1 of 68

Exploring the Therapeutic Potential of Cardamom to Attenuate the Physiological Dysfunctions

INTERNATIONAL SEMINAR-2022

Dr. Ayesha Murtaza (Ph.D.)

Assistant Professor

University of Central Punjab (UCP),PAKISTAN

PRESENTED AT

Nutrition Health Polytechnic Ministry of Health Tasikmalaya

2 of 68

Contents:

2

PART ONE

INTRODUCTION

Introduction

Cardamom
Bioactive components
Objectives
Future perspectives

Review of Literature
References

3 of 68

3

Nutrition and Immunity

During the flu season or times of illness, people often seek special foods or vitamin supplements that are believed to boost immunity.
Vitamin C and foods like citrus fruits, chicken soup, and tea with honey are popular examples.
However, a balanced diet consisting of a range of vitamins and minerals, combined with healthy lifestyle factors like adequate sleep and exercise and low stress, most effectively primes the body to fight infection and disease.

4 of 68

4

What Is Our Immune System?

Innate immunity

First-line defense from pathogens that try to enter our bodies, achieved through protective barriers. These barriers include:

Skin that keeps out the majority of pathogens
Mucus that traps pathogens
Stomach acid that destroys pathogens
Enzymes in our sweat and tears that help create anti-bacterial compounds
Immune system cells that attack all foreign cells entering the body

Adaptive or acquired immunity

The system that learns to recognize a pathogen.
It is regulated by cells and organs in our body like the spleen, thymus, bone marrow, and lymph nodes.
When a foreign substance enters the body, these cells and organs create antibodies and lead to multiplication of immune cells

Humans possess two types of immunity: innate and adaptive.

5 of 68

5

6 of 68

Cardamom

Elettaria cardamomum
“Cardamom” derived from Latin word “Cardamomum” meaning “spice plant”
Cardamom, the “Queen of Spices” is the third most expensive spices, next only to saffron and sometimes vanilla. (Singletary, 2022)
Native to Southern India and China
Cultivated in India, China and Sir Lanka
Used as flavor modifier to make the food more acceptable

6

7 of 68

7

8 of 68

Nature of plant

Cardamom is the fruit(capsule) of the plant.
The capsule are globose, or ovoid or narrowly ellipsoid to elongate in shape.
Capsules are pale green to dark green in color and on maturity seeds turn dark brown to black in color.
A single capsule contain 15 – 20 seeds.
Genus – Sitaminae

8

9 of 68

Cardamom

Used commercially in perfumes and as a flavoring in the food industry, where it can be added to meats, baked products, soups, fruit products, jellies, and pickles.
Its complex aroma is variously described as sweet, citrusy, warm, minty, pungent, and floral. (Parthasarathy et al., 2012)
It has the flexibility to enhance both sweet and savory dishes and, depending on the recipe, can be added as pods or seeds, whole or as seed powder, usually in teaspoon quantities. One pod yields 1/5 teaspoon ground cardamom, and 1 teaspoon provides 2 g powder.
Cardamom-flavored biscuits, drinks, milk, and cheese are marketed in India and other regions. In the Middle East, its powder is generously added during brewing of Gahwa or Arabic coffee and can be added to teas. Popular sweets in numerous countries contain this spice (Singletary, 2022).

9

10 of 68

Cardamom

One leaf can be added to 1 cup boiling water to produce a unique, fragrant tea.
A functional drink prepared with cardamom rhizome was reported, but routine use of the rhizome in food preparation appears less widespread (Winarsi, 2018).
Blending cardamom into Arabic coffee helps mask, in large part due to the fragrance of α- terpinyl acetate, the smoky odors of heat-induced coffee bean products introduced during preparation (Abdelwareth et al., 2021).

