Analysis of ascorbic acid, pH and antioxidants across ... · Analysis of ascorbic acid, pH and antioxidants across value to premium range supermarket apple juices at varying temperatures

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Analysis of ascorbic acid, pH and antioxidants across value to premium range supermarket apple juices at varying temperatures and periods of exposure

Warne, G., Tatham, A. Centre of Health Care and Food, Cardiff Metropolitan University, Cardiff, CF5 2YB Contact: Supervisors Surname, initial and email address

Background

Ascorbic acid is an important antioxidant and essential

vitamin used as a cofactor for the hydroxylase enzymes for proline

and lysine, which are required in the synthesis of collagen (Baum et

al, 2010). Antioxidants, such as ascorbic acid, also provide a beneficial

biological defence mechanism against free radical damage of

important biological compounds, such as DNA (Baum et al, 2010).

Apple juice naturally contains low levels of ascorbic acid because of

the high levels of malic acid and low pH, although it is often added as

preservation (Ballus et al, 2012). However, these values been shown

by: Achir et al (2015); and Baum et al (2010), to be dependent on

storage conditions and processing method.

Methods

Six samples in three price ranges of apple juice were

measured for 4-7 days after opening. The antioxidant activity was

measured using the DPPH method used by Kedare and Singh (2011),

to determine the % radical scavenging activity. Ascorbic acid was

analysed by the Iodometric method as well as by kinetic modelling

using first and zero order kinetics (Hwa and Sapei, 2011).

Discussion A similar study taken by Gevilla et al (2011), found that

pasteurised apple juice (equivalent to medium price range) contains

higher levels of both: antioxidants; and ascorbic acid, than fresh juice

(equivalent to premium range). This agreed with the observed result

for medium range apple juices for ascorbic acid and Antioxidants. The

negative effect of days, on ascorbic acid, was consistent with all 3 of

the previous studies analysed (Achir et al, 2015; Ballus et al, 2012;

Hwa and Sapei, 2011).

Conclusion In conclusion, despite the expected increased quality from

medium to premium range apple juices, medium range apple juices

were far more likely to contain beneficial antioxidants, such as

ascorbic acid. The significant negative effect of exposure on the

ascorbic acid content over the first 4 days, suggests further research

is required on juices where this value is labelled on the bottle.

References Achir, N., Belbahi, A., Dornier, M., Madani, K., Mertz, C., Remini, H. (2015). Degradation kinetic modelling of ascorbic acid and colour intensity in pasteurised blood orange juice during storage. Food chemistry. 173(1), pp 665-673.

Ballus, CA., Filho, JT., Godoy, HT., Rybka, ACP., Scherer, R. (2012). Validation of a HPLC method for simultaneous determination of main organic acids in fruits and juices. Food Chemistry. 135(1), pp 150-154. Available from: http://ainfo.cnptia.embrapa.br/digital/bitstream/item/68195/1/Artigo-Ana-Cecilia-2012-1.pdf. Accessed: 07/11/16

Baum, M., Bellion, P., Digles, J., Dietrich, H., Eisenbrand, G., Janzowski, C., Will, F. (2010). Polyphenolic apple extracts: Effects of raw material and production method on antioxidant effectiveness and reduction of DNA damage in caco-2 cells. Journal of Agricultural and Food Chemistry. 58(11), pp 6636–6642.

Hwa, L., Sapei, L. (2014). Study on the kinetics of Ascorbic acid degradation in

fresh strawberry Juices. Procedia Chemistry. 9(1), pp 62-68.

Kedare, SB., Singh, RP. (2011). Genesis and development of DPPH method of

antioxidant assay. Journal of food science and technology. 48(4), pp 412-422.

Results Medium price range apple juices had significantly more

ascorbic acid (p<0.001) and antioxidant content than premium and

value ranges. The period of exposure after opening was shown to have

an effect on the: ascorbic acid content (p<0.001); antioxidant content

(P<0.001) and the pH value (P<0.001). There were no significant

differences observed between 4°C and 19°C for: pH, ascorbic acid or

the antioxidant activity.

Figure 1: Comparison of Radical Scavenging activity (RSA %) of 6 apple juice

samples, using DPPH, at 3 different price ranges across 5 days after opening.

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Day 0

Day 1

Day 2

Day 3

Day 4

Radical Savenging activity (RSA%)

Tim

e o

f ex

po

sure

(d

ays)

Value Range Medium range Premium Range

Table 1: Calculated half-life values of Ascorbic acid concentration (mg Ascorbic

acid/100ml) in apple juice at varying price ranges, temperatures and total average

over the first 168 hours after opening.

Price Range Determined Kinetic

Model1 K value

[Ascorbic acid0] mg/100ml

Half-life (hours)

Value range First Order kinetics 0.0039 - 177.7

Medium range First Order kinetics 0.0051 - 135.9

Premium range Zero Order Kinetics 0.0492 12.022 122.2

Total average Zero Order kinetics 0.076 21.863 143.8

1 The appropriate kinetic model was determined for each of the price ranges by assessing the respective linearity’s (R2). 2 The Initial concentration was based on the trend-line intercept x=0 y=12.02 3 The Initial concentration was based on the trend-line intercept x=0 y=21.86

Figure 2: Zero Order kinetic degradation of Ascorbic acid across all apple juice over 7

days after exposure

20.21 20.04 19.8717.99

13.50

8.53y = -0.076x + 21.86

R² = 0.9155

0.00

5.00

10.00

15.00

20.00

25.00

0 24 48 72 96 120 144 168

Asv

orb

ic a

cid

co

nen

trat

ion

(m

g/10

0ml)

Time (hour)

http://ainfo.cnptia.embrapa.br/digital/bitstream/item/68195/1/Artigo-Ana-Cecilia-2012-1.pdf


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CARDIFF METROPOLITAN UNIVERSITY

SCHOOL OF HEALTH SCIENCES

DEPARTMENT OF HEALTHCARE AND FOOD

BSc (Hons) Food Science and Technology

Research Project Academic Paper

Analysis of ascorbic acid, pH and antioxidants across

value to premium range supermarket apple juices at

varying temperatures and periods of exposure

Student Number:

March 2017

Supervisor: Arthur Tatham

Attention of: Dr Ruth Fairchild

Student Declarations in Respect of Individual Work

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Statement 1

Dissertation submitted in partial fulfilment of the requirements of Cardiff Metropolitan University for the Degree of

Bachelor of Science with Honours. This work has not previously been accepted in substance for any degree and is

not being concurrently submitted in candidature for any degree.

Signed:

Date: 26/03/17

Statement 2

I declare that the whole of this work is the result of my individual effort and that all quotations from other authors

have been acknowledged. Where corrections services have been used the extent and the nature of the correction

is clearly marked in a footnote.

Signed:

Date: 26/03/17

Statement 3

I hereby give consent for my research project, if accepted, to be available in the open source repository D-space

for use inside and outside the University.

Signed:

Date: 26/03/17

Statement 4

I hereby give consent for my research data and/or portions of my report to be used by my supervisor in the

creation of academic articles for publishing, provided that I have appropriate authorship on these papers.

Signed:

Date: 26/03/17

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Abstract Purpose: The purpose of this research was to determine the antioxidant, pH and ascorbic acid content in 6

apple juice samples, at 3 different price ranges. Their stability was analysed over 7 days after opening at 4°C

and 19°C.

Design: Ascorbic acid was determined using Iodometric titration and zero and first-order kinetics.

Antioxidants were determined using the α-diphenyl-β-picrylhydrazyl and visible-light spectroscopy method

and expressed as % Radical scavenging activity.

Findings: A statistically significant effect of exposure time was observed for ascorbic acid (F=163.2; df=3.047;

P<0.001); antioxidants (F=83.029; df=2.268; p<0.001); and pH (F=27.01; df=2.554; P<0.001). The average

degradation of ascorbic acid was found to follow zero-order kinetics with a half-life of 6 days. Value range

apple juice followed first-order kinetics and had the longest half-life (7.4 days) of the 3 prices. Medium range

apple juice contained significantly more ascorbic acid than both value (MD=-38.31; p=0.002) and premium

range (MD=-31.18; p=0.008). Medium range also contained more antioxidants than both value (MD=-5.077;

p=0.002) and premium range apple juices (MD=8.373; p<0.001). No significant effect of temperature was

found in either: pH (p=0.812); antioxidants (p=0.253); or ascorbic acid. The data was however limited by the

use of just 1 sample in ambient conditions. It was clear, however, that opened apple juice deteriorated

dramatically over the shelf-life. This could affect the labelling requirements of the juices which declare

ascorbic acid levels.

Originality/value: This study was found to be unique in specifically comparing the commercial value to

nutritional benefit. New research was also made in analysing the kinetic degradation of ascorbic acid in apple

juice.

Keywords: Ascorbic acid, antioxidants, pH, storage, price, temperature

Paper type: Research Paper

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1.0 Introduction The consumption of apple juice has been shown to be important for the regulation of vital biochemical

processes as well as protection against colon cancer and free radical damage to DNA (Alvarez-Parrilla et al,

2010; Castafleda-Ovando et al, 2009; Baum et al, 2010). This has been attributed to the antioxidants and

ascorbic acid content in apple juice (Alvarez-Parilla et al, 2010; Baum et al, 2010; and Ishigami et al, 2013).

