values can increase from 77% to 95% when bovicin HC5 was added compared to no bovicin HC5 added (Carvalho and others 2008). Grape seed extracts which contain a lot of polyphenols, such as catechin (49.8%) and epicatechin (26.0%) were the major contents of the grape seed extracts, which are followed by epicatechin gallate (9.3%), procyanidin B1 (5.8%) and B2 (5.1%), epigallocatechin gallate (1.9%) and gallic acid (1.3%) (Guendez and others 2005), are regarded as natural material that can effectively inhibit the growth of vegetative cells and spore of A. acidoterrestris (Molva and Baysal 2015). Compared to control samples, 2 to 3 log decrease of the count of bacteria can be achieved when grape seed 12

27 extracts with concentration from 0.06% to 1.80% was added to contaminated juice by A. acidoterrestris (Molva and Baysal 2015). Nisin is another natural antimicrobial compound which was produced by Lactococcus lactis subsp. Lactis (Bevilacqua and others 2008). It can inhibit A. acidoterrestris cell, and by working with chelating agents (such as EDTA) or at lower ph and heat at high temperature (51 C), the ability of bacteriostatic can be improved (Nguyen and Mittal 2007; Rosa and others 2009). Supercritical carbon dioxide (SC-CO2) at temperature of 65 or 70 C and pressure of 80, 100, 120 bar with time from 10 to 40 min was proved that it can totally inactivate the spore of A. acidoterrestris and reduce the amount of spores to an undetectable level with less quality destruction (Bae and others 2009). In addition to these methods, chemical disinfectants and chlorine dioxide are also effective pathways to inactive A. acidoterrestris (Chang and Kang 2004). ε-polylysin and lauric arginate are widely used antimicrobial compounds and they are both in the GRAS (generally recognized as safe) status. ε-polylysine is edible and water-soluble antimicrobial compound (Benli and others 2011)). It has been largely used in different foods such as custard cream, potato salad, steamed cakes, fish, fish slices, sushi, boiled rice, noodles, cooked vegetable as well as soups as antimicrobial compounds (Hiraki and others 2003; Otsuka and others 1992; Chang and other 2010). Lauric arginate is colorless and odorless with a large range of antimicrobial ability which make it a very prospective food preservation (Becerril and others 2013). Gram- 13

28 negative as well as gram-positive pathogen, such as Salmonella and Listeria monocytogenes, can be effectively inhibited by it (Martin and others, 2009; Stopforth and others, 2010; Soni and others, 2010; Theinsathid and others, 2012). Because it can destroy the cell membrane and influence the metabolic process of cells (Rodríguez and others 2004; Loeffler and others, 2014). 2.5 Off-flavor of juice Off-flavor is the most common cause that a product will be rejected by consumer. Microorganisms can not only spoil products but also can produce off-odor which can affect the quality of products (Barbara and Barbara, 2007). Spoilage of juice is caused mainly by Alicyclobacillus spp by producing bad flavor. Usually, the metabolites of bacteria in juice is the main pathway to form off-flavor after a period of storage in room temperature. There are mainly two different kinds of compounds that can cause the off-flavor in juice: guaiacol and halophenols (Jensen and Whitfield 2003; Chang and Kang 2004). Halophenols include 2, 6-dibromophenol and 2, 6-dichlorophenol (Jensen and Whitfield 2003; Chang and Kang 2004). However, guaiacol is the main factor and most commonly focused compounds that can arouse the smoky or medicinal off-odor in juice associated with Alicyclobacillus spp (Chang and Kang 2004). 2.6 Flavor properties of guaiacol Guaiacol can produce sweet or burnt odor and smoky flavor, so it is always used as flavoring in food (Wasserman 1966). It is well known by its smoky or phenolic flavor. It can help promote the special flavor of roasted food products such as coffee or beef 14

