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

Copyright by

Xiaohuan Hu

2016

ii

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 20-45°C and pH 2.7-6.7 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

~105CFU/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

iii

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 (0.25-2 mg/L) to guaiacol production was highly

linear.

iv

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.

v

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.

vi

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 ......................................................................................... ４

2.1. Apple juice ............................................................................................ ４

2.1.1 Apple juice composition ..................................................................... ４

2.1.3 Method for microorganism control in apple juice .............................. ７

2.1.3.1 Physical treatment ............................................................................ ７

2.1.3.2 Heat Treatment................................................................................. ８

2.1.3.3 Antimicrobial compounds ................................................................ ８

2.1.3.4 Irradiation ......................................................................................... ９

2.2 Properties of Alicyclobacillus spp .......................................................... ９

vii

2.3 Reasons for survival of Alicyclobacillus spp in juice ........................ １０

2.4 Methods for the control of A. acidoterrestris .................................... １１

2.5 Off-flavor of juice .............................................................................. １４

2.6 Flavor properties of guaiacol ............................................................. １４

2.7 Formation pathway of guaiacol in drink ............................................ １５

2.8 The influence of substrates on guaiacol production .......................... １９

2.9 The sensory threshold of guaiacol ..................................................... ２１

2.10 The influence factors on guaiacol production .................................. ２１

2.10.1 The concentration of Alicyclobacillus spp. ................................... ２１

2.10.2 Temperature of storage ................................................................. ２２

2.10.3 Heat shock ..................................................................................... ２３

2.11 Other off-flavor compounds ............................................................ ２３

2.11.1 Chemical properties of 2, 6-dibromophenol and 2, 6-dichlorophenols

.................................................................................................................. ２４

2.11.2 The synthetic pathway of 2, 6-dibromophenol (2, 6-DBP) and 2, 6-

dichlorophenols (2, 6-DCP) ..................................................................... ２４

2.12 Detection method of guaiacol .......................................................... ２６

2.12.1 Instrumental analysis of guaiacol.................................................. ２６

a. Peroxidase enzyme colorimetric assay ................................................. ２６

b. High performance liquid chromatography with UV-diode array detection

(HPLC-DAD) ........................................................................................... ２６

viii

c. Headspace gas chromatography-mass spectrometry (HS GC-MS) ..... ２７

2.12.2 Sensory analysis ............................................................................ ２７

2.13 Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) ................ ２８

2.13.1 Principle of selected ion flow tube mass spectrometry (SIFT-MS) .. ２

８

2.13.2. Chemical characters of H3O+, NO+, and O2+ .............................. ２９

Chapter 3 - Materials and Methods .......................................................................... ３３

3.1 Yeast Starch and Glucose (YSG) solution ......................................... ３３

3.2 YSG (Yeast, Starch and Glucose) broth preparation ......................... ３３

3.3 YSG agar medium base ..................................................................... ３３

3.4 Sporulation agar medium ................................................................... ３４

3.5 Apple juice, 2-ethoxyphenol and vanillin .......................................... ３４

3.6 Peptone water ..................................................................................... ３４

3.7 Internal standard curve for YSG broth and apple juice samples ....... ３４

3.8 Selected Ion Flow Tube Mass Spectrometry Detection of Guaiacol . ３５

3.9 Culturing vegetative Alicycobacillus acidoterrestris......................... ３５

3.10 Culturing Alicycobacillus acidoterrestris spores ............................. ３６

3.11 The relationships of guaiacol and bacterial counts over time at different

temperatures in YSG broth or juice samples ........................................... ３６

3.12. The relationship of guaiacol and bacterial counts over time at different

pH in YSG broth ...................................................................................... ３７

ix

3.13 Antimicrobial compounds ................................................................ ３８

3.14 Limitation of vanillin for guaiacol production................................. ３９

3.15 Statistical Analysis ........................................................................... ３９

Chapter 4- Results and Discussion .......................................................................... ４０

4.1 The influence of temperature on bacterial growth and guaiacol

production ................................................................................................ ４０

4.2 The influence of pH on guaiacol production and bacterial growth ... ４５

4.3 Effect of antimicrobial compounds .................................................... ４６

4.4 Limitation of substrate (vanillin) on guaiacol production ................. ５１

Chapter 5-Conclusions ............................................................................................ ５２

References ................................................................................................................ ５３

Appendix: Tables and Figures ................................................................................. ６２

x

List of Figures

Figure 1.The process of forming vanillin acid from ferulic acid ............................. １６

Figure 2.The process of forming guaiacol from vanillic acid .................................. １７

Figure 3.The process of ferulic acid degradation through the producing of 4-vinyl-

guaiacol .................................................................................................................... １８

Figure 4.The pathway of guaiacol production by A. acidoterrestris FB2 ................ １９

Figure 5. Bacterial counts influenced by temperature in YSG solution (above) and

apple juice (below) ameasurements with the same letter within the same sample have

no significant difference (p ≤ 0.05) .......................................................................... ４３

Figure 6.Guaiacol production influenced by temperature in YSG solution (above) and

apple juice (below) ameasurements with the same letter within the same sample have

no significant difference (p ≤ 0.05) .......................................................................... ４４

Figure 7.The influence of pH on guaiacol production and bacterial growth in YSG

ameasurements with the same letter within the same sample have no significant

difference (p ≤ 0.05) ................................................................................................ ４６

Figure 8.The influence of antimicrobial compounds on vegetative cells (above) and

spores (below) of Alicycobacillus acidoterrestris OSYE in YSG ........................... ４９

xi

Figure 9.Relationship between the amount of vanillin (mg/L) and guaiacol

concentration in the headspace (ppb) ....................................................................... ５１