Whilst ascorbic acid is naturally low in apple juice, because of the high malic acid concentration, it is typically

added to prevent the initiation of Maillard reactions (Dong et al, 2016). This is added to attach to free radical

sugars that would normally attach to amino acids in the start of this Maillard process (Dong et al, 2016).

Ascorbic acid is an essential for various biochemical purposes; its principal purpose is the biosynthesis of

collagen (Baum et al, 2010). This requires its active form, L-ascorbic acid, for use as a cofactor with the

hydroxylase enzymes used in the hydroxylation of proline and lysine (key amino acids in collagen; Ishagimi

et al, 2013). The ascorbic acid is also required for the initial stimulation of the gene expression required for

the production of collagen (Ishigami et al, 2013).

As more ascorbic acid is oxidized, by the free radicals, it degrades to dehydroascorbic acid, which is then

hydrolyzed into the permanent 2,3-diketogulonic acid form (Hendrickx et al, 2010). These free radicals

include: lone oxygen atoms; hydrogen peroxide, iron, zinc, copper and free sugars (Ballus et al, 2012; Guo et

al, 2012). Free radicals have been shown to increase when exposed to air and ultra-violet light (Hendrickx et

al, 2010; Hwa and Sapei, 2014).

All three studies found to use kinetic modelling, found that fruit juices best fitted zero and first order

kinetics (Achir et al, 2015; Burdurlu et al, 2006; Hwa and Sapei, 2014). Therefore, both zero-order and first

order kinetics were used to analyse the degradation of ascorbic acid in apple juice. Zero-Order kinetics

describes a relationship where the concentration of ascorbic acid is affected by time (Hwa and Sapei, 2014).

However, the rate at which ascorbic acid binds to free radicals is independent of both of their concentrations

(Hwa and Sapei, 2014). First-Order kinetics, however, describes an association between ascorbic acid and

time, the rate of which is dependent on the concentration of either free radicals or ascorbic acid (Achir et al,

2015).

In premium range apple juice samples, yeasts are present and are able to aerobically metabolise the

naturally present monosaccharides (Awojuyigbe et al, 2016). Furthermore, Awojuyigbe et al (2016), found

an increase in osmophilic yeast from days 1 (2.017log10CFU/ml) to 5 (3.903log10CFU/ml). This would increase

ethanol levels and CO2, inhibiting the production of lactic acid producing bacteria, and hence limiting the

decrease in pH levels (Awojuyigbe et al, 2016).

A large number of different antioxidants have been found within apple juice, including:

hydroxycinnamic acids; flavanols; quercetin-3-glucoside; catechols; and catechins (Baum et al, 2010;

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Castafleda et al, 2009; Meca et al, 2014). These have been shown to provide multiple beneficial protective

effects from the radical oxidative species (Hendrickx et al, 2010; Baum et al, 2010). Baum et al (2010), found

that antioxidants protected deoxyribonucleic acids from oxidative stress by actively reducing free radicals. It

has been suggested that this process is initiated by ascorbic acid (Cotty et al, 2012).

2,2'-azino-bis-3-ethylbenzotiazolin-6-sulfonic acid (ABTS) assays have been used by: Baum et al (2010);

Grimi et al (2011); and Meca et al (2014), to identify the antioxidant content. This, as well as DPPH (α-

diphenyl-β-picrylhydrazyl), are typically used in conjunction with Trolox equivelents (TEAC) or equivalents of

ascorbic acid (VEAC; Meca et al, 2014; Baum et al, 2010; Gervilla et al, 2011; Grimi et al, 2011; Kedare and

Singh, 2011). However, these are more representative of samples where α-tocopherol and ascorbic acid

make up a large proportion of the total antioxidants (Gervilla et al, 2011). This is because of the differences

in interactions between DPPH and different antioxidants (Kedare and Singh, 2011). Hence, ABTS and Trolox

or ascorbic acid equivalents were not used for the apple juice samples because of their typical lack of both

Trolox and ascorbic acid (Grimi et al, 2011).

The change in colour in DPPH, from an intense violet colour to a faded, is attributed to the chromophoric

delocalised electron (Kedare and Singh, 2011). The amount to which a discolouration is observed, is

therefore directly proportional to degree of Radical scavenging activity (%RSA; Kedare and Singh, 2011). This

is because of the ability of antioxidants to donate a hydrogen to the free radicals (in this case DPPH; Alvarez-

Parrilla et al, 2010). Hence, DPPH was used because it is a reliable and effective method of determination of

the antioxidant content.

The difference in processing of apples has been shown to have an effect on both the quality and price of

apple juice (Gerville et al, 2011; Leong and Oey, 2012). All apple juices will have gone through washing and

sterilisation before being pressed with the addition of process aiding enzymes, such as: pectinases, and

polygalacturnonase (Gervilla et al, 2011). The value range apple juice would have also been through

decantation, filtration and then dehydration (Gervilla et al, 2011). All of which have been associated with

loss of quality, including reduction in antioxidants, such as ascorbic acid (Gervilla et al, 2011; Leong and Oley,

2012). The medium range juices were not filtered or from concentrate, but they would have been

pasteurised (Gervilla et al, 2011). For premium range apple juices, it is typically just pressed without

pasteurisation or filtration and therefore, it is perceived as better quality (Gervilla et al, 2011).

The intention of this research was to analyse the effects of external conditions, such as: storage

temperature; time of exposure; and financial value, on the quality of 6 apple juice samples. This quality was

assessed by the variance in ascorbic acid, pH and antioxidant capacity across these multiple external

conditions. It was also intended to determine the internal correlation between Ascorbic acid, pH and

Antioxidant capacity across all days using inferential statistical tests. One of the principal reasons for carrying

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out research into the degradation of Ascorbic acid and antioxidant capacity during storage and exposure,

was to evaluate the potential implications on the ‘on-pack’ declaration (Burdurlu et al, 2006). The effects of

storage conditions and price on pH in apple juice were analysed.

2. Materials and Methods This research was approved on 12/10/2016 by the Health Care and Food Ethics Panel, Cardiff

Metropolitan University, prior to data collection.

2.0 Materials

2.01 Fruit Juice samples

Value range (£0.50p-£1.00/litre)

- 1 litre Sainsbury’s chilled basics pure apple juice from concentrate (80p/litre): Sample A

- 1 litre Tesco’s pure apple juice from concentrate (65p/litre): Sample E

Mid-range (£1.00-£2.00/litre)

- 1 litre Tesco’s 100% pressed apple juice (£1.29/litre): Sample B

- 1 litre Sainsbury’s chilled sweet and crispy apple juice (£1.25/litre): Sample D

Premium range (£2.00-£3.00/litre)

- 1 litre Waitrose 1 cloudy pressed Cox apple juice (£2.65/litre): Sample C

- 1 litre Grove Organic fruit company apple juice (£2.94/litre): Sample F

Sample preparation

150ml of each fruit juice was taken out of the containers each day, for the ascorbic acid, pH, brix and

antioxidant capacity assays. This was carried out to simulate the increased air capacity that would be present

after the removal of an average serving (150ml).

2.02 500ml Iodine solution

5g Potassium iodide (KI), 0.268g potassium iodate (KIO3), 5ml of 18M sulphuric acid (H2SO4) and then

made up to 500ml in a volumetric flask with deionized water (H2O; Hwa and Sapei, 2014).

2.03 100ml 1% starch solution

1g of starch and 100ml of deionised water were heated until boiling whilst continuously stirring and

then allowed to cool to room temperature. This was diluted with deionised water up to 100ml in a volumetric

flask.

2.04 100ml 0.1% Ascorbic acid standard

0.1g of ascorbic acid was added to a volumetric flask and then filled to 100ml with deionised water

(0.1mg/100ml)

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2.05 100ml α-diphenyl-β-picrylhydrazyl (DPPH) solution

The α-diphenyl-β-picrylhydrazyl (DPPH) solution (2mM) was made methanol (Kedare and Singh,

2011). The DPPH deteriorates over time, hence new DPPH solutions were made on days: 0; 2; and 4 (Kedare

and Singh, 2011).

2.06 Source of reagents

All chemicals and reagents were sourced from Sigma-Aldrich Company Ltd, Gillingham, UK.

2.1 Methods

2.11 Ascorbic acid determination using Iodine titrations

The ascorbic acid content of the samples were determined (in mg/100ml) by titrating it with the

iodine solution. The 1% starch solution was used as a colour change indicator to observe the end point (Hwa

and Sapei, 2014). 25ml of each of the samples were placed in conical flasks with 10 drops of starch indicator

solution (Hwa and Sapei, 2014). This was then titrated with the iodine solution until the end point was

reached. Whilst High Performance Liquid Chromatography can be used (Leong and Oey, 2012; Albanese, et

al, 2010; and Ballus, et al, 2012), it can take up to 15 minutes per sample. The volumetric Iodine titration

method was therefore much quicker, and hence more appropriate for the number of samples that were

required to be analysed (Hwa and Sapei, 2014). All samples were analysed in triplicate.

2.12 Ascorbic acid standardisation curve

The Ascorbic acid standard was titrated with the Iodine solution using the method above, in three

different dilutions with deionised water (H2O). These were: 25mg/25ml; 12.5mg/25ml; and 5mg/25ml (Hwa

and Sapei, 2014). The Iodine solution was titrated until the end point was observed. The gradient value

(0.7851), shown in Figure: 1, was then used to subsequently calculate the ascorbic acid concentration based

on the titration volume.

