Food Enzymes in Pharmaceutical Industry:

Perspectives and Limitations 3

Iffat Zareen Ahmad, Heena Tabassum, Asad Ahmad,
and Mohammed Kuddus

Abstract
Enzymes are the chemical compounds which act as a biological catalyst and alter
the rate of biochemical reactions. There are about 3000 enzymes in our bodies
which are involved in more than 7000 metabolic reactions. Considering the
physiological and metabolic significance of the enzymes, food enzymes consti-
tute an important part of diet. There are some enzyme-rich foods that are believed
to have a different function in the human body than that of body’s own enzymes.
But, processing and cooking of foods completely degrade the enzyme contents
and leave the body deprived of these important enzymes which play a significant
role in the well-being and healthcare. Some prominent examples of high enzyme
foods are papaya, pineapple, banana, figs, and bee pollen. The sprouts are
metabolically very active, and the content of enzymes is more as compared to
seeds. The dietary enzymes commonly present in the enzyme-rich foods are
proteinases, amylases, maltases, lipases, papain, bromelain, etc. Non-processed
and uncooked foods are rich in enzymes, and their intake might decrease the
body’s burden to produce more and more of its own enzymes. As a result of some
metabolic disorders and diseases, the production of some specific enzymes is
hampered in the body. In such cases, foods which are high in enzyme contents are
the best choice for the extraction, purification, and commercialization of the

I. Z. Ahmad (*) · H. Tabassum · A. Ahmad

Department of Bioengineering, Integral University, Lucknow, Uttar Pradesh, India
e-mail: iffat@iul.ac.in
M. Kuddus
Department of Biochemistry, College of Medicine, University of Hail, Hail, Kingdom of Saudi
Arabia

# Springer Nature Singapore Pte Ltd. 2018 41

M. Kuddus (ed.), Enzymes in Food Technology,
https://doi.org/10.1007/978-981-13-1933-4_3
42 I. Z. Ahmad et al.

enzymes. These enzymes find numerous applications in pharmaceutical

industries, and there is a need for exploiting these foods for industrial
applications.

Keywords
Food enzymes · Digestive · Pharmaceutical industries · Fortified foods · Disorders

Abbreviation

ACE Angiotensin-converting enzyme

AKP Apricot kernel protein
BSA Bovine serum albumin
EAT Ehrlich ascitic tumor
EC Enzyme commission number
ELISA Enzyme-linked immunosorbent assay
GE Gastric emptying rate
GP Ginger protease
HAase Hyaluronidase
HPLC High-performance liquid chromatography
LLC Lewis lung carcinoma
LOX Lipoxygenase
NEM N-ethyl maleimide
PG Polygalacturonase
PME Pectin methyl esterase
POD Peroxidase
PPO Polyphenol oxidase
SPI Soy protein isolates
TLP Thaumatin-like protein

3.1 Introduction

Enzymes are defined as a biocatalyst that can alter the rate of a biochemical reaction. But
in the recent past, these biological molecules are the most researched entities for the novel
applications which could be conferred on them. The enzymes are proteins except for
abzymes and ribozymes. In the present-day scenario, the enzymes are applied in almost
every sphere of life. They are being applied in pharmaceutical, food, textile, detergent,
rubber, biofuel, paper, dairy, food, and medical industries to name some important ones.
They are found in every living cell and control the biochemical activities at
cellular level and functional level at large. All the important physiological functions
like digestion, circulation, respiration, excretion, and fertilization are regulated by
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 43

enzymes, and they are the most wonderful molecules of the body. However, the
body can synthesize most of the enzymes depending upon the requirement, but in
some instances, there is a deficiency of enzymes which creates disturbance in the
normal functioning and metabolism. This may be due to some disease, poor diet,
injury, or hereditary dysfunction.
The enzymes are characterized by their specificity and high catalytic power. They
have a particular temperature and pH at which there activity is maximum and get
degraded easily. Enzymes are broadly classified into six broad groups depending upon
the type of reaction which they catalyze. Each enzyme is being provided a particular
enzyme commission number (EC). The enzyme basically comprises of two parts, the
protein part which is known as apoenzyme and the nonprotein part known as the cofactor.
Together, they form the complete functional enzyme, called as holoenzyme (Fig. 3.1).
There are many sources of enzymes for their extraction on large scale to be used
in industries and for commercialization purpose. Microorganisms are being
exploited the most because of the ease by which they grow and the recovery
procedures applicable on them. But, the plants are source for some unique enzymes
which have high potential with respect to industrial applications and commerciali-
zation. Food enzymes are a new concept in the same perspective which has open up
new avenues and scope in research. In the same context, the present chapter is
designed to discuss potential dietary plant products as a source of enzymes and their
interesting applications. Since the future of nutritional enzyme research is bright, the
current work will benefit those who are working in this area of study.