10

11 of 68

Other Names

11

12 of 68

Types of Cardamom

12

13 of 68

13

Black Cardamom

White Cardamom

14 of 68

14

Cardamom Seeds

Cardamom Oil

15 of 68

Bioactive Components

Also contains Mg, Al, Si, P, S, Cl, K, Ca, Ti, Mn, Fe, Cu and Zn, with varying concentrations

15

Active ingredients	Amount
1,8-cineole	21.4%
Terpinolene	8.6%
α-terpinyl acetate	42.3%
Linalyl acetate	8.2%
Limonene	5.6%
Myrcene	6.6%

Anticholinesterase activity

Neuroprotective effect

16 of 68

Flavor Profile

Cineole contribute to pungency
Terpinyl acetate is known for its pleasant aroma
The seeds give spicy sweet flavor

(Karibasappa, 1987)

16

17 of 68

Therapeutic Potential

Nutritional challenges:

High medication cost
Side effects

Physiological disorders:

Platelet aggregation effects
Hepatic effects
Antioxidant, anti-microbial & anti-inflammatory effects
Antispasmodic effects
Gastro-protective effect
Anti-ulcer effects

17

18 of 68

Therapeutic Potential

Strategies to combat challenges

Diet based therapy
Phytochemicals enrich diet (Butt et al., 2008).

18

19 of 68

Flavor Destruction

Terpenoids, more specifically α-terpinyl acetate in cardamom oil could undergo hydrolysis, rearrangement, polymerization, and oxidative reactions
Affect flavor due to its vulnerability to acid, light, oxygen or heat
These changes can be arrested by proper encapsulation of cardamom oleoresin

(Brennand and Heinz, 2018)

19

20 of 68

Flavor Destruction

These changes increase the content of cymene which is also a terpene but with petroleum-like aroma
The variation in flavor characteristics from different sources of cardamom have been attributed to the proportion of the esters such as α-terpinyl acetate, linalyl acetate and 1,8-cineole (Korikanthimath et al., 2020)

20

21 of 68

Objectives

Optimization and characterization of cardamom through conventional and supercritical fluid extraction
Quantification of oleoresin through HPLC
Development and sensory evaluation of cardamom based tea
Investigating the hypo-cholesterolemic and hypoglycemic potential of cardamom using model feeding trial

21

22 of 68

Contents:

22

PART TWO

Review of Literature

Review of Literature

Health claims
Hepatic effects
Platelet aggregation effects
Anti-spasmodic effects

23 of 68

Review of Literature

Health claims
Antioxidant activity

Free radicals scavenging
Cardamom has medium levels (50-100mg) of antioxidant phenolics and flavonoids

Food preservation effects

Cardamom exhibited a moderate effect in the minimization of the formation of toxic histamine

23

24 of 68

Review of Literature

Hepatic effects

The influence of cardamom oil on the activities of hepatic carcinogen-metabolizing enzymes was investigated
The oil was fed at 10 micro liters per day for 14 days, and then the animals were sacrificed and their hepatic enzyme activities and sulfhydryl levels were evaluated
Cardamom oil caused a significant reduction in cytochrome P450 level activity

24

25 of 68

Review of Literature

Hypoglycemic activity of cinnamon & cardamom oil extract (100mg/kg) in streptozotocin (STZ)-induced diabetic rats caused

Significant affect on body weight
Reduce the blood glucose level

(Zhao et al., 2006)

25

26 of 68

Review of Literature

Platelet aggregation effects

An increase in concentration of cardamom has decreased malondialdehyde formation significantly
The aqueous extract of cardamom may have components, which protect platelets from aggregation and lipid peroxidation

Gastroprotective effect

Cardamom may somewhat increase gastric acid secretion

26

27 of 68

Review of Literature:

Anti-spasmodic effects

The antispasmodic activity of the oil extracted from commercial Elettaria cardamomum seeds was determined on a rabbit intestine preparation using acetylcholine as agonist, the results proving that cardamom oil exerts its antispasmodic action through muscarinic receptor blockage
A synergistic effect on drug permeation was observed when transdermal iontophoresis combined with the pretreatment of cardamom oil as a permeation enhancer