29 (Mayer and others 1999). In another hand, the spoilage off-flavor in contaminated acid products is the most widely known character of guaiacol. The spoilage off-flavor of some products such as wine (Simpson and others 1986), fruit juice (Jensen 2000), dairy products (Jensen and others 2001) as well as ice cream (Saxby 1993), are proved to be guaiacol. 2.7 Formation pathway of guaiacol in drink There are several ways that can lead to guaiacol production. It can be formed when products go through heat treatment which can decompose the phenolic compounds to produce guaiacol (Chang and Kang 2004). In fruit juice, guaiacol also can be produced because of the microbial metabolism. Microorganisms that can produce guaiacol include Alicyclobacillus acidoterrestris, Bacillus magaterium, Pseudomonas acidovorans, Rhodotorula rubra and Streptomyces setonii (Chang and Kang 2004). Alicyclobacillus acidoterrestris is the one that was first and wildly detected. In fruit juice, vanillic acid is the direct precursor of guaiacol (Figure 1, 2). Degradation of lignin which mainly comes from the plant polymer such as ferulic acid or contamination of raw product all can bring vanillic acid or vanillin into juice (Chang and Kang 2004). Lignin is one kind of cross-linked phenolic polymers which is important compound in structural tissue such as cell walls (Martone and others 2009). Lignin can decompose to conlferyl alcohol and then convert into ferulic acid during processing (Kumar and Pruthi 2014). The degradation of ferulic acid is a common method to generating vanillic acid or vanillin in juice (Figure 3). 15

30 There are two ways of ferulic acid degradation. In one way, firstly ferulic acid is hydrated and then the acetate moiety was removed through chemical reaction and then produce the vanillin acid (Figure 1). Figure 1.The process of forming vanillin acid from ferulic acid (Huang and others, 1993; Chang and Kang 2004) When the vanillin acid is formed in samples, then it will be further decomposed into guaiacol (Figure 2). Vanillin acid will change into a quinoid intermediate and then break down into guaiacol (Huang and others 1993, Chang and Kang 2004) (Figure 2). 16

31 Figure 2.The process of forming guaiacol from vanillic acid (Huang and others, 1993; Chang and Kang 2004) Ferulic acid can also be degraded into 4-vinylguaiacol and then go further to produce off-flavor guaiacol by decarboxylation (Chang and Kang 2004) or S. setonii (Max and others 2012) (Figure 3). During this process, B. coagulans will involve in this process to decompose ferulic acid into 4-vinylguaiacol and then convert 4- vinylguaiacol into vanillin during a short time (Crawford and Olson 1978, Pometto and others 1981, Huang and others 1993, Chang and Kang 2004) (Figure 3). 17

32 Figure 3.The process of ferulic acid degradation through the producing of 4-vinylguaiacol (Huang and others, 1993; Chang and Kang 2004) During the pathway of ferulic acid degradation to form guaiacol production, ferulic acid can be decomposed by Rhodotorula rubra (Huang and others 1993), Paecilomyces variotii (Rahouti and others 1989), and Sporotrichum thermophile (Topakas and others 2003). But Alicyclobacillus acidoterrestris cannot decompose ferulic acid into vanillin acid (Witthuhn and others 2012). It can only produce guaiacol when there are substrates of vanillic acid or vanillin, and the speed of 18

33 converting vanillin acid into guaiacol is much faster than vanillin (Witthuhn and others 2012) (Figure 4). Figure 4.The pathway of guaiacol production by A. acidoterrestris FB2 (Witthuhn and others 2012) 2.8 The influence of substrates on guaiacol production The pathway of guaiacol production was tested by adding 100 or 1000mg/L vanillin or vanillic acid and 5.67, or 100 mg/l ferulic acid into BAT broth with 10 6 CFU/mL final concentration of A. acidoterrestris FB2 (Witthuhn and others 2012). Samples were stored at 45 C incubator for 7 days to test the guaiacol and bacterial changes. During these 7 days, the number of bacteria have no significant difference from the number on day 0. For samples with ferulic acid, no guaiacol was produced during the storage, which proved that A. acidoterrestris FB2 cannot decompose ferulic acid into guaiacol (Witthuhn and others 2012). In samples with 100mg/L 19

34 vanillin, there are significant increase of the guaiacol concentration from days 0 to 5, and then keep stable (Witthuhn and others 2012). The 100mg/L vanillin was almost used up at day 5 and the highest concentration of guaiacol is 61.7mg/L. In 1000mg/L vanillin samples, the amount of guaiacol keep increase where the highest amount of guaiacol is 170.8mg/L, but vanillin was not used up during 7days; it is possible that guaiacol will increase until the vanillin is used up. For samples with 100mg/L vanillic acid, guaiacol increased very fast at the first 24h and then kept stable from 1 to 7 days and the highest amount of guaiacol was 67.4mg/L. In 1000mg/L vanillic acid sample, guaiacol producing was delayed initially which may because of the decrease of the amount of bacteria during the first 24h (Witthuhn and others 2012). Then the amount of bacteria as well as guaiacol kept increasing. The highest amount of guaiacol concentration for samples with 1000mg/L vanillic acid was higher than the sample with 100mg/L vanillic acid. Therefore, in 100mg/L samples, vanillic acid was used up very fast and produce a very high amount of guaiacol in a shorter time than vanillin. However, very high concentration of vanillic acid can inhibited the growth of bacteria (Witthuhn and others 2012). In fact, tyrosine is also a precursor of guaiacol (Jensen 1999). However, the amount of tyrosine in beverage is very small so the influence on juice is not obvious. The amount of it in apple juice is usually about 4.1μg/mL and in orange juice is μg/ml (Chang and Kang 2004). The synthetic way of guaiacol through tyrosine is not clear. 20