3.1.1 Significance of Food Enzymes

Plants are rich sources of enzymes, and they might be termed as “food enzymes” or
“dietary enzymes.” The plant source possesses one or more specific enzymes with
pacific substrate and catalytic function. Mostly the enzymes present in foods are
digestive in nature and improve the digestion process. They further complement the
body’s own enzymes in digestion, and they function earlier before the body’s own
digestive process begins. They might be very useful in increasing the process of food
digestion in case of a less efficient digestive system.
Proper diet and metabolism are critical for the functioning of a healthy body.
Healthcare persons should also look from this perspective while treating a disorder
since the requirement of optimum energy is the primary need of the body. A weak
body is susceptible to succumb to various types of illnesses. Enzymes are constituent of
all the living plant and animal cells. They are the facilitators of biochemical reactions.
Existence of life is not possible without the ordered metabolic reactions which take place
with the help of enzymes. Enzymes can be classified into three broad categories:

1. Food enzymes – present in raw fruits and vegetables and help in digestion as
dietary enzymes to improve the physiological process.
44 I. Z. Ahmad et al.

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Fig. 3.1 Mechanism of enzyme action

2. Digestive enzymes – synthesized in the cells to degrade complex compounds

present in food into simpler ones which can be easily transported through the
gut wall.
3. Metabolic enzymes –synthesized in the body for proper metabolism to release
energy for various functions.

Howell (1985) tried to mark the differences between plant enzymes and the
enzymes produced in the body. He was of the opinion that plant enzymes in diet
do not have the same role in human digestion as that of the body’s own digestive
enzymes. He was successful in finding the difference in function. The food enzyme
initiates food digestion in the stomach at least 1 h before the initiation of body’s
digestive system. This is the reason that these enzymes must be treated as essential
nutrients. But for this, several considerations have to be taken into account, mainly
the overcooking or heat that destroys the enzyme component of food as they are
proteinaceous in nature and easily get denatured and lose their activity. Moreover,
the preservation and the strategy of companies to remove the enzymes from food for
gaining better shelf life are seriously harmful for the body. The culture of processed,
canned, and fast foods is also responsible for the various health-related issues
especially in growing children.
Eric Schlosser’s myth-breaking survey in which he covered California’s
subdivisions along the New Jersey Turnpike which is a hub of many fast food and
the invention of recipes showed that fast food has penetrated every part of American
culture. He also unearthed the fact that the fast-food companies are creating nuisance
and efforts to bring in the young children who are the most vulnerable customers and
all the more enhance their unethical exploitation of teenagers and minors. Fast food
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 45

has encouraged the mall culture and shrunk our landscape, broadened the gap
between rich and poor, drove an epidemic of obesity, and boosted American cultural
domination abroad (Scholsser 2002).

3.1.2 Foods As a Source Of Enzymes

Different enzymes and their source are presented in Table 3.1 and Fig. 3.2.

3.1.2.1 Pineapple (Ananas comosus L.)

The pineapple is a tropical plant having a delicious edible multiple fruit consisting of
coalesced berries with an exclusive flavor and taste. The family of this plant is
Bromeliaceae and is known for several wonderful health benefits. Pineapple is being
used in traditional medicines by the tropical natives for various ailments which
include respiratory problems, coughs and colds, digestion, and obesity, as anti-
inflammatory and anticancer, for bone strengthening and eye care, and to improve
immunity and circulation. These properties may be attributed to bromelain to a great
extent as this compound has revealed specific pharmacological benefits. It has shown
anticancer, antithrombotic, anti-inflammatory, antiedematous, and fibrinolytic
activities along with improved drug absorption. The way by which bromelain
interacts with these varied biological processes is related partly to its variations in
the arachidonate pathway (Taussig and Batkin 1988). They are safe and effective
after oral administration and are nontoxic with no side effects.
The extract of pineapple is rich source of bromelain which contains many closely
related proteinases, besides other compounds. Several biochemical investigations
reveal that the therapeutic activities of bromelain are not only due to proteolytic
activity but also due to the presence of some nonprotein factors. Latest preclinical
and pharmacological researches on bromelain endorse it as a complementary tumor
therapy which could be given orally and it resembles the actions of trypsin and
chymotrypsin. They have an effect on blood and endothelial cells as they regulate the
roles of adhesion particles and also monitor the activation of different immune cells
and the production of cytokines. These enzymes are used as an alternative medicine
in the United States and Europe. Bromelain is shown to improve the cytotoxicity of
monocytes against tumor cells and acts as an immunomodulator. It helps in the
synthesis of certain cytokines such as tumor necrosis factor-a, interleukin (Il)-1b,
Il-6, and Il-8 in patients, and the action was recently confirmed with mammary tumor
patients. The data from animal models states an anti-metastatic ability and suppres-
sion of platelet accumulation during metastasis and also arrest of progression and
metastasis of tumor cells. Actually, the anti-invasive action is not dependent on the
protein degrading activity. This is the same for bromelain properties regarding its
effect on the immune system and its prospective to remove debris of burn and to
hasten healing of wound (Maurer 2001).
The study was undertaken to explore the antitumor effects of the principal
compound isolated from pineapple (Ananas comosus). For this, bromelain
(EC 3.4.22.32) was purified from pineapple stem which is a major cysteine
46 I. Z. Ahmad et al.