27

28 of 68

28

Condition	Treatment (Dose/Duration)	Outcome			References
		SD	Trend	NE

TABLE 1 - Green Cardamom Effects in Human Studies

T2DM	Seed powder (3 g/d; 8 wk)�All groups consumed with 3 glasses black tea�n = 39–42/group(gp)	Versus baseline	↓WC, ↓FBG	BW, BMI, HbA_1c, BP, blood lipids, I-CAM-1	^19,20
T2DM	Seed powder (3 g/d; 10 wk)�n = 37–38/gp	Versus placebo:�↓HbA_1c, ↓SI, ↓HOMA-IR, ↓TG, ↑SIRT1		BMI, WC, FBG, HDL, LDL, TC	²¹
Overweight/obese prediabetic women	Seed powder (3 g/d; 2 mo)�n = 40/gp	Versus placebo:�↓CRP, ↓MDA, ↓WC	↓TC, ↓LDL, ↓HOMA-IR, ↑HDL, ↑TNF-α, ↓TAC	TG, FBG, SI, IL-6, QUICKI, BMI, BW, BP, RBS-SOD, RBS-GR	^22–24
Overweight/obese NAFLD	Seed powder (3 g/d; 3 mo)�n = 43–44/gp	Versus placebo:�↓TG, ↑HDL, ↓SI, ↓HOMA-IR, ↓fatty liver, ↑irisin, ↑SIRT1, ↓CRP, ↓IL-6↓TNF-α, ↓ALT	↓LDL,	FBG, BW, BMI, TC, AST	^25,26
Overweight/obese women PCOS	Seed powder (3 g/d; 4 mo)�All groups consumed low-calorie diet�n = 95–99/gp	Versus placebo:�↓BMI, ↓%body fat, ↓TNF-α, ↓IL-6, ↓PBMC-CRP, ↓ovarian cysts, ↓androstenedione, ↓DHEA, ↓T, ↓LH, ↑FSH	↓PBMC–TNF-α	WC, TSH, CRP, PBMC-IL-6r	²⁷

29 of 68

29

TABLE 1 - Green Cardamom Effects in Human Studies

Atherosclerotic, overweight women	Rhizome powder-containing functional beverage (3 g/d; 2 mo)�Controls consumed functional beverage minus rhizome�n = 10/gp	Versus placebo:�↓IL-6, ↓CRP	↓TC, ↓LDL, ↑HDL	TG	¹¹
Pregnant women, <22 wk gestational age, mild to moderate nausea + vomiting	Seed powder (1.5 g/d; 4 d)�n = 60/gp	Versus placebo:�↓severity nausea + vomiting			²⁸
Hypertensives	Seed powder (3 g/d; 3 mo)�n = 20; no controls)	Versus baseline:�↓SBP, ↓DBP, ↑TAC, ↑blood fibrinolytic activity	↓TC, ↓TG, ↓LDL, ↓VLDL	Fibrinogen activity, HDL	²⁹
Healthy women	Arabic coffee, unfiltered. Prepared in boiling water (parts ground coffee to parts seed powder) = 3:0, 3:1, 3:2�(500 mL/d, 5 d/wk; 4 wk)�n = 10–13/gp	Versus baseline: ↑TC, ↓GGT		BP, CRP, AST, ALT, CK, LDH, TG, LDL, HDL, BP, CRP, AST, ALT, CK, LDH	³⁰

Condition	Treatment (Dose/Duration)	Outcome			References
		SD	Trend	NE

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BP, blood pressure; BMI, body mass index; BW, body weight; CK, creatine kinase; CRP, C-reactive protein; FBG, fasting blood glucose; FSH, serum follicle-stimulating hormone; GGT, γ-glutamyl transpeptidase; HbA1c, glycosylated hemoglobin; HDL, serum high-density lipoprotein cholesterol; HOMA-IR, Homeostatic Model Assessment for Insulin Resistance; ICAM-1, intercellular adhesion molecule 1; IL-6, interleukin 6; irisin, fibronectin type III domain 5; LDL, serum low-density lipoprotein cholesterol; LH, serum luteinizing hormone; NAFLD, nonalcoholic fatty liver disease; NE, no statistically significant effect; PBMC-CRP, CRP gene expression in peripheral blood mononuclear cells; PBMCIL- 6r, IL-6r gene expression in peripheral blood mononuclear cells; PBMC–TNF-α, TNF-α gene expression in peripheral blood mononuclear cells; PCOS, polycystic ovary syndrome; QUICKI, quantitative insulin-sensitivity check index; RBC-GR, red blood cell glutathione reductase activity; RBS-SOD, red blood cell superoxide dismutase activity; SI, serum insulin; SIRT1, sirtuin 1; T, serum testosterone; T2DM, type 2 diabetes mellitus; TAC, serum total antioxidant capacity; TC, serum total cholesterol; TG, serum triglycerides; TNF-α, tumor necrosis factor α; WC, waist circumference.