35 2.9 The sensory threshold of guaiacol The sensory thresholds for guaiacol are different in different conditions. The sensory threshold of guaiacol was about 20 ppb in water (Wasserman 1966). It is not very hard for olfactory evaluation to test it. Pettipher and others (1997) also find the threshold of guaiacol is about 2 ppb in some noncarbonated juice such as apple, orange juice. There were also other compounds that can produce off-odor in products such as 2, 6-dibromophenol and 2, 6-dichlorophenols. The sensory thresholds of these two compounds were low to ppt level (Chang and Kang 2004). Even though 2, 6- dibromophenol and 2, 6-dichlorophenols have a lower threshold than guaiacol, most research focus on guaiacol, which was regarded as the primary off-flavor compound. There were two reasons which make guaiacol the main role of off-flavor: (1) guaiacol has a high volatility (2) the concentration of guaiacol in juice is normal much higher than other off-flavor compounds, about 1,000 times higher than halophenols (Chang and Kang 2004) The influence factors on guaiacol production The concentration of Alicyclobacillus spp. When fruit juice was contaminated by Alicyclobacillus spp, there would be guaiacol production when the concentration of Alicyclobacillus spp. reach 10 5 CFU/mL (Pettipher and others 1997). Then with time goes by, guaiacol will kept increase and then kept stable at a higher amount (Witthuhn and others 2012). Thus the number of bacteria must reach 10 5 CFU/mL in order to produce guaiacol. 21

36 Temperature of storage Alicyclobacillus spp. growth at a range of temperature from 20 to 70 C. Depending on different species, the optimal temperature various from 42 to 60 C (Chang and Kang 2004). There is obvious difference of guaiacol between contaminated chocolate milk when stored at 8-9 C, then there was ppb; when stored at 4-5 C there was ppb guaiacol tested by GC-MS (Jensen and others 2001). One of either Alicyclobacillus acidoterrestris FB2, FB38, DSM 3922T as well as strains A. acidocaldarius FB19 and DSM 446T have been cultivated in Bacillus acidoterrestris (BAT) broth with final cell concentration of cfu/ml and supplement with 100mg/L vanillin (Witthuhn and others 2013). There were two sets of samples for each bacterium. They were stored at temperature of 25 and 45 C separately for 6d. All three strains of Alicyclobacillus acidoterrestris can produce guaiacol, and A. acidoterrestris DSM 3922T and FB38 can even produce a higher concentration of guaiacol in 25 than 45, therefore the degree of spoilage can be the same, or even more serious at lower temperature (25 ) than at higher temperatures (45 ) (Witthuhn and others 2013). However other two A. acidcaldarius strains were unable to produce guaiacol at 25 (Witthuhn and others 2013). Thus different temperatures will influence the amount of guaiacol in juice that is produced by Alicyclobacillus spp. Lower temperature also cannot be ignored which may provide a condition for more guaiacol production produced by A. acidoterrestris. 22

37 Heat shock In fruit juice, the metabolite of guaiacol will be influenced by the status of Alicyclobacillus spp. Only in vegetative status rather than dormant condition, can metabolites be produced (Chang and Kang 2004). Thus, activation of dormant bacteria is a process that can lead to spores germinate. Among lots of activation methods, exposure bacteria to sub-lethal heat is the most frequently used. Different temperatures of heat shocking, there would be different recovery speeds for bacteria at low concentration (Chang and Kang 2004). When Alicyclobacillus spp. are inoculated at low concentration, heat shocking at 80 for 10 min will lead to a higher number of bacterial germination than heat shocking at 60 for 10 min or 100 for 5 min (Walls and Chuyate 2000). While, with high concentration of bacteria, heat shock with different temperatures does not make any differences for germinating (Chang and Kang 2004). For spores of Alicyclobacillus acidoterrestris which survived pasteurization of juice, seems needs no heat shocking was needed to germinate it. It can germinate in juice with time which may because of some natural compounds in the juice that can induce them, but the speed of germinating is slower than heat shocking (Pettipher and others 1997). Thus, heat shock will definitely help to activate Alicyclobacillus spp and increase the speed of the present of guaiacol production Other off-flavor compounds Excepting to guaiacol, halophenols: 2, 6-dibromophenol (2, 6-DBP) and 2,6- dichlorophenols (2,6-DCP) are also regarded as the major cause of off-flavor in fruit juices (Jensen 1999). Both 2, 6-DBP and 2, 6-DCP is detected in spoilage fruit juice 23