Table 3.1 Food enzymes from various plant sources and their pharmacological activities
Enzyme EC no. Source Pharmacological activity References
Bromelain 3.4.22.32 Pineapple Anticancer, Taussig and
antithrombotic, anti- Batkin (1988)
inflammatory,
antiedematous, and
fibrinolytic
Proteolytic activity Maurer (2001)
Antitumor and Baez et al.
antileukemic (2007)
Proteases 3.4.22.2 Papaya Wound healing Gurung and
Skalko-Basnet
(2009)
Edema and inflammation www.newsrx.
com
Arthritis, rheumatism, Owoyele et al.
asthma, wound healing, (2008)
and antitumor effects
Pumpkin Inhibitor Krishnamoorthi
et al. (1990)
Ficin 3.4.22.3 Fig Proteolytic activity Zare et al.
(2013)
Papain 3.4.22.2 Papaya Proteolytic activity Amri and
Mamboya
(2012)
Antifungal effects Nwinyi and
Anthoni (2010)
Antiparasitic, antiseptic, Elgadiri et al.
antimicrobial, anti- (2014)
inflammatory,
antihyperlipidemic,
antihypertensive, and
antidiabetic effects
Pepsin 3.4.23.3 Papaya, Proteolytic, Vishal et al.
Papain 3.4.22.2 Fig endoesterolytic, and (2013)
Hyaluronidase 3.2.1.36 amidolytic activities
Actinidin 3.4.22.14 Kiwifruit Help in digestion Maddumage
Kiwellin 3.4.22.14 et al. (2013)
Thaumatin –
Brucin – Macassar Antibacterial activity Kumar et al.
kernels (2017)
Alpha glucosidase 3.2.1.20 Melon Substrate preference for Gao and
raffinose Schaffer (1999)
Bromelain 3.4.22.32 Pineapple Proteolytic activity, Zengion and
fibrinolytic activity Yarnell (2011)
Anti-inflammatory effects Kaye et al.
of cervical dysplasia (2012)
Menstrual problems, Romm et al.
cervical application (2010a)
Antimicrobial effects Ali et al. (2015)
(continued)
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 47

Table 3.1 (continued)

Enzyme EC no. Source Pharmacological activity References
Brucin – Fructus Antibacterial activity
bruceae
Pepsin 3.4.23.3 Cluster Gastric ulceration Kumar et al.
Fig (2010)
Lipoxygenase 1.13.11.12 Cucumber Concentrate the enzyme, Wardale and
substrate specificity, Ambert (1980)
stability, kinetics, pH, and
temperature
Amylase 3.2.1.1 Mango Maintain the temperature, Mehrnoush and
pH, metal ions, inhibitors Yazid (2013)
and surfactant agents, and
stability
Transglutaminase 2.3.2.13 Mango Food protein stabilization Lantto et al.
Tyrosinase 1.14.18.1 (2006)
β-galactosidase 3.2.1.23 Apricot Lactose intolerance Yossef and El
Beltagey (2014)
Angiotensin- 3.4.15.1 Apricot Food industry and Zhu et al. (2010)
converting nutraceuticals
enzyme (ACE)
Polyphenol 1.14.18.1 Apricot Ripening process Chevalier et al.
oxidase (1999)
Asparagus N/A Kiwifruit Improving the tenderness Ha et al. (2013)
enzyme of specific cuts of meat
Cellulase 3.2.1.4 Avocado Cellulase activity and its Kanellis et al.
Polygalacturonase 3.2.1.15 total immunoreactive (1989)
protein
Polyphenol 1.14.18.1 Avocado Ripening process Vanini et al.
oxidase (2010)
Peroxidase 1.11.1.7
Polyphenol 1.10.3.1 Banana Remove pigmentation, Archer et al.
oxidase enzymatic browning (1975)
Catalase 1.11.1.6 Beans Blanching and storage Lee et al. (1988)
Lipoxygenase 1.13.11
Polyphenol 1.14.18.1
oxidase
Peroxidase 1.11.1.7
Myrosinase 3.2.1.147 Broccoli Food preparation and Ludikhuyze
processing et al. (2000)
Betacyanine – Red beet Decolorizing Lashley and
tissue Wiley (1979)
Cyanidin-3-O- – Cherries Antioxidant, anti- Mulabagal et al.
rutinoside inflammatory, anticancer, (2009)
antidiabetic, and antiobese
properties
Bovine serum 3.4.22.67 Ginger Proteolytic activity Thompson et al.
albumin (1973)
(continued)
48 I. Z. Ahmad et al.