30 of 68

30

TABLE 2 - Summary of Green Cardamom Effects in Animal Models

Condition	Treatments Dose/Duration	Outcomes	References

Blood glucose/lipid dysregulation	Seed powder 1%–7% w/w diet; 4–8 wk	↓FBG, ↓LDL, ↓SBP, ↓SI, ±TC, ±TG, ±HDL, ±ALP, ↑blood + tissue antioxidants, improved liver morphology	^36–39
	Seed volatile oil 0.3% diet; 8 wk	↓TC, ↓LDL, ↓TG, ↓heart TC, ↑blood antioxidants, ↓blood + liver + heart LPO�NE: HDL	³⁸
	Seed extract (alcoholic SCCO₂) 55–1000 mg/kg per day PO; 1–8 wk	↓TC, ↓TG, ↓LDL, ↓SI, ↓AI, ↓FBG, ↓HOMA-IR, ↑HDL, improved liver + kidney morphology	^40–44
	Leaf extract (alcoholic) 100 mg/kg per day PO; 2 wk	↓FBG, ↓TC	⁴⁵
	Rhizome extract (alcoholic) 100 mg/kg per day PO; 2 wk	↓MDA, ↑SOD, ↓CRP, ↓IL-6	⁴⁶
GI + kidney health	Seed extract (aqueous)
	1–10 μg/mL; acute perfusion	↑Gastric acid secretion�↓MgSO₄-induced diarrhea	⁴⁷
	10 mL/kg PO; acute	NE: BaSO₄- and castor oil-induced diarrhea	⁴⁸
	Seed extract (alcoholic)�100–500 mg/kg PO; 1–4 h�1–10 mg/kg IP; 6 h	↓Ethanol- and aspirin-induced gastric lesions�↑Urine vol, ↑Na⁺/K⁺ excretion	^49,50�⁵¹
	Seed extract (petroleum ether) 12.5–150 mg/kg PO; 1–4 h	↓Ethanol- and aspirin-induced gastric lesions	^49,50
	Seed volatile oil 12.5–150 mg/kg PO; 1–4 h	↓Ethanol- and aspirin-induced gastric lesions	^49,50
Inflammation + pain	Seed volatile oil
	133–400 μL/kg IP; 1 h	↓Carrageenan-induced paw edema, ↑analgesic activity	⁵²
	10 mg/mL PO; 1 h	NE: carrageenan-induced paw edema	⁵³
	0.3% w/w diet; 1 mo	NE: carrageenan-induced paw edema	⁵³
	Seed extract (hexane) 50–100 mg/kg IP; acute	↓Carrageenan-induced paw edema�↓Paw tissue levels of COX-2 + IL-6 + TNF-α + iNOS-mediated NO generation�↑SOD, ↑CAT, ↑GSH	⁵⁴
	Seed powder�5% w/w diet; 1 mo	NE: carrageenan-induced paw edema	⁵³
			⁵³
			⁷⁷

31 of 68

31

TABLE 2 - Summary of Green Cardamom Effects in Animal Models

Condition	Treatments Dose/Duration	Outcomes	References

Cancer	Seed powder
	0.5 mg/d PO; 8 wk	↓Chemical-induced colonic ACF, ↓colon cell proliferation, ↓colon COX-2 + iNOS expression, ↑colon + liver GST, ↓colon + liver LPO	^55,56
	0.5 mg/d PO; 16 wk	↓Chemical-induced skin cancer, ↓liver LPO, ↑liver GSH	⁵⁷
	500 mg/kg PO; 12 wk	↓Chemical-induced skin cancer, ↓skin TBARS + COX-2, ↓skin Nrf-2 expression + Keap-1 expression, ↑skin GSH + GPx + SOD + CAT	⁵⁸
	500 mg/kg PO; 24 wk	↓Chemical-induced stomach cancer, ↑liver GST + GPx + SOD + CAT + GSH, ↓liver LPO	⁵⁹
	Seed extract (alcoholic) 100 mg/kg PO; 10 d	↓Ehrlich ascites tumor weight + size�↑Tumor SOD + GPx + mRNA expression of Bax + caspase-3/caspase-8/caspase-9	⁶⁰
	Seed volatile oil 100–200 mg/kg PO; 24 wk	↓Chemical-induced liver cancer�↓TNF-α, ↓IL-1β, ↓AFP, ↓ALT, ↓AST�↑Liver SOD + CAT + GSH + GPx + GR	⁶¹