Figure 1: Iodometric standardisation line using three known dilutions of ascorbic acid (5, 12.5 and

25mg/100ml)

19.5

10

4.1

y = 0.7851xR² = 0.9996

0

5

10

15

20

25

0 5 10 15 20 25 30

Iod

ine

titr

atio

n v

olu

me

(ml)

Ascorbic acid standard (mg/25ml)

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2.13 Antioxidant Capacity assay

The antioxidant capacity was measured using VIS-spectrometry at a wavelength of 517nm on a visible

light (200-1100nm wavelength range) spectrophotometer (CE-1021 Spectrophotometer, Cecil Instruments

Limited, Cambridge, UK; Kedare and Singh, 2011).

0.5ml of each of the apple juice samples were added to separate cuvettes containing 3.5ml of

methanol, as well as into a blank of 3.3ml of methanol (Kedare and Singh, 2011). 0.3ml of the DPPH solution

was then added to the cuvettes containing the samples (excluding the blank) and a control cuvette of 3.5ml

of ethanol. These were inverted and then kept in the dark for 30mins (Kedare and Singh, 2011). Before

adding the samples to the spectrophotometer, they were rapidly inverted to prevent the pulp from settling.

Without this inversion, prior to analysis, it was found that the pulp would diffract the light beam, causing the

absorbance values to continually fluctuate. For each of the samples, the absorbance value was zeroed on

the blank cuvette, and the control was measured before analysing the respective samples (Kedare and Singh,

2011). This DPPH method was used because of its reliability of measuring antioxidants. All samples were

analysed in triplicate and measured on their Radical Scavenging Activity (RSA%; Kedare and Singh, 2011).

RSA%= (1 −𝐴𝑠

𝐴𝑐) × 100

As= absorbance value of the sample

Ac= Absorbance value of the control

2.14 Brix

The °Brix was measured using a sugar refractometer (Digit 0-32 ATC, Medline Scientific, Chalgrove,

UK) with a range of 0-32°Brix, measured at 20°C (Albanese, et al, 2010).

2.15 pH

The pH was measured using a digital pH metre (Hydrus 300, Fisher Scientific Ltd., Loughborough, UK)

with a calibration at pH of 4, measured at 20°C (Albanese, et al, 2010).

2.16 Storage

Sample: A was kept at ambient temperature (19°C) for the 4-day duration to assess the difference in

storage temperature on the ascorbic acid, antioxidant and pH levels once opened. Samples B-F were kept in

refrigerated conditions (4°C) for the 4-day duration once opened.

2.17 Analysis of data

Descriptive statistics were found using Excel 2016 (Microsoft Excel workbook 2016 for Windows,

Microsoft, Redmond). The data was analysed using SPSS 23.0 (IBM SPSS statistics 23.0 for windows, IMB

corporation, New York) for the following tests; Pearson’s correlation on the association between ascorbic

acid, pH and RSA%; Repeated measures general linear model Anova was used for comparing temperature

and ascorbic acid, pH and RSA%, price value and ascorbic acid, pH and RSA%; and all samples and ascorbic

acid, pH and RSA%. All tests with probability of chance factors (p) of less than 0.05, were deemed statistically

significant.

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3. Results 3.1 Statistical correlation between: pH, antioxidant capacity, and ascorbic acid, across all apple juice samples

The null hypothesis, ‘there is no association between antioxidant capacity, ascorbic acid content and

pH’, was assessed using 3 Pearson’s correlation tests (SPSS statistics 23.0; n=90). It was found that there was

a significant (p<0.001) medium positive correlation (r=0.368) between the antioxidant capacity (RSA%) and

the acidity (pH). The correlation between ascorbic acid and pH, however, was not statistically significant

(p=0.470). There was a statistically significant (p=0.012) small positive correlation (r=0.265) between

ascorbic acid and RSA%. The null hypothesis was therefore kept for the relationship between ascorbic acid

and pH, but it was rejected for: pH and RSA% and ascorbic acid and RSA.

3.21 Statistical analysis on the effect of days on antioxidant content across all apple juice samples

A ‘repeated measures general linear model’ test (SPSS 23.0) was carried out to assess the null

hypothesis, ‘there is no statistically significant change in the radical scavenging activity (RSA%) in apple juice

across the five 5-day period of exposure’. There was a large and statistically significant effect of the period

of exposure (days) on the antioxidant content across all apple juices (Wilks Lambda (λ)=0.011; p<0.001;

partial eta squared (η)=0.989). This was verified by the Greenhouse Giesser correction (F=83.029; df=2.268;

p<0.001). This was partly attributed to the significant increase in antioxidants between days 1 and 5 (MD: -

2.491; p<0.001). Therefore, the decision was to reject the null hypothesis.

Figure 2: Comparison of Radical Scavenging activity (RSA %) of 6 apple juice samples, using DPPH, at 3

different price ranges across 5 days after opening.

3.31 Statistical analysis on the effect of days on ascorbic acid content across all apple juice samples

A ‘repeated measures GLM’ test (SPSS 23.0) was used to test the validity of the null hypothesis, ‘there

is no statistically significant change in the ascorbic acid content in apple juice across the five-day period of

exposure’. The time of exposure (days) had a statistically significant negative effect on the ascorbic acid

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Day 0

Day 1

Day 2

Day 3

Day 4

Radical Savenging activity (RSA%)

Tim

e o

f ex

po

sure

(d

ays)

Value Range Medium range Premium Range

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concentration (F=163.2; df=3.047; P<0.001). Furthermore, a statistically significant decrease in ascorbic acid

values was observed between day 7 and the initial day of exposure (day 0; MD=-11.676+/-0.358; p=<0.001).

The null hypothesis, therefore, was rejected.

3.32 Kinetic modeling of ascorbic acid degradation in apple juice

The ascorbic acid values, recorded as the average across all samples, showed slightly higher linearity

with the zero-order kinetic model (R2= 0. 9155) compared to first-order kinetics run on the same data set

(R2= 0. 9031). The average apple juice samples were found to follow zero order kinetic modeling. From this,

the half-life (t1/2) of Ascorbic acid, across all apple juice samples, was determined.

Equation 1: Zero-Order Kinetics Ascorbic acid Half life

𝑡12

=[𝑉𝑖𝑡𝑎𝑚𝑖𝑛 𝐶0]

2𝑘

Equation 2: First-Order Kinetics Ascorbic acid Half life

𝑡12

=𝐿𝑛(2)

𝑘

Where ‘t’ is time (hours), ‘Ascorbic acid0’ is the initial concentration of ascorbic acid in mg/100ml and ‘k’ is

the reaction rate coefficient represented by the negative gradient (-m).

Table 1: Calculated half-life values of Ascorbic acid concentration (mg Ascorbic acid/100ml) in apple juice at

varying price ranges, temperatures and total average over the first 168 hours after opening.

Price Range Determined

Kinetic Model1

R2 of kinetic model K value

[Ascorbic acid0]

mg/100ml

Half-life (hours)

Half-life (days)

Zero Order

First Order

Value range First Order kinetics 0.933 0.958 0.0039 - 177.7 7.4

Medium range First Order kinetics 0.854 0.865 0.0051 - 135.9 5.7

Premium range Zero Order

Kinetics 0.939 0.860 0.0492 12.022 122.2 5.1

Total average Zero Order kinetics 0.9155 0.9031 0.076 21.863 143.8 6.0 1 The appropriate kinetic model was determined for each of the price ranges by assessing the respective linearity’s (R2). 2 The Initial concentration was based on the trend-line intercept x=0 y=12.02 (Appendix: 2; Figure: 9). 3 The Initial concentration was based on the trend-line intercept x=0 y=21.86 (Figure: 2)

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Figure 3: Zero Order kinetic degradation of Ascorbic acid across all apple juice over 7 days after exposure

3.33 Ascorbic acid, antioxidant capacity and pH for all apple juice samples from the initial day of opening

The ascorbic acid content for samples A-F was found to range between 1.698mg/100ml (samples D

and F) and 74.55mg/100ml (sample B). The mean value across all samples was found to be 20.21+/-25.74.

The antioxidant content was found to range from 75.70% (sample C) to 95.64% (sample D), with a mean

value of 87.59+/-6.42%. The pH had a mean of 3.44+/-0.12 and a range of: 3.220 (sample c) to 3.563 (sample

E).

3.4 pH

3.41 Range of pH values across all apple juices over the 5 day period

The range of pH, on the initial day of opening (Day 0), was recorded at: 3.220 (sample C) to 3.563

(sample E) with an average of 3.440+/-0.12. Days 1, 2, 3 and 4 had average values of: 3.431 +/-0.12; 3.456+/-

0.11; 3.420+/-0.12; and 3.450+/-0.11 respectively. The combined average pH value across all samples and

days was determined to be 3.450 +/- 0.12.

3.42 Statistical analysis on the effect of days on the acidity (pH) of apple juices

The effect of exposure on pH was carried out using a ‘repeated measures general linear model test

(SPSS 23.0). The exposure time (days) had a large and statistically significant effect on the pH values (F=27.01;

df=2.554; P<0.001). This a significant increase was also observed over the 4 days after exposure (MD:-0.064;

p<0.001). The null hypothesis, ‘there is no statistically significant change in the pH content in apple juice

across the five 5-day period of exposure’, was therefore rejected.