Table 3.1 (continued)

Enzyme EC no. Source Pharmacological activity References
Proteases 3.4.22.2 Ginger Proteolytic activity Nafi et al.
(2013)
Peroxidase 1.11.1.7 Garlic Blanching activity Fante and
Polyphenol 1.14.18.13 Norena (2012)
oxidase
Inulinase 2.1.7
Lipase 3.1.1.3 Garlic Food storage activity Mashayekhi
Protease 3.4.21.112 et al. (2016)
α-Amylase 3.2.1.1
Peroxidase 1.11.1.7 Grape Antioxidant activity Troiani et al.
Polyphenol 1.10.3.1 (2003)
oxidase
Thioglucosidases 3.2.1.147 Mustard Flavor enhancer Mackay and
and Hewitt (1959)
cabbage

Fig. 3.2 Structure of various food enzymes from plant sources

proteinase, and its pharmacological properties were investigated. The aqueous

extraction with buffering was carried out for the extraction of bromelain from
stems of mature pineapple plants and purified by ion exchange chromatography
using cation. Further characterization of bromelain was carried out by reverse-phase
high-performance liquid chromatography (HPLC), SDS-PAGE, and N-terminal
sequencing. The in vivo antitumor/antileukemic effects were also estimated on
various tumor cell lines. Bromelain was given through intraperitoneal administration
after 24 h of inoculation with tumor cell in experimental sets in which 5-fluorouracil
was taken as positive control. The antitumor effect was determined by the improve-
ment in the rate of survival (% survival index) after the treatments in different sets. It
was seen that all the animals induced with tumor had a marked increase in survival
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 49

index except for MB-F10 melanoma after bromelain treatment. The best survival
index was obtained in mice bearing EAT ascites which was even better than 5-FU.
Bromelain reduced the metastasis in lung cells considerably induced by LLC
transplantation, as seen with 5-FU (Baez et al. 2007).
Bromelain, because of its proteolytic activity, showed stimulation of fibrinolysis
by enhancing the production of plasmin. It has also shown the prevention of kinin
production and the inhibition of platelet aggregation (Glaser and Hilberg 2006).
Since its action is a general anti-inflammatory response, it is used as a painkiller, and
it is not given with food as it acts as a digestive enzyme. It is also used as a sports
medicine as it improved contractile function of muscle during the recovery after
workout and altered period of muscle pain with respect to placebo (Miller et al.
2004).
Bromelain improved the function and reduced aching in acute knee pain and knee
osteoarthritis (Brien et al. 2004). Bromelain might be helpful to reduce the postsur-
gical healing period and intensities of edema, pain, and ecchymoses (Orsini 2006).
Studies on animal model indicated that bromelain inhibits fibrinogen synthesis and
increases fibrinolytic activity (Lotz-Winter 1990; Zengion and Yarnell 2011).
Animal and human models have been used for in vivo studies of bromelain for
inflammatory disease (Hale et al. 2005; Majima et al. 1996; Majima et al. 1997;
Ogino et al. 1996). Bromelain acts through various possible mechanisms such as
inhibition of plasma exudation by inhibiting the release of bradykinin at the location
of inflammation via declination of the plasma kallikrein system and inhibition of the
arachidonic acid pathway (Felton 1980; Kumakura et al. 1988; Taussig and Batkin
1988; Uchida and Katori 1986). Advantageous anti-inflammatory properties have
also been seen in patients suffering from HIV and cancer (Kaleef et al. 1996). It has
shown inhibitory effects on Chlamydia infection. Medication of the sexual partner
(with antibiotics) was also considered necessary for the success of the study.
Bromelain is a significant proteolytic component in the management of cervical
dysplasia (Kaye et al. 2012; Romm et al. 2010b).
The treatment of cervical patients with bromelain improved symptoms in nullip-
arous women with severe primary dysmenorrhea. All patients got instant relief of
their primary dysmenorrhea. An indefinite number of patients with disabling dys-
menorrhea were treated with bromelain solution. Among 64 patients who have
undergone treatment, 40 got instant relief. Only fair to poor results were obtained
in patients with secondary dysmenorrhea owing to other gynecologic diseases
(Romm et al. 2010a).
The study evaluated the antimicrobial effect of crude bromelain extract from
pineapple fruit (Ananas comosus L.) on the microorganisms isolated from fresh and
overnight kept meat at varied temperatures and pH. The extraction of bromelain was
carried out from pineapple fruit followed by its estimation. Six bacterial strains,
namely, Proteus spp., Corynebacterium spp., B. subtilis, S. pyogenes, and two
dissimilar strains of E. coli., were isolated and identified by the traditional
techniques. The antimicrobial activity of raw bromelain extract was evaluated by
the disk diffusion method. One strain of E. coli exhibited maximum zone of inhibi-
tion, but the other strain was resistant. Corynebacterium spp. was least inhibited of
50 I. Z. Ahmad et al.

all the tested strains and showed temperature-dependent activity. Proteus spp.
displayed inhibition, but the activity was not temperature-dependent. B. subtilis
and S. pyogenes were resistant to bromelain extract at all tested temperatures in
neutral pH media. B. subtilis, S. pyogenes, and E. coli were completely inhibited at
pH 10.0. The crude enzyme showed improved action against Proteus spp. at pH 10.0
but was unable to inhibit the growth of Corynebacterium spp. Crude bromelain
appeared to be more active in the inhibition of Gram-positive bacteria as compared
to Gram-negative. Crude bromelain could be an effective antimicrobial agent against
E. coli and Proteus (Ali et al. 2015).