Neurological function	100–400 mg/kg PO; 8 wk	↓Diabetes-induced learning and memory deficits, ↓brain Aβ_1–42 deposits + p-tau–positive cells + AChE + GSK-3β	⁴²
	200–800 mg/kg IP; 30 min	↓Anxiety-like behavior	⁷²
	Seed powder
	10%–20% w/w diet; during pregnancy to postnatal 15 d	Offspring: ↑learning and memory, ↓eye opening, ↓neuromotor maturation, ↓weight gain, ↑forebrain 5-HT + DA	⁷³
	5%–20% w/w diet; during pregnancy to postnatal 22 d	Offspring: ↓weight gain + eye opening�♂: ↑Aggressive behavior�♀: ↓Aggressive behavior	⁷⁴
	10%–20% w/w diet; during pregnancy to postnatal 15 d	Offspring: ↑nonsocial threat and attack behavior, ↓social and defense behaviors + motor activity�♂: ↑Progesterone�♀: ↑Testosterone	⁷⁵

32 of 68

32

TABLE 2 - Summary of Green Cardamom Effects in Animal Models

Condition	Treatments Dose/Duration	Outcomes	References

Infection	Seed volatile oil 258 mg/kg PO; 6 d	↓Intestinal burden of bacterial pathogens, ↓inflammation, ↓proinflammatory mediators in intestine, kidney, and lungs	⁷⁶

Toxicity	Seed extract (aqueous)
	100–200 mg/kg PO; 30 d	↓Chemical-induced myocardial injury, ↓myonecrosis, ↓edema, ↓inflammation	⁶²
	100–200 mg/kg PO; 3 wk	↓Gentamycin-induced kidney damage, ↓serum urea + creatinine	⁶³
	200 mg/kg PO; 5 wk	↓DOX cardiotoxicity, ↓heart apoptosis + oxidative stress + inflammation, ↑heart angiogenesis	⁶⁴
	Seed extract (alcoholic)
	250–1000 mg/kg PO; 15 d	↓Chemical-induced amnesia. ↓brain LPO, ↑brain GSH + CAT + SOD	⁶⁵
	Seed extract (ethyl acetate) 100–200 mg/kg PO; 5 wk	↓CCl₄-induced liver injury, ↓GPT, ↓GOT, ↓ALP, ↓liver LPO, ↑liver SOD, ↑liver GST + SOD gene expression	⁶⁷
	Seed volatile oil
	5–100 mg/kg PO; 13 d	↓DOX-induced immunosuppression, ↑WBCs, ↑CD4⁺ cells, and CD8⁺ cells	⁶⁸
	100–200 mg/kg PO; 42 d	↓Al-induced neurotoxicity, ↓behavioral deficits, ↓brain AChE + oxidative stress + amyloid β plaque	⁶⁹
	Seed powder
	0.2% w/w diet; 12 mo	↓Pan masala–induced lung injury	⁷⁰
	0.2% w/w diet; 9 mo	↓Pan masala–induced testicular damage	⁷¹

33 of 68

Review of Literature

Side effects:

Dermatitis

Chronic contact dermatitis has occurred with repeated exposure to cardamom
In a case report, the patient had positive patch test reactions to cardamom and certain terpenoid compounds present in the dried ripe seeds of cardamom