Figure 10.The influence of antimicrobial compounds on vegetative cell of

Alicycobacillus acidoterrestris ATCC in YSG ........................................................ ６５

Figure 11.The influence of antimicrobial compounds on spores of Alicycobacillus

acidoterrestris ATCC in YSG .................................................................................. ６５

Figure 12.The influence of antimicrobial compounds on vegetative cell of

Alicycobacillus acidoterrestris ATCC in juice ........................................................ ６６

Figure 13.The influence of antimicrobial compounds on spore of Alicycobacillus

acidoterrestris ATCC in juice .................................................................................. ６６

Figure 14.The influence of antimicrobial compounds on vegetative cell of

Alicycobacillus acidoterrestris OSYE in YSG ........................................................ ６７

Figure 15.The influence of antimicrobial compounds on spore of Alicycobacillus

acidoterrestris OSYE in YSG .................................................................................. ６７

Figure 16.The influence of antimicrobial compounds on vegetative cell of

Alicycobacillus acidoterrestris OSYE in Juice ........................................................ ６８

Figure 17.The influence of antimicrobial compounds on spore of Alicycobacillus

acidoterrestris OSYE in Juice .................................................................................. ６８

xii

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 ............................................................ ５０

Table 5.Difference between the initial (0h) and final bacterial counts (48h for YSG or

72h for juice) among different conditions ............................................................... ６２

Table 6. Difference between the initial (0h) and final (48h for YSG and 72h for juice)

guaiacol concentration among different conditions ................................................. ６３

Table 7.Volatile Compounds Measured in Headspace Scan Volatile Compound ... ６４

Table 8.The influence of storage time in water bath on volatilization .................... ６４

xiii

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.

1

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). 42-60°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 2.5-6.0 (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

2

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 oil-

water 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, gram-

positive 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

3

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).

4

Chapter 2- literature review

2.1. Apple juice

2.1.1 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

5

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)

6

Table 3. Mean, Standard Deviation (SO), Coefficient of Variation (CV), minimum

and maximum for apple juice mineral content (Mattick and Moyer 1983)

2.1.2. 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

7

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, hand-

picked 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.

2.1.3 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.

2.1.3.1 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).

8

2.1.3.2 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.

2.1.3.3 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).

9

2.1.3.4 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 freeze-

concentrated 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 42-60°C. They also can grow in a wide

range of pH, from 2.5-6.0 (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

10

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

11

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

12

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

13

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-

14

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

15

(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).

16

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).

17

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).

18

Figure 3.The process of ferulic acid degradation through the producing of 4-vinyl-

guaiacol (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

19

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, 66.67 or 100 mg/L ferulic acid into BAT broth with

106CFU/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

20

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 3.4–13.5

μg/mL (Chang and Kang 2004). The synthetic way of guaiacol through tyrosine is not

clear.

21

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).

2.10 The influence factors on guaiacol production

2.10.1 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 105CFU/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 105CFU/mL in order to produce guaiacol.

22

2.10.2 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 150-190 ppb; when stored at 4-5°C there

was 50-70 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 101-103cfu/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.

23

2.10.3 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.

2.11 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

24

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.

2.11.1 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).

2.11.2 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

25

(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

26

could be also one kind of microorganism that related to the generation of these two

off-odor compounds.

2.12 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.

2.12.1 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

27

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).

2.12.2 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.

28

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).

2.13 Selected Ion Flow Tube Mass Spectrometry (SIFT-MS)

2.13.1 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

29

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).

2.13.2. 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

30

Španěl 1996; Smith and Španěl 2005) are ideal ions for SIFT-MS. These ions do not

react with major air molecular such as N2, O2, H2O, CO2, or Ar; and only through

slow termolecular association so that can these ions react with these molecules (Smith

and Španěl,1996 a, c; Španěl and Smith, 1996b; Smith and Španěl 2005). Negative

ions are also tested for the possibility of being the precursors. As for OH-, it cannot

react with organic molecules as quickly as H3O+, and it is also does not have

sufficient amount of its molecular to form the current of negative ions (Smith and

Španěl 2005). Thus, the negative ions will decrease the accuracy of the instrumental

analysis (Smith and Španěl 2005). More information regarding the reaction properties

can be seen as follows:

A. H3O+ reaction

As for this ion, H3O+ reaction with molecules M will produce a stable product of

MH+. The main reaction of H3O+ ion is H3O

+ + M→ (M-H)+ + H2O according to the

function of][

2AkN

iDNi

dt

dNj

i

. But these compounds of MH+ also can

decompose or re-synthesize to form other compounds such as (M-OH)+, and (M-OR)+

( R is radical such as CH3 and C2H5). And (M-OH)+ comes from the reaction of MH+

with H2O. Tertiary alcohols (M-OH)+ can be produced when further react with H3O+

molecular (Španěl and Smith 1997; .Smith and Španěl 2005). There are also other

compounds that will be produced because of further complex reaction, such as the

ester reactions: H3O+ + C3H7COOCH3 →C3H7COOCH3H

++ H2O (90%) →C3H7CO+

31

+ H2O +CH3OH (10%) (Španěl and Smith 1998; Smith and Španěl 2005); and react

with aliphatic hydrocarbons or halocarbons such as H3O+ + C10H22 + He

→H3O+(C10H22)

+ He (Smith and Španěl 2005); when moisture gas currents- such as

human breath-was tested, there may form cluster because of too much water:

H3O++nH2O+ X→H3O

+ (H2O)n +X,

where X is a third media that can stabilize the cluster (Smith and Španěl 2005).