3.1.2.2 Papaya (Carica papaya L.)

Carica papaya is also known as papaya, chichpu, melon, papaw, pawpaw, and
mamao tree (http://www.rain-tree.com/papaya.htm#.WaUyNfVOLVI). It is gener-
ally cultivated for its young leaves, shoots, and fruits which are a part of Indian
recipes and eaten as a vegetable, and its ripe fruit is very tasty besides being used as a
popular beverage (Hewitt et al. 2002). Papaya is known for a range of medicinal
properties and has shown a positive effect against bacterial infections (Wimalawansa
1981). It is also shown to have wound healing activity which could be attributed to
phagocytic engulfment of bacteria (Gurung and Skalko-Basnet 2009). The different
parts Carica papaya have shown antibacterial activities against Staphylococcus
aureus, Bacillus subtilis, Bacillus cereus, Escherichia coli, Enterobacter cloacae,
Proteus vulgaris, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas
aeruginosa, and Shigella flexner (Yismaw et al. 2008). Papain is the main enzyme
present in Carica papaya, and it is shown to have a great potential of herbal drug in
monitoring both edema and inflammation accompanying with surgical operations
(Kumar et al. 2017). It has also shown therapeutic properties in patients with
inflammatory disorders of the liver, intestine, and eye (Rakhimov 2000). Some
diseases linked with arthritis, rheumatism, asthma, and wound healing can be
cured using leaf extract of Carica papaya (Owoyele et al. 2008). The aqueous
extract of Carica papaya has also shown promising antitumor effects on human
lymphocytes (Otsukia et al. 2010).
Carica papaya L. is a natural source of papain which is synthesized from the latex
of raw papaya fruits. It is a plant proteolytic enzyme belonging to the cysteine
proteinase family containing cysteine protease enzyme. The enzymes catalyze the
breakdown of proteins comprising polypeptide chains and therefore play a vital role
in varied biological phenomenon involving normal body functions to diseased
conditions, drug formulation and development, and applications in food industry
such as meat tenderizers. Papain has a typical structure which is responsible for its
specific function explaining the proteolytic action of this enzyme and its mechanism
of action (Amri and Mamboya 2012). Effects of extracts from Carica papaya
L. containing papain were shown to reduce mycelial growth of various fungi
including Rhizopus spp., Aspergillus spp., and Mucor spp. (Nwinyi and Anthoni
2010). Papaya is a fruit herb belonging to the family Caricaceae of tropical and
subtropical regions. It is a polygamous plant which is known for its nutritional and
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 51

medicinal properties. Its fruits are low in calories and are a rich source of vitamins,
minerals, and fiber (Boshra and Tajul 2013).
The extracts of C. papaya showed positive antimicrobial, antiparasitic, antiseptic,
anti-inflammatory, antihyperlipidemic, antidiabetic, and antihypertensive effects
(Elgadiri et al. 2014). The papain enzyme extract showed degradation activity on
levetiracetam and granisetron HCl drug compounds which are harmful to cellular
systems (Hitesh et al. 2012). The study was undertaken on pepsin, papain, and
hyaluronidase to explore the principle of enzyme assays and kinetics, mechanism of
enzyme action, effect of pH, and inhibitor on enzyme activity and its significance in
controlling diseases. The proteolytic, endoesterolytic, and amidolytic activities of
papain were studied. Enzyme-linked immunosorbent assay (ELISA) for hyaluroni-
dase (HAase) has also been done (Vishal et al. 2013).
The proteolytic enzymes of papaya latex have been extensively studied for long
(Bergmann and Fruton 1941; Hwang and Ivy 1951). Later, Balls and coworkers
stated the purification of papain (Balls et al. 1937; Balls and Lineweaver 1939) and
chymopapain (Jansen and Balls 1941) from fresh papaya latex. As shown by earlier
researchers (Bergmann and Fruton 1941), semi-purified extracts of papain hydrolyze
different synthetic peptide derivatives; the purified enzyme in crystal form also
showed similar activity. Papain also exhibited strong esterase activity, and in this
manner it showed similarity (Kimmel and Smith 1954).

3.1.2.3 FIG (Ficus carica)

The latex of fig tree is a rich source of ficin (EC 3.4.22.3) which is a cysteine
endoproteolytic protease, and it occurs in several isoforms. The research was done
for the evaluation of autolysis of ficin as the data in this regard was lacking. Ficin
was purified, and its autolysis was evaluated by HPLC chromatogram data and
ultrafiltrations at varying temperatures and storage duration. High temperatures
lead to autolysis of all ficins, while only two ficins were prone to autolysis at low
temperatures. Increased storage time leads to the degradation of ficins. The data
revealed that the number of HPLC peaks in latex protein fractions of Ficus carica
cv. Sabz was different from earlier studied fig cultivars. Cysteine was the important
amino acid present on the active site of ficins as its proteolytic activity was inhibited
by specific cysteine protease inhibitors. The data of zeta potential was negative for
the first two peaks eluted, whereas that of other peaks was positive (Zare et al. 2013).