33

34 of 68

Conclusion

The limited number of human trials evaluating cardamom’s effects on blood glucose and lipid dysregulation provides inconsistent results.
Animal studies do provide preliminary evidence that different extracts of green cardamom can modify aberrant glucose and lipid metabolism and biomarkers of inflammation.
Thus, future clinical trials of participants with cardiometabolic disorders should consider examining multiple doses of cardamom seed powder and its essential oil for periods of administration greater than 3months.
Larger well-controlled studies of subjects stratified by similar health issues are needed. Despite few adverse effects observed from intake of up to 3 g/d cardamom seed powder in the trials to date, continuing monitoring of any adverse events during extended periods of seed powder or essential oil intake is recommended. Further confirmation and characterization of cardamom's potential anti-inflammatory mechanisms of actions in humans are warranted.
More high-quality clinical trials elucidating mechanisms of action, intake levels, safety, and health outcomes of cardamom consumption are needed before evidence-based dietary recommendations can be developed.

34

35 of 68

Future Perspectives:

Genotoxic effects
Allergic effects
Analgesic effects
Quantification of active ingredient (HPLC)

On the basis of antioxidant potential through HPLC quantification
Oleoresin (Hu et al., 2021)

35

36 of 68

Changes in enzymatic activity of polyphenol oxidase (PPO) and its structural modification in apple juice subjected to high pressure carbon dioxide (HPCD)

INTERNATIONAL SEMINAR-2022

Dr. Ayesha Murtaza (Ph.D.)

Assistant Professor

University of Central Punjab (UCP),PAKISTAN

PRESENTED AT

Nutrition Health Polytechnic Ministry of Health Tasikmalaya

37 of 68

Apple Juice

Problem during juice processing

Polyphenol oxidase

Thermal and non-thermal Technologies

PART ONE

INTRODUCTION

38 of 68

38

Explored the effects of different phases of carbon dioxide under HP-CO2 treatment on aggregation, conformational changes and inactivation mechanism of polyphenol oxidase from apple juice.

To study the comparison of thermal and HP-CO2 treatment on quality and stability of apple juice.

To investigate the aggregation, conformational changes and inactivation mechanism of polyphenol oxidase enzyme in apple juice after thermal treatment

Characterization of phenolic profile using HPLC from apple juice after thermal and HP-CO2 treatment.

Research Objectives

01

02

04

03

39 of 68

39

Need of the day

Mostly consumers prefer minimal processed fruit juices with good nutritional value, natural appearance and fresh like characteristics without adding preservatives

Apple Juice

40 of 68

40

Endogenous Enzymes

Polyphenol oxidase (PPO)

Peroxidase (POD)

Pectin methylesterase (PME)

PPO-the major culprit

Apple, Pear, Peach, Mango, Strawberries, avocados, lettuce, potatoes

Browning

Either desirable (bakery) or undesirable
Alters the appearance and nutritive value, thus lower consumer acceptability.
Enzymatic browning mainly caused by the endogenous enzyme

41 of 68

Improper handling, storage and processing cause browning.

Fruits and vegetables

Enzymatic

Non-Enzymatic

Oxidation of phenolic compounds

Sensory properties

Bad

Problems during juice processing

41

42 of 68

42

“Copper” containing molecule, mainly responsible for browning reactions in fruits
Copper catalyzing the oxidation of phenol by

Oxidation of monophenols to O-diphenol
Reduction of O-diphenol to O-quinone, responsible for polymerization of brown pigments

Quinones may also non-enzymatically react with phenolic compounds to produce brown pigments “Melanoidin”

Polyphenol Oxidase (PPO)

43 of 68

43

Structurally, PPO has 2 copper ion in its active site, surrounded by six histidine and one cysteine residues
Changes in active site affect the activity of PPO protein
Cu ions play role in oxidation-reduction process by leading transition of active site among met-, oxy-, and deoxy-forms in a cyclic manner.
During each cycle, catechols are oxidized and water molecule is formed by reducing molecular oxygen, resulting in formation of browning caused quinine products.