Sometimes there would be a further reaction in a humid environment which can

reaction with (M-H)+ or (M-OH)+ to form MH+(H2O)n.

B. NO+ reaction

As for NO+, there are several kinds of reactions, but usually they do not occur at the

same time. All kinds of reactions can be seen as follow:

(1) charge transfer, producing M+·

NO+ + C6H5CH3→C7H8+·+NO·

(2) H+ transfer

When NO+ ions react with aldehydes or ethers, (M-H) + can be produced (Španěl

and Smith 1997b; Španěl and Smith 1998b; Smith and Španěl 2005).

NO++CH3CHO→CH2CHO+ +HNO

(3) OH- transfer

NO+ + (CH3)3COH→ (CH3)3C++HNO2

(4) charge transfer, producing NO+M

32

In this kind of reaction, there would be transform of alkoxide ion simultaneously

which will form NO+M and (M-OH)+ ions in some of ester reactions (Španěl and

Smith 1998a, Smith and Španěl 2005).

NO+ +CH3COOCH3 +He→NO+CH3COOCH3 + He→CH3CO+ +CH3NO2 + (He)

C. O2+ reactions

The IE of O2 molecules is relative higher than most of organic compounds (Lias and

others 1988; Smith and Španěl 2005). Thus it is much easier for O2+·to react with

most of organic molecules. As for the reaction for O2+·, no-dissociative charge

transfer reaction and dissociative charge transfer reaction are two main kinds of

reactions:

O2+·+CH3COCH3→CH3COCH3

+·+O2 (60%)

O2+·+CH3COCH3→CH3CO++CH3

·+O2 (40%)

Some small molecules such as NO, NO2 and CS2 which can hardly react with NO+

or H3O+ can be tested by O2

+· (McIntosh and Smith 1988; Smith and Španěl 2005).

These reactions can be seen as follows:

O2+·+NO·→NO++O2

O2+·+NO2

·→NO2+ +O2

O2+·+CS2→CS2

+·+O2

According to the describing of all precursors’ reaction, it would be very important

to find the suitable ions which can accurately reflect the reaction with analysts

without or with less other molecules’ influence (Smith and Španěl 2005).

33

Chapter 3 - Materials and Methods

3.1 Yeast Starch and Glucose (YSG) solution

YSG solution was used to culture bacteria. YSG solution was prepared by mixing

0.2% yeast extract (MSDS, NJ, USA), 0.2% soluble starch (J. T. Barker, NJ, USA)

and 0.1% dextrose (Difco, MD, USA) in distilled water in a 900mL pyrex glass bottle

and adjusting the pH to 3.7. This solution was sterilized by autoclave (30min,

120.5°C) and then stored at 25°C.

3.2 YSG (Yeast, Starch and Glucose) broth preparation

YSG broth was prepared by mixing YSG solution with 8% sucrose (Difco, MD,

USA) with a final pH of 3.7. This solution was sterilized by autoclave (30min,

120.5°C) and then stored at 25°C. The YSG broth was used for sample preparation,

since it is a near ideal environment for bacterial growth.

3.3 YSG agar medium base

The YSG agar medium base was used for counting the number of bacteria and

culturing the bacterial colonies. This agar medium was prepared by adding 1.5% agar

(Difco, MD, USA) to YSG solution, sterilizing and cooling to about 55°C before

poured into Petri dishes and cooling until it solidified (Silva and others 1999).

34

3.4 Sporulation agar medium

Sporulation agar medium were prepared by mixing the following two solutions after

being sterilized separately then pouring in Petri dishes before it cooled down. The first

solution was prepared by mixing 1g yeast extract; 0.2 g (NH)2SO (Fisher Scientific

Fair Lawn, NJ, USA), 0.25 g CaCl2 (Fisher Scientific Fair Lawn, NJ, USA), 0.5 g

MgSO (Fisher Scientific Fair Lawn, NJ, USA), 1 g glucose; 0.6 g KH2PO (Fisher

Scientific Fair Lawn, NJ, USA) with 500 ml distilled water, and adjusting to pH 4.0

with 6mol/L HCl. The second solution was prepared by mixing 20 g agar with 500 ml

distilled water.

3.5 Apple juice, 2-ethoxyphenol and vanillin

Apple juice (Old Orchard, LLC Co, Sparta, MI, USA) was used for this research

with 1.068g/mL 2-ethoxyphenol (ACROS Organics, Fair Lawn NJ, USA) as an

internal standard. 10mg/L vanillin (Sigma-Aldrich, Louis MO, USA) was used as the

substrate which can be decomposed by bacteria to produce guaiacol.

3.6 Peptone water

0.1% peptone (HIMEDIA Laboratories Pvt. Ltd, Mumbai, India) was prepared with

distilled water and then sterilized. 0.1% peptone water was used for serial dilutions

before counting the number of bacteria.

3.7 Internal standard curve for YSG broth and apple juice samples

Guaiacol (Sigma-Aldrich, Louis MO, USA) was prepared at four different

concentrations: 0, 282, 1129, 5645 ppm in YSG broth or apple juice. One hundred

35

milliliters YSG broth or apple juice with five concentrations of guaiacol were placed

into 500mL-sterilized Pyrex glass bottles with 3 replicates for each group and sealed

well with open top screw cap with polytetrafluoroethylene (PTFE)-faced silicone

septa (Corning Life Sciences, Tewksbury, MA, USA). One-milliliter filtered and

diluted 2-ethoxyphenol (1.069ug/mL) (ACROS Organics, Fair Lawn NJ, USA) was

added into every sample as an internal standard and these samples were held in a

37°C water bath for 1h to volatilize guaiacol and 2-ethoxyphenol in the solution. The

concentration of guaiacol in the headspace were tested by SIFT-MS.