3.1.2.4 Kiwifruit (Actinidia deliciosa)

Kiwifruit has shown allergic reactions, and the three major allergens have been
studied including actinidin, kiwellin, and thaumatin-like protein (TLP) (Maddumage
et al. 2013). Kiwifruit has been used for long to aid in gastric digestion and has been
an edible fruit of choice since ancient times. This might be attributed to the
proteolytic enzyme called actinidin. In the current study, an in vitro experiment
was performed to evaluate the activity of kiwifruit proteases (actinidin) on the
digestion of a variety of normal food proteins under simulated gastric conditions.
The physiological digestive system was mimicked by using green kiwifruit extract
containing actinidin, and its digestive potential on different proteins derived from
52 I. Z. Ahmad et al.

soy, meat, milk, and cereals was evaluated and compared with pepsin at pH 1.9. The
band pattern of denaturing gel, SDS-PAGE, showed degradation of complete protein
which resulted into various peptide patterns in kiwifruit extract as compared to those
observed after digestion with pepsin alone. It can be concluded that, in in vitro
conditions, actinidin present in kiwifruit extract improved the digestion of some food
proteins better than that with pepsin alone (Kaur et al. 2010).
Actinidin belongs to the category of cysteine protease enzyme present in fruits
including kiwifruit. Dietary actinidin affects the protein digestion in the stomach to
great extent, and it is also believed that this digestive effect is supplemented by the
gastric emptying rate (GE). A study was conducted on a rat model to evaluate the
influence of dietary actinidin on GE and gastric digestion of six dissimilar dietary
protein sources which included beef muscle, gelatin, gluten, soy protein isolate
(SPI), whey protein isolate, and zein. The two diets, one supplemented with green
kiwifruit (Actinidia deliciosa cv. Hayward) containing actinidin and the other gold
kiwifruit (Actinidia chinensis cv. Hort16A) which does not contain actinidin, were
supplied to the experimental rats. The real-time GE and the digestion of protein in
the stomach were evaluated, and the data showed that the dietary actinidin increased
the gastric digestion of beef muscle maximally followed by gluten and SPI. An
increase in the GE of the diets having beef muscle and zein was also observed
(Montoya et al. 2014).
The inclusion of green kiwifruit in the diet has been supposed to help in the
digestion of dietary proteins present in milk, meat, fish, eggs, legumes, and cereal
proteins, and this was attributed to the proteolytic enzyme actinidin. It is already
shown that green kiwifruit and actinidin can improve the digestion in the upper tract.
Kiwifruit extract alone without any digestive enzymes has shown to affect the
protein digestion in foods such as yogurt, cheese, fish, and raw eggs (Kaur and
Boland 2013).
Seven different cultivars of Chinese kiwifruit were studied for protease and milk-
coagulation activity, and protein patterns were matched. The cultivar Xuxiang
showed actinidin with largest protease and milk-clotting activity which was a
cysteine protease characterized by a pH of 3.5 and temperature of 40  C. It showed
better tenderness muscle proteins of animals after the treatment with actinidin. Also,
angiotensin I-converting enzyme (ACE) inhibitory peptides were achieved from five
plant-derived proteins using actinidin (Zhang et al. 2017).

3.1.2.5 Brucea javanica (L.) Merr.

The pepsin hydrolysis of the dried fruits of Brucea javanica (L.) Merr. leads to the
production of a new antibacterial peptide to which Streptococcus pyogenes showed
sensitivity. The extract was characterized by reverse-phase HPLC, and antibacterial
peptides showed activity against Gram-negative and Gram-positive bacteria when
analyzed by SDS-PAGE and nano-LC-MS/MS. One chemically synthesized
11-amino acid-long peptide having a molecular mass of 116,831 Da showed the
most powerful antimicrobial effect against S. pyogenes, and it was called as Brucin.
Its antibacterial activity was 16 times and 125 times greater than penicillin G and
chloramphenicol, respectively (Sornwatana et al. 2013).
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 53

3.1.2.6 Cluster Fig (Ficus racemosa Roxb.)

The ethanol extract of Ficus racemosa was studied for its anti-ulcer activity against
gastric ulcer induced by pyloric ligation, aspirin, and ethanol in rats. The extract of
fruits exhibited a dose-dependent decrease in the occurrence of ulcers in rat models
(Kumar et al. 2010).