3.5 Price value comparison of apple juice samples across 5 days of exposure for Ascorbic acid, pH and %

Radical scavenging activity

One-way Anova tests and repeated measures general linear model tests were carried out with a

Tukey post hoc test (SPSS 23.0). These were used to test the validity of the null hypothesis, ‘There is no

correlation between the amounts of ascorbic acid, pH and antioxidant potential, and the price of apple

20.21 20.04 19.87

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0 24 48 72 96 120 144 168

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Page | 13

juices’. The multiple comparison analysis showed no significant differences in pH between value and mid-

range apple juices (MD=0.002; p=0.999). There were, however, significant differences in pH between value

and premium range (MD=0.198; p<0.001) and between mid and premium range apple juices (MD=0.197;

p<0.001).

For ascorbic acid, there were significant differences in value and mid-range (MD=-38.31; p=0.002),

and mid and premium range (MD=31.18; p=0.008), across the 7-day period. There was, however, no

significant difference between value and premium range apple juices (MD=-7.133; p=0.705).

A statistically significant difference was found in the RSA% value in all price ranges, including: value

and premium range (MD=3.296; p=0. 040); value and mid-range (MD=-5.077; p=0.002); and mid and

premium range (MD=8.373; p<0.001) apple juices. The null hypothesis was therefore only kept for pH in

value and mid-range apple juices as well as for the ascorbic acid content of value and premium range.

3.6 Statistical analysis between ambient (19°C) and refrigerated (4°C) apple juice samples for Ascorbic acid,

RSA% and pH over the first 4 days after opening

Repeated measures general linear model test was carried out (SPSS 23.0), to assess the null

hypothesis, ‘The storage temperature has no effect on the pH, antioxidant capacity (%RSA) or ascorbic acid

content’. The pairwise comparison analysis showed no significant differences between ambient (19°C) and

refrigerated apple juice samples for pH values (p=0. 812). Ascorbic acid also showed no significant difference

between the two temperatures (p=0.214) for the first 7 days after opening. No statistical significance was

observed for the RSA% of the apple juice (p=0.253). The null hypothesis was kept.

4.0 Discussion

The Intention of the research was to identify the effect of storage conditions, price range and

exposure time on the acidity (pH), ascorbic acid (ascorbic acid) and Antioxidant content (%RSA) of various

apple juice samples. It was also intended to observe the correlation between Ascorbic acid, pH and

Antioxidant capacity over the first 5 days after opening.

4.1 Correlation between ascorbic acid, antioxidant activity and pH

The small positive association, found between: ascorbic acid and antioxidants, was attributed to the

antioxidant properties of ascorbic acid (Castafleda-Ovando et al, 2009). This was consistent with the studies

taken by: Ignat et al (2011); and Castafleda-Ovando et al (2009), which found that ascorbic acid had a small

antioxidant potential.

A medium positive correlation between %RSA and pH was observed. This was comparable with the

findings of: Castafleda-Ovando et al (2009); and Dong et al (2014), which showed that the antioxidant

capacity increased with increasing pH. This has been attributed to a decrease in scavenging activity caused

by changes to the structure of antioxidants, at increased concentrations of hydrogen ions (Castafleda-

Page | 14

Ovando et al, 2009). This study shows that ascorbic acid was found to be independent of the pH, which

suggests that the acidity of the solution was unchanged by the dehydrogenation of ascorbic acid (Ballus et

al, 2012).

These associations are important in the understanding of what influences important nutritional and

quality attributes in juice. Because all juices had a small observed standard deviation in pH (3.440+/-0.12),

this study was not accurately representative of the effect of pH on the antioxidant values. Further research,

therefore, on the influence of pH on the antioxidant content, across varying dilutions of fruit juices, should

be carried out. This could help with the understanding of the exact nature of antioxidants and how more

antioxidants can be incorporated into fruit juice.

4.2 The effect of storage temperature of apple juice samples on their ascorbic acid, antioxidant capacity and

pH

The observed difference between ambient and refrigerated conditions was not significant for either:

pH; ascorbic acid; or the antioxidant capacity. This was consistent with the findings of Gutierrez-Mendez et

al (2014; using apple juice), which also showed no significant difference, in either antioxidant capacity or pH,

between two storage temperatures (4°C and 20°C).

However, research carried out by: Achir et al (2015); Guiterrez-Mendez et al (2014); and Hwa and

Sapei (2014), found that that ascorbic acid decreased significantly more at room temperature (19-28°C)

compared to refrigerated conditions (4-8°C). The principal reason for not observing differences in ascorbic

acid, between the two temperatures, was determined to be the stark differences in initial concentration

between the different apple juice samples.

For future studies, pairs of samples (one at 4°C, the other at 20°C), with the same initial ascorbic acid

concentration, should be used. Testing the differences between ambient storage and refrigerated storage

provides information on how the temperature is influencing the stability and quality of apple juice. Further

testing should also compare both opened and unopened fruit juices in refrigerated conditions, directly after

industrial production. This would avoid the limitation of potential differences in remaining unopened shelf-

life between different studies.

4.3 The effect of exposure to air over 4 days on the antioxidant content of apple juice samples

A change in all three variables were observed over the respective periods of exposure. The increase

in antioxidants, observed over the 4 days after opening, contradicts studies such as: Guitierrez-Mendez et al

(2014), who found significant reductions in antioxidant capacity (using DPPH) of apple juice over 34 days.

Reduction of radical scavenging ability (using DPPH) has also been seen after just 2 weeks in filtered (juice

and pulp measured separately) golden delicious apple juice (Camangi et al, 2015).

The potential reason for this was a difference in the methods used. In this research, however, four of

the apple juice samples contained pulp dispersed in the juice. The rapid inversion of the cuvettes did help to

Page | 15

disperse the pulp, but only suspended the particulates without dissolving them. Therefore, there was still a

potential for unwanted diffraction, which could have affected the absorbance readings (Kedare and Singh,

2011). It was concluded that since research, taken by Camangi et al (2015) found antioxidants in the pulp,

removing it via filtration would also give an inaccurate reading of the antioxidant content.

The interference of pulp in the DPPH method was an important issue which needed to be addressed,

because of its presence in a wide range of fruit juices (Camangi et al, 2015; Guitierrez-Mendez et al, 2014; Li

et al, 2015). Furthermore, it has been shown that rapid inversion did not completely solve this issue. It has

also been shown that antioxidants exist within this pulp; this could affect the results if it is removed by

centrifugation or filtration (Camangi et al, 2015; Guitierrez-Mendez et al, 2014; Li et al, 2015). Further

analysis of antioxidant content, using DPPH, should be developed to ensure even dispersion of the pulp.

4.4 The effect of exposure to air over 4 days on the pH value of apple juice samples

Whilst a significant effect of days on the pH value was found, the values fluctuated over the 5-day

period showing troughs on days 1 (3.431+/-0.12) and 3 (3.420+/-0.12; Table: 2). The significant increase from

the original pH of 3.44 to 3.45 on day 4 (Table: 2), was consistent with the results from Awojuyigbe et al

(2016). Awojuyigbe et al (2016) showed that apple juice slightly increased in pH after 4 days after opening.

However, a significant difference was not observed by Guiterrez-Mendez et al (2014), in unopened samples

in the first 6 days (Guiterrez-Mendez et al, 2014).

It was found that these troughs in pH were similar to those observed in the total bacterial count

(Awojuyigbe et al, 2016). This association could be attributed to lactic acid producing bacteria (Awojuyigbe

et al, 2016). However, the removal of 150ml every day might have allowed aerobic metabolism in the

naturally present yeast, to have inhibited the reproduction of lactic acid producing bacteria (Awojuyigbe et

al, 2016). The importance of this is that it demonstrates that the acidity does not increase over the period,

hence the quality of apple juices, with regards to acidity, is unlikely to be affected (Meca et al, 2014). Future

research should look into the production of ethanol in apple juice to identify its role in reducing quality over

the shelf-life.

4.5 The effect of exposure to air over 7 days on ascorbic acid concentration in apple juice samples

The time of exposure had a large negative effect on the total ascorbic acid content. This is a similar

result to the studies taken by: Achir et al (2015; using orange juice), who found that the ascorbic acid content

decreased after 5 days at both 4°C and 20°C; and Guiterrez-Mendez et al (2014), who found that over the

first 6 days the ascorbic acid content decreased from 21.18mg/100ml to 4.71mg/100ml at 4°C. This was

agreed by all four of the other studies found on the degradation of ascorbic acid over time (Ballus et al, 2012;

Burdurlu et al, 2006; Hwa and Sapei, 2014; Leong and Oey, 2012).

Across all apple juice samples, the ascorbic acid degradation was observed to follow the zero order

kinetic model. This suggests that the degradation rate was independent of free radical and ascorbic acid

Page | 16

concentration (Hwa and Sapei, 2014). This is consistent with the findings of Hwa and Sapei (2014), who found

that the degradation of ascorbic acid across all strawberry juices followed zero order kinetic modelling. Achir

et al (2015; using blood oranges) and Burdulu et al (2006; using citrus juices), however, found the ascorbic

acid degradation followed first-order rate kinetics across all samples. Achir et al (2015) also found the half -

life of ascorbic acid was 13.9 days in refrigerated conditions (4°C). This was contradictory to the half-life

observed for the average of all apple juice samples (6.0 days).