3.8 Selected Ion Flow Tube Mass Spectrometry Detection of Guaiacol

Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) (Voice 200; Syft

Technologies Ltd., Christchurch, New Zealand) was used for guaiacol detection under

selected ion mode (SIM). Guaiacol reacted with precursor ion of NO+ with m/z of 124

to produce C7H8O2+. 2-ethoxyphenol was tested by precursor ion O2

+, with m/z of 138

to produce C8H10O2+. Volatiles had a 60 sec scan processing. The volatiles selected

for headspace measurement and SIFT-MS settings are listed in Appendix (Table 4)

3.9 Culturing vegetative Alicycobacillus acidoterrestris

Colonies of pure frozen Alicycobacillus acidoterrestris OSYE and ATCC were

aseptically removed and added to sterile YSG Agar Medium Base and cultured at

37°C for 2 d, then 1conly of bacteria was aseptically removed from YSG agar

medium base into a 50mL centrifuge tube with 30mL YSG solution. The two

centrifuge tubes were stored at 37°C for 2 days until there was turbidity. These

cultures were used to inoculate YSG broth and juice samples.

36

3.10 Culturing Alicycobacillus acidoterrestris spores

Solutions of Alicycobacillus acidoterrestris OSYE and ATCC were aseptically

spread on sterile spore agar medium base and cultured at 45°C for 4d, then aseptically

removed from medium base with a sterilized microscope slide into 2ml centrifuge

tube with sterilized water to a final volume of 1ml. The spore solution was

centrifuged (15 min, 16.1*1000rcf (relative centrifugal force), 4°C), the supernatant

was decanted and the sediment was re-suspended in 50% (v/v) aqueous ethanol for 30

min, to destroy any vegetative cells. The spore suspension was re-centrifuged, the

supernatant was discarded and the sediment was re-suspended in sterile water

(repeated three times). The final sediment was re-suspended in sterile distilled water

and heated 10 min at 80°C, and the number counted and then stored at 4°C for use.

3.11 The relationships of guaiacol and bacterial counts over time at different

temperatures in YSG broth or juice samples

Samples were prepared by adding 100mL YSG broth or apple juice, 1mL of

Alicycobacillus acidoterrestris OSYE, or Alicycobacillus acidoterrestris ATCC, and

10mg/L vanillin solution into a 500mL sterilized Pyrex glass bottle. Fifteen samples

were prepared and divided into five groups with 3 replicates in each group and stored

in an incubator. In order to test the influence of temperature, samples were stored at

20, 25, 37, 45°C in separate incubators. Before testing, 1mL 2-ethoxyphenol was

added into each bottle as the internal standard. These samples were placed into a 37°C

water bath for 1h to volatilize guaiacol and 2-ethoxyphenol, and the headspace was

37

tested by SIFT-MS. The guaiacol concentration in the headspace was determined by

dividing the concentration of 2-ethoxyphenol in headspace and then multiplying it by

the average 2-ethoxyphenol concentration in the headspace for all samples.

At every temperature, samples were tested at 5 different times (YSG broth: 20°C: 0,

211.5, 282.5, 304.5, 326.5h; 25°C：0, 70, 82, 101, 146h; 37°C: 0, 18, 26, 42, 56h;

45°C: 0, 18.5, 25.5, 42.5, 74h; Apple Juice: 20 :0, 22, 31, 33, 35d; 25°C:0, 35, 48, 57,

59d; 37°C: 0, 66, 90.5, 114.5h; 45°C: 0, 69, 76, 100, 119h) according to the bacterial

growth.

After sample testing by SIFT-MS, the number of bacteria in every bottle was

counted where the limitation is 10 CFU/ml. 100uL solution was taken from one

sample, and then diluted to 10-1, 10-2, 10-3, 10-4, 10-5 times of original concentration

with 0.1 % peptone water. 100uL from each diluted solution was spread onto YSG

agar plates. All plates were stored at 37°C for 2 d for bacterial growth and then plates

with 20 to 200 bacterial colonies were selected to count the average number of

bacteria.

3.12. The relationship of guaiacol and bacterial counts over time at different pH in

YSG broth

YSG solution was adjusted to different pH (2.7, 3.7, 4.7, 6.7) with 6M HCL.

Samples were stored at 37°C for bacterial growth. For every pH level, guaiacol

concentration in the headspace and bacterial counts were tested 7 times and there

38

were 3 replicates for every point at different times according to bacterial growth

speed.

3.13 Antimicrobial compounds

Lauric arginate and ɛ-polylysine were tested for their minimum inhibitory

concentrations (MICs) using the method of Clinical and Laboratory Standards

Institute (CLSI 2006). Lauric arginate was diluted to 4.7, 9.4, 18.8, 37.6, 75.2ug/mL

and then 100uL of these solutions were added to the cells of a 94 cells culture plate

with 1uL Alicycobacillus acidoterrestris ATCC or OSYE culture added. ɛ-Polylysine

was diluted to 37.5, 75,150, 300, 600 ug/mL and measured in the same way as lauric

arginate. The 96-cell cultural plate was stored at 37°C for 24h to check whether there

was bacterial growth with microtiter plate reader

Samples used to test the effect of antimicrobial compounds were prepared by

adding their MICs to 100 mL YSG broth or apple juice with 1mL of 10mg/L vanillin

solution and 1mL vegetative cells or 0.1ml spores (Alicycobacillus acidoterrestris

ATCC or OSYE) separately with final concentration ~103CFU/mL. In every batch,

there were three groups of samples: samples with ɛ-polylysine added, samples with

lauric arginate added as well as a control group (no antimicrobial compound was

added). During the test of every batch, guaiacol concentration in the headspace as

well as bacterial counts were recorded.