38 by Alicyclobacillus acidoterrestris (Baumgart and others 1997, Borlinghaus and Engel 1997). But there is still no affirmative answer to whether these halogenated phenolic compounds can be produced by Alicyclobacillus acidoterrestris or not. It is also possible that these compounds are presented in juice just because of chemical contamination Chemical properties of 2, 6-dibromophenol and 2, 6-dichlorophenols 2, 6-dibromophenol and 2, 6-dichlorophenols usually present a similar flavor as guaiacol. Actually, most common way of halogenated phenolic compound contamination was coming from disinfectant taints at a level below 1 ppb (Saxby 1993). In fact, even though at a very low level of 2, 6-DBP and 2, 6-DCP, they still can produce off-odor and influence the quality of products, because they have much lower threshold than guaiacol (Chang and Kang 2004) The synthetic pathway of 2, 6-dibromophenol (2, 6-DBP) and 2, 6- dichlorophenols (2, 6-DCP) There are two ways that will lead to food contamination by 2, 6-DBP and 2, 6-DCP: chemical contamination and microbial synthetic pathway (Chang and others 2004). As for the chemical way, bromophenol and chlorophenol are easily formed by the reaction of phenol with halogen solutions (Chang and others 2004). During the process of fruit, disinfectants will be used to clean fresh fruits as well as in the process of diluting the juice. Some recycled paper also contains high level of chlorophenolic compounds, thus if these materials are used for packaging, it will also can bring chlorophenolic compounds to products and then produce food contamination 24

39 (Mottram 1998). Thus during these steps, when phenols containing water contacts with halogen solution, then a quick product of halophenols will be formed. If halophenols were not removed from the system, then it will continue to pollute the final products (Chang and others 2004). Even though phenol is important factors that lead to halophenol contamination, it is not a necessary compound in this kind of contamination. When there was no phenol in carrots, but if carrots were treated with sodium hypochlorite and then heat them to 121 C, then 2, 6-DCP can also been produced (Adams and others 1999). In fact, free phenol has not detected in fresh vegetable In the microbial synthetic pathway, the contaminations were produced because of the bacterial biosynthesis, instead of external disinfection contamination. In the process, some main reactants were the phenolic precursor, hydrogen peroxide, halide ions and haloperoxidase (Neidleman and Geigert 1986). In the biosynthetic process, microbial haloperoxidase can make contribution to promote the process, even without metal ions or cofactors to speed up reactions (Picard and others 1997). From these aspects, fruit juice should be a very suitable environment to form the off-odor halophenol, because even though in the juice there are trace quantities of phenolic compounds, hydrogen peroxide and halide ions, haloperoxidases produced by bacteria can still promote the happen of this reaction (Chang and Kang 2004). Strains of A. acidoterrestris are capable to produce halogenation. Therefore A. acidoterrestris 25

40 could be also one kind of microorganism that related to the generation of these two off-odor compounds Detection method of guaiacol There are two categories for the guaiacol detection: instrumental analysis and sensory analysis. Instrumental analysis focuses on quantifying the amount of guaiacol. The sensory analysis is usually applied to detect the presence or absence of guaiacol Instrumental analysis of guaiacol a. Peroxidase enzyme colorimetric assay Peroxidase enzyme colorimetric assay will show different colors which means different concentrations of guaiacol (Sheu and Chen 1991, Bahçeci and Acar 2007). UV/Vis spectrophotometer will be combined with it to measure the color changes at 420nm (Sheu and Chen 1991, Bahçeci and Acar 2007). Different colors will lead to different signals. After that, the guaiacol concentration can be calculated depending on the standard curve. But the disadvantage is that the high concentration of vanillic acid can also influence the color of solution and then produce a discoloration of the buffer. Thus, sometimes it just cannot gain an obvious color change or useful signal in UV/Vis spectrophotometer (Witthuhn and others 2012). Therefore, most of the time, this method will only be applied to detect the presence or absence of guaiacol. b. High performance liquid chromatography with UV-diode array detection (HPLC- DAD) For this method, sample need to be filtered first and then injected it into HPLC with a silica C18 column (Witthuhn and others 2012, Chang and Kang 2004). Since 26