The degradation of ascorbic acid has been attributed to the ability of ascorbic acid to reduce free

radicals, such as: free hydroxyl groups and single oxygen atoms (Baum et al, 2010). Another impact on

ascorbic acid content in the fruit juices is the natural availability of the ascorbic acid oxidase enzyme, which

catalyses the reaction of paired oxygen with ascorbic acid making DHAA (Li et al, 2011).

The importance of this research was that it demonstrates how the ascorbic acid degradation could

affect other fruit juices, where the ascorbic acid content is labelled ‘on pack’. The partial limitation of this

data, however, was the use of a sample kept in ambient conditions (19°C) and 5 kept at 4°C. The issue with

this was that the temperature could have different effects on the ascorbic acid, antioxidant and pH contents

(Guitierrez-Mendez et al, 2014). Future studies could include identifying any correlations between ethanol

concentration and both forms of ascorbic acid (ascorbic acid and dehydroascorbic acid).

4.6 The effect of commercial value and processing on ascorbic acid, antioxidants in apple juices.

The apple juice at value range followed first order kinetics, suggesting that the rate of degradation is

affected by the concentration of either the: free radicals, or ascorbic acid (Achir et al, 2015). This was in

agreement with the study found by: Achir et al (2015), who found that the ascorbic acid degradation in

orange juice followed first order kinetics when filtered and pasteurised.

The value range apple juices had the longest ascorbic acid half-life of 7.4 days (Table: 1), despite

starting out with the least amount (Figure: 2). This was considerably less than the half-life of 13.9 days,

observed by Achir et al, 2015). This implies that juices which have been through the least processing

(premium juices) are more likely to contain more free radical producing compounds (Hwa and Sapei, 2014).

Furthermore, it has been suggested by: Hendrickx et al (2010), that the ascorbic acid enzyme is also

inactivated during pasteurization treatments and hence improves the bioavailability of ascorbic acid.

Despite this, higher levels of both: antioxidants and ascorbic acid, were observed in medium range

juices. This agrees with: Gervilla et al (2010), who found that unfiltered (cloudy) apple juice, which had been

pasteurised, had significantly higher antioxidant capacity (769-902µM TEAC) than fresh juice (334-421µM

TEAC). This was also found for ascorbic acid concentrations between fresh (0.044mg/100ml) and pasteurised

(0.46mg/100ml; Gervilla et al, 2011). The potential implications of this is that the combination of

Page | 17

pasteurisation and lack of filtration, in medium range apple juices, provides optimal conditions for nutritional

benefit (Hendrickx et al, 2010; Gervilla et al, 2011).

Studies were not found to detail price ranges of fruit juices, however, comparisons were made for

the different processing conditions they would have under gone. This limits the applications of this study,

because the exact nature of processing conditions were not able to be observed. Future studies, should

explore if the commercial value of other fruit juices effects the levels of beneficial antioxidants and ascorbic

acid.

Conclusion

To conclude, this study found important evidence on the effects of different storage conditions, and

commercial value, on the: pH, ascorbic acid and antioxidants. Ascorbic acid, for instance, was found to be

volatile in opened apple juice; half of the ascorbic acid content was determined to have degraded after just

6 days. Furthermore, the temperature was observed to have no significant effect on pH, antioxidants, or

ascorbic acid, despite contrasting evidence found by: Burdulu et al (2006). It was also found that the middle

price range contained significantly more beneficial antioxidants and ascorbic acid.

Page | 18

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Appendix 1: Raw data Antioxidant activity (% Radical scavenging activity)

Table 3: Absorbance value and calculated Radical scavenging activity (%RSA) of apple juice, using the DPPH

method, on the initial day of opening (day 0).

Absorbance values

Sample A Sample B Sample C Sample D Sample E Sample F Average

1st absorbance 0.112 0.219 0.390 0.064 0.180 0.165 0.188

2nd absorbance 0.156 0.216 0.384 0.068 0.183 0.164 0.195

3rd absorbance 0.116 0.184 0.398 0.073 0.172 0.134 0.180

Control 1.584 1.508 1.608 1.568 1.624 1.180 1.512

1st %RSA 92.929 85.477 75.746 95.918 88.916 86.017 87.544

2nd %RSA 90.152 85.676 76.119 95.663 88.732 86.102 87.092

3rd %RSA 92.677 87.798 75.249 95.344 89.409 88.644 88.128

Average %RSA 91.919 86.317 75.705 95.642 89.019 86.921 87.588

SD 1.54 1.29 0.44 0.29 0.35 1.49 0.52


method, after 1 day of exposure (day 1). Sample A Sample B Sample C Sample D Sample E Sample F Average

1st absorbance 0.090 0.040 0.080 0.041 0.080 0.308 0.107

2nd absorbance 0.104 0.049 0.073 0.035 0.078 0.313 0.109

3rd absorbance 0.091 0.030 0.070 0.032 0.068 0.309 0.100

Control 1.446 1.184 1.212 1.188 1.478 1.544 1.342

1st %RSA 93.776 96.622 93.399 96.549 94.587 80.052 92.064

2nd %RSA 92.808 95.861 93.977 97.054 94.723 79.728 92.813

3rd %RSA 93.707 97.466 94.224 97.306 95.399 79.987 93.386

Average %RSA 93.430 96.650 93.867 96.970 94.903 79.922 92.754

SD 0.54 0.80 0.42 0.39 0.43 0.17 0.66


method, after 2 days of exposure (day 2). Sample A Sample B Sample C Sample D Sample E Sample F Average

1st absorbance 0.145 0.055 0.107 0.136 0.182 0.364 0.165

2nd absorbance 0.156 0.077 0.112 0.130 0.215 0.328 0.170

3rd absorbance 0.141 0.058 0.092 0.099 0.211 0.297 0.150

Control 1.844 1.546 1.488 1.822 1.842 1.378 1.653

1st %RSA 92.137 96.442 92.809 92.536 90.119 73.585 90.030

2nd %RSA 91.540 95.019 92.473 92.865 88.328 76.197 89.738

3rd %RSA 92.354 96.248 93.817 94.566 88.545 78.447 90.948

Average %RSA 92.010 95.903 93.033 93.322 88.997 76.076 90.239

SD 0.42 0.77 0.70 1.09 0.98 2.43 0.63

Page | 21



1st absorbance 0.328 0.079 0.230 0.076 0.121 0.209 0.174

2nd absorbance 0.400 0.153 0.163 0.091 0.130 0.182 0.187

3rd absorbance 0.364 0.151 0.186 0.072 0.141 0.217 0.189

Control 1.336 1.162 0.980 0.982 1.378 0.955 1.132

1st %RSA 75.449 93.201 76.531 92.261 91.219 78.115 84.646

2nd %RSA 70.060 86.833 83.367 90.733 90.566 80.942 83.527

3rd %RSA 72.754 87.005 81.020 92.668 89.768 77.277 83.351

Average %RSA 72.754 89.013 80.306 91.887 90.518 78.778 83.841

SD 2.69 3.63 3.47 1.02 0.73 1.92 0.70



1st absorbance 0.300 0.133 0.086 0.069 0.165 0.112 0.144

2nd absorbance 0.271 0.100 0.080 0.063 0.202 0.099 0.136

3rd absorbance 0.330 0.128 0.129 0.072 0.244 0.096 0.167

Control 1.616 1.504 1.316 1.428 1.592 1.292 1.458

1st %RSA 81.44 91.16 93.47 95.17 89.64 91.33 90.11

2nd %RSA 83.23 93.35 93.92 95.59 87.31 92.34 90.68

3rd %RSA 79.58 91.49 90.20 94.96 84.67 92.57 88.58

Average %RSA 81.42 92.00 92.53 95.24 87.21 92.08 89.79

SD 1.83 1.18 2.03 0.32 2.48 0.66 1.09

Ascorbic acid content

Table 8: Iodometric titration volumes and Ascorbic acid content (ascorbic acid; mg/100ml) of apple juice, on

the initial day of opening (day 0).

Sample A Sample B Sample C Sample D Sample E Sample F Total average

1st titration volume (ml) 0.50 14.70 4.40 3.60 0.40 0.30 3.983

2nd titration volume (ml) 0.40 14.40 3.60 4.20 0.30 0.30 3.867

3rd titration volume (ml) 0.40 14.80 4.80 3.60 0.30 0.40 4.050

1st Ascorbic acid (mg/100ml) 2.547 74.895 22.418 18.342 2.038 1.528 20.295

2nd Ascorbic acid (mg/100ml)

2.038 73.366 18.342 21.399 1.528 1.528 19.700

3rd Ascorbic acid (mg/100ml) 2.038 75.404 24.455 18.342 1.528 2.038 20.634

Average (mg/100ml) 2.208 74.555 21.738 19.361 1.698 1.698 20.210

SD 0.29 1.06 3.11 1.77 0.29 0.29 25.74

Page | 22

Table 9: Iodometric titration volumes and Ascorbic acid content (ascorbic acid; mg/100ml) of apple juice,

after 1 day of exposure (day 1).







2.038 76.423 19.361 17.323 1.019 2.038 19.700


Average (mg/100ml) 2.547 74.725 19.361 20.719 1.189 1.698 20.040

SD 0.51 1.56 2.04 3.11 0.29 0.59 25.82


after 2 days of exposure (day 2).