Structure of PPO protein

44 of 68

44

Structure-PPO protein

Active site of PPO

45 of 68

Most common food processing method to achieve enzymatic inactivation is “Thermal Processing”
Combination of temperature & time must be optimized to increase the effectiveness in enzymatic inhibition
Biggest drawback of this traditional processing can cause undesirable changes in food flavor, color, and texture, and can destroy heat-sensitive nutritional attributes such as vitamins

ThermalMethod

45

46 of 68

46

Non-thermal processing market has globally grown exponentially in the last few years and estimated to reach USD 1,224.2 Million by 2022 (Global Forecast, 2022)
Non-thermal technologies are receiving wide acceptability from the government and federal bodies

Non-thermal processing

47 of 68

47

NONTHERMAL

PROCESSING

Shelf Life Extension

Innovative Fresh Products

Minimally Processed

Green Technology

Unwanted Enzyme Inactivation

Pathogen Inactivation

48 of 68

High pressure carbon

dioxide (HPCD)

Pulsed electric field

Ultrasonic processing

Non-thermal

Processing

Emerging Non-thermal processing

48

49 of 68

49

Without exposing food to detrimental heat influence , it retains the physical, sensory, and nutritional qualities that are more closer to fresh foods.
CO₂ which is non-flammable, non-toxic and physiologically safe, used to inactivate enzymes.
Combination of pressure, temperature and time is used to avoid negative quality effects on fruits juices.

Pressure range 4 to 30 MPa
Temperature range 20 ºC–55 ºC

HPCD

Method

50 of 68

50

Different factors affect the enzyme inactivation
HPCD Pressure
HPCD Temperature
Treatment time
Initial pH
Processing system & storage time
CO2 states

Gaseous State 25 ºC and 40 ºC at 5 MPa
Liquid State 25 ºC and 40 ºC at 10 MPa
Critical State 31.1 °C-7.38 MPa
Super-critical state 40 °C and 55 °C at 25 MPa

Factors affecting enzyme inactivation by HPCD

51 of 68

51

METHODOLOGY

Apple juice

HPCD treatment

CD spectral Analysis

Fluorescence analysis

Quality Attributes

Electrophoresis analysis

PPO Extraction & Purification

Structural Analysis

PPO activity Assay

BD Analysis

Color Measurement

pH

TSS

PSD Analysis

52 of 68

52

Effect of HPCD on the activity of crude PPO

PPO retained most of its RA in gaseous & liquid states even at 40 ºC

Transition from a critical to a supercritical state cause high CO2 diffusibility and solubility, resulting in intensive structural modifications which leads to PPO inactivation.

Critical state at 31.1 ºC, 7.38 MPa, its activity drastically decreased to 64.88%.

Fig. 1. (A) Residual activity of PPO (%).

RESULTS

53 of 68

53

Browning degree of HPCD treated juice during storage

Super critical state could reduce the BD of juice during storage, indicating high pressure can prevent browning by inactivation PPO enzyme.

HPCD had an inhibitory effect on the browning of apple juice content.

Fig. 2. Browning degree during storage at 4 °C.

54 of 68

54

No significant change in pH and brix. Thus, sweetness and acidity of apple juice after CO2 treatment will not have a significant impact on the taste.
In the critical state (31.1 ºC for 7.38 MPa), sudden change in color occurred while super critical state at 55 ºC for 25 MPa had the lowest △E value

pH, △E and brix of apple juice after HPCD treatment

Parameter	Control	25 °C 5MPa	40 °C 5MPa	25 °C 10MPa	31.1 °C 7.38 MPa	40 °C 10MPa	40 °C 25MPa	55 °C 25MPa
pH	3.60±0.05^a	3.60±0.11^a	3.58±0.01^a	3.61±0.02^a	3.62±0.056^a	3.56±0.02^a	3.58±0.051^a	3.59±0.03^a
△E	6.87±0.03^a	6.71±1.76^a	5.97±0.15^b	4.93±0.08^c	3.30±0.73^d	3.22±0.12^d	2.91±0.57^e	2.03±0.40^f
Brix°	10.25±0.18^a	10.22±0.83^a	10.25±0.57^a	10.25±1.21^a	10.25±0.01^a	10.25±0.05^a	10.26±0.5^a	10.24±0.12^a

55 of 68

55

Effect of HPCD on the PSD analysis

8.23 %

29.8 %

Dissociation

Aggregation

36.6 %

At critical state two peaks depolymerized into 3 peaks, suggesting dissociation and aggregation along with formation of polydisperse enzyme microstructure.