3.1.2.7 Pumpkin (Cucurbita maxima)

Pumpkin seeds have been shown to be a source of a serine protease which is
involved in blood clotting and is a protein inhibitor of trypsin and activated
Hageman factor (Factor XII) by means of trypsin-affinity chromatography and
reverse-phase HPLC. The protein comprises of 68 amino acid residues and 1 disul-
fide bridge and showed a high level of sequence homology to the Potato I inhibitor
family (Krishnamoorthi et al. 1990).

3.1.2.8 Melon (Cucumis melo)

Three α-galactosidases were reported from the melon (Cucumis melo) fruit tissue,
and they were partially purified for the characterization of these enzymes. The form I
enzyme which was unique showed desired activity with raffinose and marked
activity with stachyose. There was a weak product inhibition by galactose in
comparison to other counterparts which also confer physiological importance. The
activities of these enzymes were studied at different phases of fruit growth using
raffinose and stachyose as substrates The data showed that activity of form I enzyme
increased initially during ovary development and fruit set, in comparison to the other
α-galactosidase enzymes, which showed decreased activity. In the ripened mesocarp
which contained great amount of sucrose, the alkaline form I enzyme was the major
α-galactosidase which was present (Gao and Schaffer 1999).

3.1.2.9 Cucumber (Cucumis sativus)

The cellular localization of lipoxygenase (LOX) enzyme from cucumber fruit was
studied, and high LOX activity was obtained in the intact protoplasts isolated from
peel and flesh tissues. More LOX activity was observed in the peel than in flesh
tissues, and it was shown in both that this activity was associated with the vacuoles.
The cucumber LOX enzyme showed similarity with the potato and tomato enzymes
(Wardale and Ambert 1980).

3.1.2.10 Mango (Mangifera indica)

Amylase is one of the industrially important enzymes applied in food, detergent,
pharmaceutical, pulp, and paper industries. Amylase can be extracted from mango
(Mangifera indica cv. Chokanan) peel which might be a prospective source of
amylase. The amylase having a molecular weight of 42 kDa was obtained from
mango peel which was stable at high temperature as more than 85% of the activity
was reserved at temperatures of 20–55  C and pH of 7.0 for 20 min. Activity of the
enzyme was markedly enhanced in the presence of Ca2+ but reduced in the presence
of Zn2+ and Cu2+. The enzyme activity was completely deactivated in the presence
54 I. Z. Ahmad et al.

of carbodimine and p-chloromercuribenzoic acid, whereas iodoacetamide did not

show any effect (Mehrnoush and Yazid 2013).

3.1.2.11 Apple (Malus pumila)

The effect of polyphenol oxidase (PPO) present in apple powder was studied on the
meat homogenate structure. Mushroom tyrosinase was not able to affect gel forma-
tion. But, apple pulp powder, a by-product of an industrial method, may contain
suitable enzyme activities for food protein stabilization (Lantto et al. 2006).

3.1.2.12 Apricot (Prunus armeniaca L.)

Apricot seed is a source of β-galactosidase, and after purification and dialysis, high
enzyme yield was obtained. The isolated enzyme showed an optimum temperature
of 70  C and pH of 5. It was also stable for 30 min when incubated at 55–70  C of
temperature range, and its activity was improved by Ca+2. No marked difference in
flavor and consistency was seen in treated free lactose white cheese and non-treated
cheese (Yossef and El Beltagey 2014).
The six varieties of commercially available proteases (Flavourzyme, Neutrase,
Protamex, Alcalase, Proleather FG-F, and papain) were used to hydrolyze apricot
kernel protein (AKP). Alcalase was chosen for the enzymatic treatment of AKP to
produce ACE inhibitory peptide. The ACE inhibitory peptides were further purified
by gel filtration column which could find applications in food and nutraceutical
industries (Zhu et al. 2010). A homologous PPO probe from apricot (Prunus
armeniaca var. Bergeron) fruit was prepared by reverse transcriptase-polymerase
chain reaction experiment and was used to isolate a full-length PPO cDNA. The gene
was greatly expressed in young, unripe green fruit and was turned off early during
ripening (Chevalier et al. 1999).

3.1.2.13 Asparagus (Asparagus officinalis)

The enzyme extracts from two fruits, that is, kiwifruit and asparagus, were studied to
find out their potential for the hydrolysis present in beef proteins in connective tissue
and topside myofibrillar extracts. The kiwifruit protease extract was proved better at
hydrolyzing myofibrillar and collagen proteins than the asparagus protease extract
(Ha et al. 2013).

3.1.2.14 Avocado (Persea americana)

The hydrolytic enzymes present in cell walls and total protein content were evaluated
with and without ethylene during ripening of avocado fruits (Persea americana Mill.
cv. Hass). The less O2 atmosphere prohibited the increase in the activities of
cellulase, polygalacturonase, and acid phosphatase in avocado fruits, whereas low
O2 atmosphere did not show these effects (Kanellis et al. 1989).
The study was undertaken to find out the activity of PPO and peroxidase enzymes
in avocado pulps, from the northwest area of Paraná-Brazil. The enzymes were
extracted from avocado pulp of Choquete, Fortuna, and Quintal varieties, in unripe
and ripe stages of ripening. A reduction in polyphenol oxidase activity was seen in
all the varieties on increasing the temperature and time. Fortuna and Choquete
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 55

varieties exhibited the minimum PPO activity in the ripe stage. In Choquete variety,
soluble peroxidase activity was observed in the unripe fruit, whereas ionically bound
peroxidase activity was seen during alteration from unripe to ripe stage of maturation
(Vanini et al. 2010).