2.038 73.366 17.323 21.399 1.019 2.038 19.530


Average (mg/100ml) 2.208 75.235 17.323 21.738 1.189 1.528 19.870

SD 0.29 2.06 1.02 1.56 0.29 0.51 26.07









1.019 69.291 17.323 21.399 1.274 1.019 18.554


Average (mg/100ml) 1.189 66.573 16.304 21.059 1.613 1.189 17.988

SD 0.29 3.11 1.02 0.59 0.39 0.29 23.141

Page | 23









1.528 51.968 13.247 11.209 1.528 1.528 13.501


Average (mg/100ml) 1.189 48.232 16.304 12.907 1.359 1.019 13.501

SD 0.29 3.58 3.67 2.12 0.29 0.51 16.783



Sample A Sample B Sample C Sample D Sample E Sample F Total

average





2nd Ascorbic acid (mg/100ml) 1.019 38.721 4.585 5.095 1.019 1.019 8.576


Average (mg/100ml) 0.934 38.382 4.925 4.755 1.189 1.019 8.534

SD 0.15 1.56 0.29 0.29 0.29 0.00 13.467

Soluble solids content (°Brix)

Table 14: Soluble solids content (°Brix) of apple juice, on the initial day of opening (day 0).


1st reading 11.40 11.50 10.90 11.90 11.60 11.90 11.53

2nd reading 11.50 11.70 10.70 12.00 11.40 12.00 11.55

3rd reading 11.60 11.40 10.80 12.10 11.50 11.90 11.55

Average 11.50 11.53 10.80 12.00 11.50 11.93 11.54

SD 0.10 0.15 0.10 0.10 0.10 0.06 0.01

Page | 24

Table 15: Soluble solids content (°Brix) of apple juice, after 1 day of exposure (day 1).


1st reading 10.9 10.9 10.1 11.2 11.0 10.5 10.77

2nd reading 11.1 10.7 10.2 11.0 10.8 10.3 10.68

3rd reading 10.9 10.8 10.1 10.9 10.6 10.8 10.68

Average 10.97 10.80 10.13 11.03 10.80 10.53 10.71

SD 0.12 0.10 0.06 0.15 0.20 0.25 0.05

Table 16: Soluble solids content (°Brix) of apple juice, after 2 days of exposure (day 2).


1st reading 11.1 10.8 9.9 11.0 10.5 11.2 10.75

2nd reading 11.1 10.9 10.0 11.2 10.8 10.9 10.82

3rd reading 11.2 10.6 10.1 11.3 10.9 11.1 10.87

Average 11.13 10.77 10.00 11.17 10.73 11.07 10.81

SD 0.06 0.15 0.10 0.15 0.21 0.15 0.06



1st reading 11.2 10.8 10.1 11.4 11.0 11.6 11.02

2nd reading 11.1 10.9 9.9 11.6 10.9 11.3 10.95

3rd reading 10.9 10.8 10.0 11.6 11.0 11.4 10.95

Average 11.07 10.83 10.00 11.53 10.97 11.43 10.97

SD 0.15 0.06 0.10 0.12 0.06 0.15 0.04



1st reading 10.4 11.5 10.4 11.7 11.3 10.9 11.03

2nd reading 10.4 10.9 9.9 11.8 11.0 11.1 10.85

3rd reading 10.7 11.1 10.2 11.7 11.1 11.2 11.00

Average 10.50 11.17 10.17 11.73 11.13 11.07 10.96

SD 0.17 0.31 0.25 0.06 0.15 0.15 0.10

Page | 25

Acidity (pH)

Table 19: Acidity (pH) of 6 apple juice samples, on the initial day of opening (day 0).


average

1st reading 3.41 3.50 3.18 3.50 3.55 3.32 3.41

2nd reading 3.48 3.53 3.25 3.53 3.58 3.38 3.46

3rd reading 3.50 3.51 3.23 3.54 3.56 3.37 3.45

Average 3.463 3.513 3.220 3.523 3.563 3.357 3.44

SD 0.05 0.02 0.04 0.02 0.02 0.03 0.03

Table 20: Acidity (pH) of 6 apple juice samples, after 1 day of exposure (day 1).


average

1st reading 3.48 3.53 3.20 3.55 3.46 3.35 3.43

2nd reading 3.48 3.47 3.23 3.48 3.55 3.40 3.44

3rd reading 3.49 3.50 3.21 3.45 3.56 3.36 3.43

Average 3.483 3.500 3.213 3.493 3.523 3.370 3.43

SD 0.006 0.03 0.02 0.05 0.06 0.03 0.004

Table 21: Acidity (pH) of 6 apple juice samples, after 2 days of exposure (day 2).


average

1st reading 3.46 3.53 3.28 3.51 3.60 3.40 3.46

2nd reading 3.48 3.52 3.23 3.54 3.56 3.40 3.46

3rd reading 3.43 3.50 3.25 3.52 3.58 3.41 3.45

Average 3.457 3.517 3.253 3.523 3.580 3.403 3.46

SD 0.03 0.02 0.03 0.02 0.02 0.006 0.008



average

1st reading 3.45 3.54 3.20 3.49 3.54 3.40 3.44

2nd reading 3.41 3.47 3.20 3.50 3.54 3.37 3.42

3rd reading 3.42 3.47 3.19 3.49 3.52 3.36 3.41

Average 3.427 3.493 3.197 3.493 3.533 3.377 3.42

SD 0.02 0.04 0.01 0.01 0.01 0.02 0.02



average

1st reading 3.48 3.52 3.31 3.55 3.66 3.48 3.50

2nd reading 3.52 3.55 3.30 3.58 3.63 3.50 3.51

3rd reading 3.48 3.53 3.29 3.55 3.64 3.50 3.50

Average 3.493 3.533 3.300 3.560 3.643 3.493 3.50

SD 0.02 0.02 0.01 0.02 0.02 0.01 0.01

Page | 26

3.01 3.00 2.99 2.89

2.60

2.14

y = -0.0055x + 3.144R² = 0.9031

0

0.5

1

1.5

2

2.5

3

3.5

0 24 48 72 96 120 144 168 192

LnC

(m

g A

sco

rbic

aci

d/1

00

ml)

Time (hours)

Appendix 2: Processed data Sugar content (°Brix)

Table 24: Soluble solids content of 6 apple juice samples across 5 days after opening (°Brix).

Apple Juice Day 0 Day 1 Day 2 Day 3 Day 4

Sample A 11.50 (+/-0.10) 10.97 (+/-0.12) 11.13 (+/-0.06) 11.07 (+/-0.15) 10.50 (+/-0.17)

Sample B 11.53 (+/-0.15) 10.80 (+/-0.10) 10.77 (+/-0.15) 10.83 (+/-0.06) 11.17 (+/-0.31)

Sample C 10.80 (+/-0.10) 10.13 (+/-0.06) 10.00 (+/-0.10) 10.00 (+/-0.10) 10.17 (+/-0.25)

Sample D 12.00 (+/-0.10) 11.03 (+/-0.15) 11.17 (+/-0.15) 11.53 (+/-0.12) 11.73 (+/-0.06)

Sample E 11.50 (+/-0.10) 10.80 (+/-0.20) 10.73 (+/-0.20) 10.97 (+/-0.06) 11.13 (+/-0.15)

Sample F 11.93 (+/-0.06) 10.53 (+/-0.25) 11.07 (+/-0.15) 11.43 (+/-0.15) 11.07 (+/-0.15)

Total average 11.54 10.71 10.81 10.97 10.96

Average of triplicates

Ascorbic Acid content

Table 26: Ascorbic acid concentration (mg Ascorbic acid/100ml) of six apple juice samples over the first 168

hours after opening in both refrigerated (4°C) and Ambient (19°C) conditions.

Hours after opening

Sample A (19°C)

Sample B (4°C)

Sample C (4°C)

Sample D (4°C)

Sample E (4°C)

Sample F (4°C)

0 2.21 74.56 21.74 19.36 1.70 1.70

24 2.55 74.73 19.36 20.72 1.19 1.70

48 2.21 75.23 17.32 21.74 1.19 1.53

72 1.19 66.57 16.30 21.06 1.61 1.19

96 1.19 48.23 16.30 12.91 1.36 1.02

168 0.93 38.38 4.93 4.76 1.19 1.02

Figure 4: First Order kinetic degradation of Ascorbic acid across all apple juice samples over the first 168

hours after opening.

Page | 27

1.951.87

1.70

1.401.27

1.06

y = -0.0057x + 1.9315R² = 0.9329

0.00

0.50

1.00

1.50

2.00

2.50

0 24 48 72 96 120 144 168 192ASc

orb

ic a

cid

co

nce

ntr

atio

n (

C;

mg

AA

/10

0m

l)

Time (hours)

Figure 5: First Order kinetic degradation of Ascorbic acid in value range apple juice over the first 168 hours

after opening.

Figure 6: Zero Order kinetic degradation of Ascorbic acid in value range apple juice over the first 168 hours

after opening.