39

3.14 Limitation of vanillin for guaiacol production

Vanillin was added to samples at different concentrations (0.00, 0.25, 0.05, 1.00,

2.00mg/L). There were three replicates for samples of each concentration of vanillin.

Samples were stored at 37°C, and tested at 24h when guaiacol reached its highest

point according to preliminary tests. 95% confidence interval was used to determine

the minimum concentration of guaiacol in the headspace that is significantly different

from the background.

3.15 Statistical Analysis

One-way analysis of variance (ANOVA) and Tukey’s post hoc analysis (RStudio

Statistical Software, RStudio, Inc, Richmond Hill, ON, Canada) were used to

determine statistical difference. P value of 0.05 was used for indicating significant

difference.

40

Chapter 4- Results and Discussion

4.1 The influence of temperature on bacterial growth and guaiacol production

Research was conducted in YSG (yeast starch and glucose) broth and apple juice

samples with A. acidoterrestris ATCC and OSYE. A. acidoterrestris OSYE was

isolated from commercial juice samples. It is thermophilic and acidophilic bacteria,

therefore, it can survive the pasteurization process of juice in the form of spores and

then produce the off-odor guaiacol (Chang and Kang 2004). A. acidoterrestris ATCC

is a well-studied and has been reported to produce the off-odor guaiacol in juice

(Gocmen and others 2005). YSG broth is a media with ideal conditions for bacterial

growth.

Temperature did not influence the final concentration of bacteria when they reached

the stationary phase in the sample, but did influence the time for bacterial

concentration to reach its stationary phase (Figure 5). Bacterial growth curves were

very similar for samples at 37 and 45℃, however bacteria at 25 or 20℃ took longer to

reach the exponential phase in YSG broth. A. acidoterrestris is thermophilic and the

optimum temperature for its growth is from 42 to 60°C (Chang and Kang 2004).

There are ω-alicyclic fatty acids in the membrane of A. acidoterrestris, which protect

the bacteria from high temperature and low acid conditions (Chang and Kang 2004).

41

The higher temperatures (37 or 45℃) increase the speed of metabolism due to the

increased activity of enzymes, but it does not influence the final concentration of

bacteria in the stationary phase as long as there are enough nutrients (carbon source,

nitrogen source, minerals, growth factors), and water. Others also reported that A.

acidoterrestris strains grew slower at 25 than 45°C in yeast starch glucose (YSG)

broth (Witthuhn and others 2013). Alicyclobacillus acidoterrestris 2498 can live and

grow at 25, 35, and 44°C but it cannot grow at 4°C in apple juice or orange juice

(Pettipher and others 1997).

In apple juice, there was no bacterial growth at 20 and 25°C (Figure 5). Some

natural compounds such as polyphenols in juice can inhibit the growth of bacteria

(Brodbeck and others 2009). The yeast extract, glucose and starch in YSG broth are

important nutritions which support the growth of bacteria. Therefore, in a perfect

media (such as YSG broth), temperature can only delay bacterial growth, while in

nutrient limited media such as apple juice, storage at room temperature can inhibit the

growth of bacteria. At the same temperature, bacteria grew slower in apple juice than

in YSG solution (Figure 5, 6).

Temperature did not influence the maximum amount of guaiacol produced, but did

influence the time for guaiacol production to reach its highest concentration (Figure

6). Guaiacol production is highly related to the bacterial growth rate. Bacteria grew

faster at 37 or 45°C than 20 or 25°C, leading to different time (18, 18.5, 282.5, 70h)

until guaiacol production under different temperature conditions (Figure 5, 6). There

42

was guaiacol production at all temperatures which were tested in YSG broth.

Guaiacol production occurred at almost the same time (18, 18.5h) at 37 or 45°C, but

longer time (282.5, 70h) were needed for guaiacol production at 20 or 25°C. Guaiacol

formation accelerated when incubation temperature increased from 32 to 50°C

(Chang and others 2015). Others also reported that in YSG broth, Alicyclobacillus

acidoterrestris can produce guaiacol at 25 or 45°C, and longer time was needed to

produce guaiacol at 25 than 45°C (Witthuhn and others 2013). On the other hand, a

different researcher also found no guaiacol formation at 25°C, in K broth with

different strains (Alicyclobacillus isolates1016 and 1101) during 48h (Chang and

others 2015).

There was no significant difference in the maximum amount of guaiacol produced

in YSG or juice samples or at different temperatures (Figure 5, 6). The growth media

only influenced the growth rate of bacteria, therefore leading to longer time needed

for guaiacol to reach its maximum in juice compared to YSG. In apple juice, no

guaiacol production occurred at 20 and 25°C, because there was no bacterial growth.

But there was bacterial growth at 37 or 45°C in apple juice which lead to guaiacol

production in the samples.