41 different compounds will have different retention times, thus, the peak area of a specific compound with a fixed retention time point can be calculated through standard curve. This method is much accurate than peroxidase enzyme colorimetric assay method, but tedious sample preparation is needed (Witthuhn and others 2012). c. Headspace gas chromatography-mass spectrometry (HS GC-MS) Headspace gas chromatography-mass spectrometry (HS GC-MS) has ever used to detect guaiacol produced by Alicyclobacillus spp strains at different temperatures. Though it is an expensive and time-consuming method, the advantage is that it is very high accurate, sensitive detection method with relative less sample preparation (Witthuhn and others 2013). The detection limit is 0.5ug/L (ppb) (Witthuhn and others 2013) Sensory analysis The detection threshold for volatile compounds guaiacol are the level which the different samples can be separated correctly by panel without being able to describe the off-flavor as disinfection or medicine (Witthuhn and others 2012; Chang and Kang 2004). The recognition threshold is the concentration of guaiacol at which the different samples can be recognized correctly and the medicinal flavor of guaiacol also can be described (Chang and Kang 2004). The sensory threshold of guaiacol in juice is a little higher than guaiacol threshold in water (20ppb). In fact, the special smell of guaiacol is very obvious thus most consumers are able to detect the presence of guaiacol even without sensory training. 27

42 Thus this kind of sensory test worth nothing for qualification, and most of time, sensory test can be utilized to confirm the presence of guaiacol (Chang and Kang 2004) Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) Principle of selected ion flow tube mass spectrometry (SIFT-MS) SIFT-MS is a new and fast analytical technique which are mainly used for real-time quantification of the trace gases both in air and breath (Smith and Španěl 2005). Three precursor ions: H3O +, NO +, or O2 + in SIFT-MS are used to react with aimed trace volatiles by chemical ionization reaction to test the concentration of volatile compounds (Munch and Barringer 2014). When testing the sample, the three precursor ions can react with volatiles in headspace in a fixed time, and then the reaction can be detected and counted depending on the change of the concentration of precursor ions by mass spectrometer, therefore, the concentration of volatiles can be detected (Smith and Španěl 2005). Most of the time, only one of the ions, which shows the lowest reaction, was used for calculating the concentration of one compound. Absolute concentrations of volatile compounds in one sample can be tested by SIFT-MS down to ppb levels (Smith and Španěl 2005). In the SIFT, the three positive ions (H3O +, NO +, or O2 + ) are generated from ion mixtures. The information of mass-to-change ratio can be obtained by the detection of quadrupole mass filter (Smith and Španěl 2005). The reaction ions will be injected into the fast-flowing inert carrier gas (usually pure helium under a pressure of at most 100Pa) by a venturi-type orifice (diameter usually 1-2mm). They will be carried as a 28

43 cold ion current with carrier gas along the flow tube. The speed of ions follows the Maxwellian velocity distribution related to the change of temperature (normally 300K) of carry gas (Smith and Španěl 2005). The ions for mass analysis are sampled by the pinhole orifice from the flowing current (the diameter at most 0.3mm) into a differentially-pumped quadrupole mass spectrometer (Smith and Španěl 2005). After that they can be analyzed by channeltron multiplier/pulse counting system. The rate of coefficient and ion products for the injected ions and trace volatile gas can be determined according to the reduction of injected ion current and the increase of the product ion which can be measured by the downstream mass spectrometer system. Then the rate coefficient of the reaction can be calculated according to series of procedures. The main focus of SIFT-MS analyses is the ions change of precursor and the produce of ion products by downstream mass spectrometry detection system. Depending on these principles, SIFT-MS can achieve the real-time quantification of trace gases in a complex circumstance (Smith and Španěl 2005) Chemical characters of H3O +, NO +, and O2 + The most important principles to select appropriate ions is that the ions should hardly react with major air molecules such as CO2,O2, N2, but they should be easily to react with trace gases which will be tested (Smith and Španěl 2005). After a long time s study, it has been found that H3O + (Španěl and Smith, 1995; Španěl and others 1995; Smith and Španěl 1996b; Smith and Španěl 2005), NO +, O2 + (Smith and 29