0.670.62

0.53

0.34

0.24

0.06

y = -0.0039x + 0.6767R² = 0.958

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 24 48 72 96 120 144 168 192

LnC

(m

g A

sco

rbic

aci

d/1

00

ml)

Time (hours)

Page | 28

3.85 3.87 3.88 3.78

3.42

3.07

y = -0.0051x + 3.9947R² = 0.8653

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 24 48 72 96 120 144 168 192

LnC

(m

g A

sco

rbic

aci

d/1

00

ml)

Time (hours)

46.96 47.72 48.4943.82

30.57

21.57

y = -0.1731x + 51.625R² = 0.8542

0.00

10.00

20.00

30.00

40.00

50.00

60.00

0 24 48 72 96 120 144 168 192

Asv

orb

ic a

cid

co

ne

ntr

atio

n (

mg/

10

0m

l)

Time (hour)

Figure 7: First Order kinetic degradation of Ascorbic acid in medium range apple juice over the first 168


Figure 8: Zero Order kinetic degradation of Ascorbic acid in medium range apple juice over the first 168


Page | 29

2.462.35

2.242.17 2.16

1.09

y = -0.0078x + 2.6067R² = 0.8598

0

0.5

1

1.5

2

2.5

3

0 24 48 72 96 120 144 168 192

LnC

(m

g A

sco

rbic

aci

d/1

00

ml)

Time (hours)

11.72

10.53

9.438.75 8.66

2.97

y = -0.0492x + 12.022R² = 0.9391

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0 24 48 72 96 120 144 168 192

Asv

orb

ic a

cid

co

ne

ntr

atio

n (

mg/

10

0m

l)

Time (hour)

Figure 9: First Order kinetic degradation of Ascorbic acid in premium range apple juice over the first 168


Figure 10: Zero Order kinetic degradation of Ascorbic acid in premium range apple juice over the first 168


Page | 30

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

SAMPLE A

SAMPLE B

SAMPLE C

SAMPLE D

SAMPLE E

SAMPLE F

Radical scavenging ability (%RSA)

Ap

ple

juic

e s

amp

les

Day 1 Day 2 Day 3 Day 4 Day 5

DPPH

Figure 11: Change in Radical Scavenging activity of DPPH (RSA %) of 6 apple juice samples over time (Days)

Page | 31

Appendix 3: Declared information on pack Table 27: Information declared on Samples A-F (Pre-packed supermarket apple juices)

Juice Storage Storage

once opened

Best before date

Shelf-life

once opened

Cloudy/ Clear

Sugars (grams)

per 100ml

Salt (g) per 100ml

From concentrate

Sample: A

Ambient (19-22°C)

Ambient 14/03/17 4 days Clear 8.9g 0.1g Yes

Sample: B

Refrigerated (3-8°C)

Refrigerated 17/03/17 5 days Cloudy 11.2g 0.1g No

Sample: C



Sample: D



Sample: E


Refrigerated 15/03/17 5 days Clear 9.6g 0.06g Yes

Sample: F



Page | 32

Appendix 4: Statistics tests using SPSS 23.0 Figure 12: Output of Pearson’s correlation test for pH and Antioxidant values across all samples over the first

4 days after opening.

Figure 13: Output of Pearson’s correlation test for pH and Ascorbic acid across all samples over the first 4

days after opening.

Page | 33

Figure 14: Output of Pearson’s correlation test for %RSA and Ascorbic acid across all samples over the first 4

days after opening.

Figure 15: Output of one way Anova, on the effect of exposure time (Days) on the antioxidant content

(%RSA) of 6 apple juice samples.

Page | 34

Figure 16: Output of a repeated measures general linear model analysis, with Greenhouse Giesser

correction and Tukey post hoc analysis, on the effect of exposure time (Days) on the antioxidant content

(%RSA) of 6 apple juice samples.

Mauchly’s sphericity assumption was violated (p=0.015) and there was a low (<0.75) Greenhouse Giesser value (g=0.567);

therefore, the Greenhouse Geisser correction was used.

Page | 35

Figure 17: Output of a repeated measures general linear model analysis, with Greenhouse Giesser

correction and Tukey post hoc analysis, on the effect of exposure time (Days) on the Ascorbic acid content

(mg/100ml) of 6 apple juice samples.



Page | 36

Figure 18: Output of a one sample t-test on the variance of the Ascorbic acid content (mg/100ml) between 6

apple juice samples, against the RNI (80mg).

Figure 19: Output of a one sample t-test on the variance of the Ascorbic acid content (mg/100ml) across the

8-day period, against the RNI (80mg).

Page | 37

Figure 20: Repeated measures general linear model analysis, with Greenhouse Giesser correction and Tukey

post hoc analysis, on the effect of exposure time (Days) on the acidity (pH) of 6 apple juice samples.



Page | 38

Figure 21: Repeated measures general linear model analysis, using Tukey post hoc analysis, on the effect of

apple juice price range on the acidity (pH) of 6 apple juice samples.


apple juice price range on the Ascorbic acid content (mg/100ml) of 6 apple juice samples.


apple juice price range on the Antioxidant content (%RSA) of 6 apple juice samples.

Page | 39

Figure 24: Repeated measures general linear model analysis, on the effect of storage temperature (between

4°C and 19°C) on the Acidity (pH) of 6 apple juice samples.


4°C and 19°C) on the Ascorbic acid content (mg/100ml) of 6 apple juice samples.


4°C and 19°C) on the Antioxidant content (%RSA) of 6 apple juice samples.

Page | 40

Appendix 5: Risk Assessments RISK ASSESSMENT FORM 1

1A

MEMBER OF STAFF Arthur Tatham

LOCATION OF WORK: T201 DURATION OF

ACTIVITY: 5 days

ACTIVITY TITLE: Ascorbic acid, pH and Antioxidant potential

NATURE OF WORK: Lab based analysis

PEOPLE INVOLVED:

Will the work detailed above involve any of the following hazards?

YES NO

1 Hazards of fire or explosion N

2 Hazards arising from the use of known corrosive, toxic or carcinogenic

compounds Y

3 Biohazard arising from viruses, microorganisms the manipulation of

genetic material or human tissue N

4 Administering a treatment with the potential for adverse reactions N

5 Ingesting or inhaling a substance with the potential for adverse

reactions N

6 Physical or ergonomic hazards arising from radiation, high voltage, high

pressure, ultra-sonics, lasers, exercising etc N

7 Other hazards not listed above e.g. noise, driving, violence, aggression,

lone working, working in an unfamiliar environment, smoking, home

working etc

N

1B

STAFF MEMBER’S CERTIFICATION OF ASSESSMENT

If you have answered no to the seven questions above and are satisfied with the following statement, you

should sign and date it, setting a date not more than 12 months hence for the work to be reassessed.

I have assessed/reassessed* the work outlined above with respect to the School's Health & Safety Policy.

I undertake to review this assessment if the nature of the work should change, or if any unforeseen hazards should be encountered.

*This assessment will be reviewed not later than

*This project was completed on

Signed: (Staff member)

Print name:

Date:

* - delete or complete as necessary

CARDIFF METROPOLITAN UNIVERSITY, CSHS, RISK ASSESSMENT FORM 2

Page | 42

Cardiff Metropolitan University

Cardiff School of Health Sciences

RISK ASSESSMENT FORM 2

NAME:

TITLE OF

WORK

ACTIVITY:

Ascorbic acid, pH and Antioxidant potential analysis

LOCATION(S)

OF WORK

(Room Numbers):

T201

This form MUST BE COMPLETED prior to the commencement of any work involving risks to

health from a hazardous substance or activity so that a suitable and sufficient assessment of

health risk is made.

Special Situations:

1. Any member of staff or student who may be or is pregnant, or who has recently givenbirth, or is immunocompromised or may be susceptible to harm is required to inform theDean of School before undertaking any practical work. This information will be treatedwith strictest confidence and is required to protect the interests of the person concerned.

2. Before commencing any work involving samples of human origin or human participants,you are obliged to get a favourable decision from the School’s Research EthicsCommittee.

3. Before commencing any work involving samples of human origin, you must contact theUniversity’s Designated Individual (under the Human Tissue Act).

4. Before commencing any work involving genetically modified organisms, or the geneticmodification of cells or materials, you must contact the Chair of the School’s GeneticManipulation Safety Committee.

5. If this procedure involves transferring materials into/out of Cardiff Metropolitan University,you must obey the School’s ‘Transportation of Biohazardous Materials Policy’.

Page | 43

6. If this procedure involves transporting materials between Cardiff Metropolitan University’slaboratories, you must obey local Codes of Practice.

This assessment should be reviewed immediately if there is any reason to suppose that the

original assessment is no longer valid due to significant changes in the work activity, arising for

example, from the introduction of new hazardous substances, new personnel, changes in

procedures or reported ill-health. Otherwise, the assessment should be reviewed annually.

GUIDANCE NOTES FOR COSHH RISK ASSESSMENT

Material Safety Data Sheets (MSDS)

A substance should be regarded as hazardous to health if it is hazardous in the form in which it occurs

in the work activity, including by products and waste residues. All information regarding a substances

hazard risks may be obtained through their Material Safety Data Sheets (MSDS).

MSDS Information may be obtained in the following ways:

1) MSDS information is supplied with every new purchase of a product2) Most MSDS information on any product can usually be found on the internet.

Useful Website Addresses

http://www.fisher.co.uk (useful for the majority of chemicals purchased through UWIC)

http://www.sigmaaldrich.com (Another popular chemical supplier for UWIC)

http://www.vwrsp.com (The worlds largest chemical manufacturer)

http://www.promega.com (For molecular Biology/Genetic Related Substances)

http://www.invitrogen.com (For molecular Biology/Genetic Related Substances)

http://www.atcc.com (A useful website for all Cultures cell lines, bacterial, plant and animal)

http://www.nctc.com (A National bacterial culture supplier)

Search the manufacturer’s web site or use any search engine (e.g. google), type in a substancefollowed by MSDS and search.