43

Figure 5. Bacterial counts influenced by temperature in YSG solution (above) and

apple juice (below) ameasurements with the same letter within the same sample have

no significant difference (p ≤ 0.05)

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250 300

Aver

age

nu

mb

er o

f b

act

eria

in

YS

G(l

og10C

FU

/mL

)

Time (h)

37℃

45℃

25℃

20℃

limit of detection

abde abef

bcdef

bcdefbde

bdebdebde

ff bcef

gg

hh h

defdef

a

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250 300

Aver

age

nu

mb

er o

f b

act

eria

in

ju

ice

(Log10C

FU

/mL

)

Time (h)

37 ℃

45 ℃

25℃

20℃

limit of detectionc

b

b

c

b b

abaa

c

c

44

Figure 6.Guaiacol production influenced by temperature in YSG solution (above) and

apple juice (below) ameasurements with the same letter within the same sample have

no significant difference (p ≤ 0.05)

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300

Ad

just

ed a

ver

age

gu

aia

col

con

cen

trati

on

in

hea

dsp

cace

of

YS

G

(pp

b)

Time (h)

37 ℃

45 ℃

25 ℃

20 ℃

h

bc

e

h

g

de

bce

h

f

bc

ab

h h

bc

a

abab

b bcbc

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300

Ad

just

ed a

ver

age

gu

aia

col

con

cen

trati

on

in

hea

dsp

cace

of

juic

e

(pp

b)

Time (h)

37 ℃

45 ℃

25℃

20℃

f

d

bb

c

f

e

aba

ff

45

4.2 The influence of pH on guaiacol production and bacterial growth

Adjusting the pH to 2.7-6.7 did not influence the final microbial count in the

stationary phase or guaiacol concentration, but did influence the time for bacteria or

guaiacol to reach the stationary phase (Figure 7). Higher pH (6.7) inhibited bacterial

growth thus no guaiacol was produced. Lower pH (2.7) delayed bacterial growth and

guaiacol production was delayed. Bacterial growth in the exponential phase occurred

the fastest at pH 3.7 and 4.7 with corresponding guaiacol production. Others reported

that guaiacol can be produced by Alicyclobacillus isolate 1016 in a pH range of 3.5 to

4.5 and Alicyclobacillus isolate 1101 in a pH range of 3.5 to 5.0 (Chang and others

2015). The maximum concentration of guaiacol was the same but required different

time to reach their maximum at different pH (Chang and others 2015). Longer time

was needed at pH 3.5 than pH 4 or 4.5 for Alicyclobacillus isolate 1016 and 1101 to

produce guaiacol; and no guaiacol production occurred at pH 5 (Chang and others

2015).

Guaiacol production started when bacterial counts exceeded 104 (about 105) (Figure

7). It has been reported that only when the number of A. acidoterrestris reach about

105~106cfu/ml, can it produce guaiacol (Pettipher and others 1997).

46

Figure 7.The influence of pH on guaiacol production and bacterial growth in YSG ameasurements with the same letter within the same sample have no significant

difference (p ≤ 0.05)

4.3 Effect of antimicrobial compounds

Lauric arginate and ɛ-polylysine had bactericidal and bacteriostatic effects on the

vegetative cells and spores of Alicycobacillus acidoterrestris ATCC and

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250 300 350

Bact

eria

nu

mb

er (

log c

fu/m

L)

Time (h)

pH 4.7

pH 3.7

pH 2.7

pH 6.7

Limit of detectionfgg

gg

a

g

ded

g

g gfg

ee

e

cd

abcd

bca

bcd

fg

g

abcd

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Gu

aia

col

con

cen

trati

on

(p

pb

)

Time (h)

pH 4.7

pH 3.7

pH 2.7

pH 6.7

a abbcdc

cdc

abc dde

f ffff ff f f f ff

f

ff

47

Alicycobacillus acidoterrestris OSYE in YSG broth and apple juice (Table 4).

However, there were turbidity in lauric arginate YSG samples and ɛ-polylysine in

apple juice samples for all tree replicates, which may because of some chemical

reactions which lead to the sedimentation.

Lauric arginate has a cationic head from L-arginine and a nonpolar tail from lauric

acid. It adheres to oil-water interfaces and crosses the cell membrane into the

cytoplasm, where it breaks the inter cell environmental balance and inhibits metabolic

processes (Rodríguez and others 2004). ɛ-Polylysine has a positive charge in solution,

thus because of electrostatic adsorption and osmotic pressure between cell

membranes, cationic ɛ-polylysine enters the cell membrane and disrupts the balance

of neutral charge in the cytoplasm causing damage to the cell (Shima and others

1984).

In control samples, the increase in bacterial counts during storage showed few

differences between juice and YSG broth or for different strains with spores or

vegetative cells (Table 4). In control samples, bacterial counts increased to a high

level (106 CFU/ml) and produced guaiacol. Bacterial counts in many samples had an

initial decrease in the first 6h due to adaptation to the new environment. An example

graph is showed in Figure 8. However, after 6h, bacterial counts increased in the

control.

With added antimicrobial compounds at their MICs values (9.4ug/ml lauric

arginate, 75ug/ml ɛ-polylysine), bacterial counts decreased to the detection limit of

48

10CFU/mL, and no guaiacol was produced in the samples (Table 4). Only in the YSG

solution with lauric arginate added, did the spore count of Alicycobacillus

acidoterrestris OSYE increase (Figure 8, Table 4). YSG broth is an ideal growth

media for bacterial growth, and spores are resistant to stressful environments because

of the coat, cortex and dipicolinic acid in core that can protect the core. Since the MIC

value was determined on vegetative cells, it is clear that the MICs were not high

enough to inhibit spore germination in an ideal media.