At supercritical state the main part transferred to 531 nm, indicating the aggregation of large particles.

PPO molecules deformed, aggregated and conformational changes in the critical and supercritical states.

Fig. 2. Dynamic light scattering analysis showed particle size distribution (PSD) pattern of native and HPCD-treated PPO.

56 of 68

Effect of HPCD on the secondary structure

Value of the slots gradually decreased as temperature and pressure increased, resulting the loss of α-helix conformation.

Negative peak in the critical state and the supercritical state decreased compared with that of native PPO.

Fig. 3. Circular dichroism (CD) spectra of the native and HPCD -treated PPO.

56

57 of 68

57

Effect of HPCD on the tertiary structure of PPO

Critical state dramatically decreased the fluorescence intensity at λ_max of 312

induced evident blue shifts

Supercritical state induced the unfolding of PPO molecules, causing a decrease in the fluorescence intensity and the disruption of the tertiary structure

A series of blue shifts was observed as HPCD intensity increased with decreased in fluorescence intensity

RESULTS

Fig. 4. Fluorescence spectra of native and HPCD treated PPO at various treatments showing correlation analysis between blue shift and inactivation rate.

58 of 68

Secondary structure contents

Treatments	Secondary Structure contents (%)
Treatments	α-Helix	β-sheet	β-turn	Random coil
Native	23.56±0.61^a	40.81±1.43^c	18.41±0.98^c	17.22±0.64^c
25 °C 10 MPa	20.42±0.40^b	41.21±1.03^b	19.44±1.56^b	18.93±1.31^b
31.1 °C 7.38 MPa	12.21±0.73^c	43.11±0.62^ab	21.85±0.42^ab	22.83±1.87^ab
55 °C 25 MPa	10.50±1.49^d	44.12±0.35^a	22.01±0.20^a	23.37±0.98^a

x

Loss of α-helix caused a rearrangement by increasing the negative ellipticity of the spectra
Supercritical CO2 state leading to secondary structure deformation and resulting in the reduction of activity

RESULTS

58

59 of 68

59

(I) SDS-PAGE); (II) Native PAGE stained with Coomassie Brilliant Blue R-250; (III) Native PAGE stained with catechol.

Super-critical state showed complete loss of enzyme activity because of protein degradation

Native

25 °C,10 MPa

31.1 °C,7.38 MPa

55 °C,25 MPa

Native

25 °C,10 MPa

31.1 °C,7.38 MPa

55 °C,25 MPa

Native

25 °C,10 MPa

31.1 °C,7.38 MPa

55 °C,25 MPa

60 of 68

60

Proposed Mechanism of PPO by HPCD inactivation

61 of 68

61

CO2 in critical and super critical states could effectively inactivate the PPO enzyme in apple juice
PSD pattern revealed that structural modification of PPO leads to initial dissociation and subsequent aggregation
Decreased α-helix and β-turn contents and increased β-sheet & Random coil contents confirmed the rearrangement and denaturation of the secondary structure

Conclusion

62 of 68

HPCD induced blue shifts in λmax with large decreases in fluorescence intensities resulting disruption of tertiary structure, exposing PPO to more polar environment, consequently reducing the activity of PPO enzyme
The application of supercritical state of CO2 under HPCD would become an important and green processing technology to obtain safe and nutritious food products

62

Conclusion

63 of 68

Future Directions

FUTURE DIRECTIONS

51

64 of 68

64

FUTURE DIRECTIONS

Further research is necessary to determine the influence of combination of non-thermal treatments on the enzyme inactivation and quality improvements of juices to propose more effective treatments applicable on the industrial scale.

52

The inactivation mechanism of other enzymes as result of thermal and non-thermal treatment should also be focused in future studies

Economics of different processing methods should be assessed to reinforce their commercial usage.

PPO should be broadly studied to determine its usage i.e. biotechnological applications in food industry, pulp and paper industry, textile industry, medicine and environmental technology.

Recently, the use of PPO to treat industrial waste water by removing phenols should also be addressed.

65 of 68

65

Publications

66 of 68

66

67 of 68

67

68 of 68

68