What is regarded as a hazard?

(a) Any substance which is listed as very toxic, toxic, harmful, or irritant is a substance hazardous to

health.

(b) Any substance which has either an MEL (Maximum Exposure Limit) or OES (Occupational Exposure

Standard) given in the HSE Guidance Note EH.40 is a substance hazardous to health. Appropriate

precautions, such as the use of fume hoods/masks, must be taken when handling any substance known to

have an MEL/OES.

(c) A dust of any kind is a substance hazardous to health when present in a "substantial

concentration". See the Approved Code of Practice, para 2(f) and HSE Guidance Note EH.40.

(d) Any chemical which may be flammable, corrosive, oxidising, or explosive is a substance

hazardous to health.

(e) Any substance with any carcinogenic, mutagenic or teratogenic effects is classed as hazardous

to health.

http://www.fisher.co.uk/

http://www.sigmaaldrich.com/

http://www.vwrsp.com/

http://www.promega.com/

http://www.invitrogen.com/

http://www.atcc.com/

http://www.nctc.com/

Page | 44

What is regarded as a Biohazard?

(a) Micro-organisms which create a hazard to the health of any person, where the hazard arises out

of or in connection with a work activity. Hazard classification of pathogens is given in the booklet

"Categorisation of Pathogens etc.", by the Advisory Committee on Dangerous Pathogens.

(b) Tissue Culture Cell Lines of animal or human origin are regarded as being hazardous to health.

Many cell lines are potentially carcinogenic, having originated from malignant tissue extracts.

Some cell lines are transfected with known viruses to create established cell lines. Hazard

classification of most cell cultures are given by the American Type Culture Collection (ATCC)

(c) All human and animal blood, blood products, tissues, certain body fluids and clinical specimens.

(d) Certain recombinant/genetically modified products.

Other Advice:

Note: Any special training required to ensure that persons involved in the work activity can operate

safely should be described. This is particularly important so that persons can understand and

comply effectively with the scheme of work, where this has been formulated.

Note: The level of any supervision must always be appropriate to the competence of the individuals

involved in the work activity.

CARDIFF METROPOLITAN UNIVERSITY, CSHS, RISK ASSESSMENT FORM 2

Page | 45

1) Description of procedure:

A staff member must take overall responsibility for all aspects of the work activity.

Outline of overall procedure:

500ml Iodine solution: 5g Potassium Iodide KI, 0.268g Potassium iodate KIO3, 30ml of 3M sulphuric acid (H2SO4) and filling up to 500ml of deionized

water into a volumetric flask

Ascorbic acid assay: The Ascorbic acid (C6H8O6) concentration of Samples A-F were determined (in mg/100ml) by titrating it with the Iodine solution. A 1:99 starch to distilled water (H2O) solution was used as a colour change indicator to observe the end point. 25ml of each of the Samples were placed in conical flasks as well as 2ml of the starch indicator solution. This was then titrated with the Iodine solution until there was an observed colour change and the end point was reached.

DPPH Solution: The α-diphenyl-β-picrylhydrazyl (DPPH- C18H12N5O6) solution was made to a concentration of 60µM using ethanol. 4.732mg was needed for 200ml

DPPH Assay: The antioxidant capacity was measured using VIS-spectrometry at a wavelength of 517nm on a Spectrophotometer. 50µl of 3 of each of

the apple juice samples were added to separate cuvettes as well as 50µl of a positive control sample of ascorbic acid and 50µl of α-diphenyl-β-

picrylhydrazyl (DPPH) blank control of deionized water. 3.5ml of the DPPH solution was then added to each Cuvette and the absorbance was measured.

Technique Associated chemicals, microorganisms and other potentially hazardous activities

Person(s) undertaking technique e.g. staff, student, visitor

Level of training or supervision required*

Iodine solution Potassium Iodide, Potassium Iodate, and Sulphuric acid

George Warne: student None

Ascorbic acid Assay Iodine solution George Warne: student None

DPPH Solution DPPH Ethanol


DPPH Assay DPPH Ethanol


*Must be completed when an activity scores ≥ 4 (Section 3)

Page | 46

2) Risk Rating Matrix

SEVERITY LIKELIHOOD

DESCRIPTOR EXPLANATION SCORE

X

DESCRIPTOR EXPLANATION SCORE

Damage to

property

Incident resulting in no injury but causing damage to

property or equipment 1 Inconceivable Cannot imagine incident will occur. Beyond belief. 1

Minor

injury/illness

Injury/illness not requiring application of first aid and not

involving absence from work/study 1 Remote Conceivable, but highly unlikely that incident will occur. 1

Medical attention Injury/illness requiring medical attention 2 Unlikely Doubtful that incident will occur 2

Major

injury/illness

Injury/illness resulting in more than 3 days absence from

work/study 3 Possible Feasible chance that incident will occur 3

Fatal

injury/illness Injury/illness causing death to an individual 4 Probable Credible chance that incident will occur 4

Multi-fatalities Injury/illness causing death to more than one person 5 Certainty Incident will definitely occur. Sure to happen. 5

NB: In the above tables, the severity and likelihood multiplication factors 0 have been replaced with 1 to enable the recording of near miss / damage incidents.

RISK INDEX TABLE (WORK ACTIVITIES)

SCORE ACTION

12 – 25 Unacceptable intolerable level of risk, not to proceed unless control measures can be implemented to eliminate or reduce risk to an acceptable level.

8 - 10 High level of risk still requiring documented risk reduction strategies (see section 4 and 5)

4 - 6 Moderate level of risk, consequences to be fully considered when completing risk assessment

2- 3 Low level of risk but still requiring periodic monitoring and review.

1 No action required

Page | 47

3A) Hazardous Chemicals: (Use the Risk Rating Matrix (Section 2 ) when completing this table and use extra lines if required))See guidance notes at front for sources of data

No Name Concentration Quantity

Signal Word CLP Pictogram

Hazard Statements

Risk Control Measures

Disposal of Waste Residue

Contingency (Fire, Spillage, Eyewash etc)

Chemical Incompatibilities

1

Sulphuric Acid 3M at 30ml (made from 5ml of 18M solution)

Danger Corrosive

Gloves Goggles Wash hands after handling

Dilute with water, pour into sink

Deionized water and soap

H2O2 Sodium hydroxide Calcium hydroxide

Risk Rating Without Control S = 3 L = 3 RI = 9 Risk Rating With Control S = 3 L = 1 RI = 3

2

100% Ethanol made to a 10% solution

Danger Highly flammable Gloves Keep away from flames


CO2 fire extinguisher Rinse infected areas

Oxidising agents


3

DPPH 60µM 47mg in 200ml ethanol

Danger Very toxic

Gloves Goggles Wash hands after handling


Call poison centre if ingested Rinse infected areas

Oxidising agents


4

5g of 100% Potassium Iodide made to a 0.015M solution in 500ml

Warning Causes eye irritation Wear goggles

Dilute and use paper towels then place in yellow bins

Rinse eyes Metals Reducing agents


Page | 48

5

0.268g of 100%

Potassium

iodate made to

a 0.6mM

solution in

500ml

Danger

Corrosive

Acute toxicity

Serious eye damage

Oxidises fires

Gloves

Goggles

Closed container

Dilute with water,

pour into sink

Rinse eyes for

several minutes

with water

Use sand on fires

Alakali metals

Arsenic

Sulphur


Page | 49

4)

Risk Evaluation of the Overall Procedure (with controls)

Select the most hazardous activity described in Section 3 to use as the basis for the Risk Level Index

(Be aware that certain combinations of substances may have synergistic/antagonistic hazardous effects and should be considered when completing this section.)

Score of Severity 3

Score of Likelihood 4

Risk index (score of severity x score of likelihood) 6

Yes No

Does the risk index (score of severity x score of likelihood) exceed a total score of 10 points?

If yes, then this is an unacceptable/intolerable level of risk and the activity must not proceed. x

Page | 50

5A) Certification Of Assessment

Section A of this form must be signed by a member of staff, before the activity can proceed

Honours/Masters project and PhD/MPhill dissertation students must sign section B before their supervisor signs section B

A Staff B Student

Print Name Print Name

Signature Signature

Date Date

Once section 6A of this form has been signed, the staff member has taken responsibility for all aspects of the work activity.

Once section 6B is signed, the student is acknowledging that they have received a copy of the assessment and that they understand it. The student

is also agreeing to the controls measures as described in the assessment.

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5B) Review

Use this section for an annual review only. If the work activity changes, complete a new form.

See notes regarding review (page 1 of this document).

Date Student

Signature

Staff Member

Signature

Date Student

Signature

Staff Member

Signature

ONE SIGNED COPY OF THIS ASSESSMENT MUST BE READILY AVAILABLE (IN THE LABORATORY/AREA OF WORK), FOR AS LONG AS IT IS RELEVANT

A SECOND SIGNED COPY SHOULD BE KEPT WITH THE STAFF MEMBER RESPONSIBLE FOR THE TEACHING SESSION OR SUPERVISION OF

* HONOURS/MASTERS PROJECTS AND PHD/MPHIL DISSERTATIONS

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Appendix 6: Ethical approval

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sm76889

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