Others reported that the more lauric arginate in the sample, the greater the reduction

of L. monocytogenes in the sample (Soni and others 2010). ɛ-Polylysine also been

reported to have a marginally better inhibition against L. monocytogenes at pH of 5.5

than 7 (Najjar and others 2007).

49

Figure 8.The influence of antimicrobial compounds on vegetative cells (above) and

spores (below) of Alicycobacillus acidoterrestris OSYE in YSG

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 10 20 30 40 50

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Polylysine

Lauric

arginatelimit of

detection

aa

bbbb

cce cecd

cdfe

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50

Gu

aia

col

co

nce

ntr

ati

oin

(p

pb

)

Time (h)

Control

polylysine

lauric

arginate

b

ccc cc c c c c c

a

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 10 20 30 40 50

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Polylysine

Lauric

arginatelimit of

detection

ab

c

ddd de e

f g g

0102030405060708090

100

0 10 20 30 40 50Gu

aia

col

co

nce

ntr

ati

oin

(p

pb

)

Time (h)

Control

polylysine

lauric

arginate

a

bc

ddd ddd d d d

50

Table 4. Difference between the initial (0h) and final (48h for YSG and 72h for juice)

bacterial counts and guaiacol concentration

difference in bacterial count

(log cfu/ml)

difference in guaiacol

concentration (ppb)

Samples Control lauric

arginate

ε-

polysine Control

lauric

arginate ε-polysine

Juice with

spore of

ATCC

2.59 ab -2.10ed -2.08defgh 72.1AB 0.79D 0.88D

Juice with

spore of

OSYE

1.89bc -1.67defg -0.91d 90.0A 0.63D 0.63D

Juice with

vegetative

cell of

ATCC

1.55bc -1.83defg -1.58defgh 33.2C 1.38D -0.71D

Juice with

vegetative

cells of

OSYE

2.09abc -1.28defgh -1.10de 49.8C -0.71D -0.42D

YSG with

Spore of

ATCC

1.27c -1.03de -2.59gh 69.4B 0.57D 0.93D

YSG with

Spore of

OSYE

3.22a 2.28abc -2.37fgh 74.8AB 48.0C 0.17D

YSG with

Vegetative

cells of

ATCC

2.20abc -1.83defg -1.43defg 72.6AB 0.04D 0.54D

YSG with

Vegetative

cells of

OSYE

2.60ab -2.16efgh -3.13h 84.8AB 0.53D -0.08D

log population difference= Initial log cfu/ml– final log cfu/ml

ameasurements within a sample with the same letter are not significantly different

51

4.4 Limitation of substrate (vanillin) on guaiacol production

The correlation between the substrate (vanillin) concentration and guaiacol

production was highly linear in the range of 0.25-2mg/L vanillin (Figure 9). Others

have also reported that higher substrate concentration produced higher maximum

guaiacol: 100mg/L vanillin can produce about 61.7mg/L guaiacol while 1000mg/L

vanillin can produce about 170.8mg/L guaiacol (Witthuhn and others 2012).

Therefore, carefully controlling the amount of substrate can reduce the maximum

amount of guaiacol production in a sample. SIFT-MS was able to detect down to

0.1mg/L vanillin added or 3.6ppb of guaiacol in the headspace which produced a

significant difference from the background.

Figure 9.Relationship between the amount of vanillin (mg/L) and guaiacol

concentration in the headspace (ppb)

y = 7.7704x + 2.7935

R² = 0.9983

0

5

10

15

20

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

Gu

aia

col

con

cen

trati

on

(p

pb

)

Vanillin's concentration (mg/L)

52

Chapter 5-Conclusions

Temperature didn’t influence the final concentration of bacteria in the stationary

phase or concentration of guaiacol produced, but did influence bacterial growth rate

which determined the time until guaiacol production occurs. Temperature inhibited

bacterial growth at 25 and 20°C in apple juice but not at 37 and 45°C. pH 6.7

completely inhibited bacteria thus there is no guaiacol production; and pH 2.7 delayed

bacterial growth, thus leaded to delay on guaiacol production. However, bacteria grew

well at pH 3.7 and 4.7. Compared to apple juice, YSG broth is more ideal for bacterial

growth. Guaiacol was produced when the bacterial count reached about 105 CFU/mL.

9.4 and 75ug/ml were the minimal inhibitory concentrations for lauric arginate and

ɛ-polylysine respectively on Alicycobacillus acidoterrestris OSYE and ATCC. Lauric

arginate and ɛ-polylysine have bactericidal and bacteriostatic effect on Alicycobacillus

acidoterrestris vegetative cells and spores, except lauric arginate does not inhibit the

spores of Alicycobacillus acidoterrestris OSYE in YSG solution. When adding lauric

arginate to YSG samples and ɛ-polylysine to apple juice samples, there was turbidity,

which will influence the appearance of samples. Vanillin concentration was highly

related to guaiacol production, therefore carefully control the concentration of vanillin

can also reduce guaiacol production.

53

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Appendix: Tables and Figures

Table 5.Difference between the initial (0h) and final bacterial counts (48h for YSG or

72h for juice) among different conditions

Control

2.59ab Juice spore of ATCC

1.89bc Juice spore of OSYE

1.55bc Juice vegetative cell of ATCC

2.09abc Juice vegetative cells of OSYE

3.22a YSG Spore of OSYE

2.60ab YSG Vegetative cells of OSYE

2.20abc YSG Vegetative cells of ATCC

1.27c YSG Spore of ATCC

lauric

arginate

-2.10ed Juice spore of ATCC

-1.67defg Juice spore of OSYE

-1.83defg Juice vegetative cell of ATCC

-1.28defgh Juice vegetative cells of OSYE

2.28abc YSG Spore of OSYE

-2.16efgh YSG Vegetative cells of OSYE

-1.83defg YSG Vegetative cells of ATCC

-1.03de YSG Spore of ATCC

ε-polysine

-2.08defgh Juice spore of ATCC

-0.91d Juice spore of OSYE

-1.58defgh Juice vegetative cell of ATCC

-1.10de Juice vegetative cells of OSYE

-2.37fgh YSG Spore of OSYE

-3.13h YSG Vegetative cells of OSYE

-1.43defg YSG Vegetative cells of ATCC

-2.59gh YSG Spore of ATCC

63

Table 6. Difference between the initial (0h) and final (48h for YSG and 72h for juice)

guaiacol concentration among different conditions

Control

72.1AB Juice spore of ATCC

90.0A Juice spore of OSYE

33.2C Juice vegetative cell of ATCC

49.8C Juice vegetative cells of OSYE

74.8AB YSG Spore of OSYE

84.8AB YSG Vegetative cells of OSYE

72.6AB YSG Vegetative cells of ATCC

69.4B YSG Spore of ATCC

lauric

arginate

0.79D Juice spore of ATCC

0.63D Juice spore of OSYE

1.38D Juice vegetative cell of ATCC

-0.71D Juice vegetative cells of OSYE

48.0C YSG Spore of OSYE

0.53D YSG Vegetative cells of OSYE

0.04D YSG Vegetative cells of ATCC

0.57D YSG Spore of ATCC

ε-polysine

0.88D Juice spore of ATCC

0.63D Juice spore of OSYE

-0.71D Juice vegetative cell of ATCC

-0.42D Juice vegetative cells of OSYE

0.17D YSG Spore of OSYE

-0.08D YSG Vegetative cells of OSYE

0.54D YSG Vegetative cells of ATCC

0.93D YSG Spore of ATCC

64

Table 7.Volatile Compounds Measured in Headspace Scan Volatile Compound

Compound Reagent Reaction rate Mass

(m/z)

Produce

guaiacol NO+ 2.5E-9 124 C7H8O2+

2-ethoxyphenol O2+ 2.5E-9 138 C8H10O2

+

Table 8.The influence of storage time in water bath on volatilization

Time (h)

Guaiacol

in solution

(g/mL)

2-

ethoxyphenol

concentration in

solution (g/mL)

Guaiacol in

headspace (ppb)

2-ethoxyphenol

concentration in

headspace (ppb)

0.50 1.129 1.068 14.80 16.29

1.00 1.129 1.068 14.04 15.51

1.50 1.129 1.068 16.04 15.26

65

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

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 10 20 30 40 50

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Lauric

arginatePolylysine

limit of

detection

aa

bbbc

d d d

eff

0

10

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30

40

50

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70

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90

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aia

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pb

)

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lauric

arginate

polylysine

a

b

cc cc cc

cc cc

0.0

1.0

2.0

3.0

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6.0

7.0

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nu

mb

er o

f b

act

eria

(lo

gcf

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l)

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Polylysine

Lauric

arginate

limit of

detection

aa

bbbb

cdd

e e e

0

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50

60

70

80

0 10 20 30 40 50

Gu

aia

col

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nce

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ati

oin

(p

pb

)

Time (h)

Control

polylysine

lauric

arginate

a

b

ccc

ccc c c cc

66

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

0.0

1.0

2.0

3.0

4.0

5.0

0 10 20 30 40 50 60 70 80

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Polylysine

Lauric

arginatelimit of

detection

a

bbbbb

c

dd d dd

0

10

20

30

40

50

0 10 20 30 40 50 60 70 80

Gu

aia

col

co

nce

ntr

ati

oin

(p

pb

)

Control

polylysine

lauric

arginate

bbb

bbb bbb bb

Time (h)

a

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 10 20 30 40 50 60 70 80

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Lauric

arginate

Polylysine

limit of

detection

a

bbcc

b dddf

c

e

0

10

20

30

40

50

60

70

80

90

0 10 20 30 40 50 60 70 80

Gu

aia

col

co

nce

ntr

ati

oin

(p

pb

)

Time (h)

Control

lauric

arginate

Polylysine

a

bbb bb

bbb

b b b

67

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

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 10 20 30 40 50nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Polylysine

Lauric

arginatelimit of

detection

aa

bbbb

cce cecd

cde

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50

Gu

aia

col

con

cen

tra

tio

in (

pp

b)

Time (h)

Control

polylysine

lauric

arginate

b

ccc cc c c c c c

a

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 10 20 30 40 50

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

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Lauric

arginatePolylysine

limit of

detection

ab

c

ddd de e

f g g

0102030405060708090

100

0 10 20 30 40 50

Gu

aia

col

co

nce

ntr

ati

oin

(p

pb

)

Time (h)

Control

lauric

arginatepolylysine a

bc

ddd d

dd

d d d

68

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

0.0

1.0

2.0

3.0

4.0

5.0

0 10 20 30 40 50 60 70 80

nu

mb

er o

f b

act

eria

(lo

gcf

u/m

l)

Time (h)

Control

Lauric

arginatePolylysine

limit of

detection

a

bbbb

bc

ccc

c c

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70 80

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)

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lauric

arginatepolylysine a

bb

b bbb

bb b b b

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

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Polylysine

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arginatelimit of

detection

a

dd d

bbcb

c

b b

bce

0102030405060708090

100

0 10 20 30 40 50 60 70 80

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arginate

bbb bbb

bbb

a

b b