3.1.2.15 Banana (Musa acuminata)

An experiment on the characterization of banana PPOs showed that they (o-
diphenol:oxygen oxidoreductase, EC 1.10.3.1) are accountable for the enzymatic
browning in many fresh fruits and vegetables during an injury. Banana PPO is
responsible for the catalytic oxidation of many orthodiphenols to the respective
quinones which react nonenzymatically to produce melanin pigments. The dietary
melanins are synthesized by polymerization and/or condensation of the quinones
with amino acids, peptides, or proteins (Archer and Palmer 1975).

3.1.2.16 Beans (Phaseolus vulgaris L.)

The study was undertaken to see the effects of various types of blanching on enzyme
activity and the alterations in its properties in green beans during cold storage. The
data was collected at various temperature and time durations at both small- and large-
scale bases. The samples were also analyzed for antioxidant activities including
catalase, lipoxygenase, PPO, and peroxidase and alterations in the sensory quality as
evaluated by taste panel, color, and firmness. The blanching effects varied from the
deactivation of most of the enzymes, and the activity also varied (Lee et al. 1988).

3.1.2.17 Broccoli (Brassica oleracea)

The effects of MgCl2, ascorbic acid, pH, temperature, and pressure were studied on
myrosinase activity of broccoli. A mixture of MgCl2 and ascorbic acid increased the
activity of enzyme, and the pH optimal was found to be between 6.5 and 7, and the
optimum temperature was 30  C. Low pressure increased the activity slightly,
whereas high pressure decreased the activity. This study can be beneficial in
monitoring the glucosinolate hydrolysis in food and processing research
(Ludikhuyze et al. 2000). The presence of betacyanine decolorizing enzyme has
been confirmed in raw beet tissue. The effects of temperature and pH on the enzyme
activity were studied (Lashley and Wiley 1979).

3.1.2.18 Cherries (Prunus cerasus)

The study was conducted on Royal Anne and Bada cherries and it was observed
that they soften constantly throughout ripening and cold storage. However, main
alterations in texture took place in the course of a 2-week duration which
corresponded with sharp increments in weight, volume, soluble solids,
polygalacturonase (PG), and pectin methyl esterase (PME) activity. No changes
ensued among the varieties in the activities of enzymes responsible for softening at
any time during sampling. PME activity was observed during the first week of
sampling, and PG and β-gal activities were seen after the second and fifth week,
separately. The combined activities of PG, PME, and β-gal seem to be necessary for
softening of cherry (Barreit and Gonzalez 1994).
56 I. Z. Ahmad et al.

3.1.2.19 Ginger (Zingiber officinale Roscoe)

The extraction and characterization of proteolytic enzyme was done from Zingiber
officinale Roscoe (ginger) by homogenization in phosphate buffer. The enzyme was
purified by dialysis and lyophilized. The data showed that the ginger protease
(GP) was totally inhibited by divalent metallic ions, such as Cu2+ and Hg2+ ions,
and a thiol-blocking agent, n-ethyl maleimide (NEM), showing its cysteine protease
nature. The enzyme showed temperature optima at 60  C, and the pH optima ranged
from 6 to 8. Detergents also affected the activity of the enzyme in which its activity
was enhanced by sodium dodecyl sulfate and there was slight reduction by Tween
80 and Tween 20 (Nafi et al. 2013).
The protein degrading activity of ginger was evaluated on bovine serum albumin
(BSA), collagen, and actomyosin as substrates. The enzyme extract was prepared
from rhizome, and high proteolytic activity was observed with BSA at a pH range of
4.5–6, and the proteolytic degradation of collagen was much more than that of
actomyosin. The proteolytic active principle Zingiber officinale Roscoe is known
as “zingibain,” and it is more beneficial than papain and ficin as it showed higher
proteolysis of collagen in comparison to actomyosin. Moreover, as compared to
bromelain, zingibain has greater temperature optima (Thompson et al. 1973).

3.1.2.20 Garlic (Allium sativum L.)

The kinetics of enzyme inactivation of peroxidase, PPO, and inulinase and changes
in the color were studied in garlic cloves during steam blanching at 100  C and water
at 80 and 90  C. The best blanching conditions were in steam for 4 min, during
which no variation in texture was observed, the enzyme activities were decreased,
and the samples became light in color as the blanching time increased (Fante and
Norena 2012).The alterations in the carbohydrates, enzymes, and pigments were
evaluated in Allium sativum L. cv. Azarshahr bulbs during storage from harvest to
sprouting. The data showed that the contents of starch, lipase, and protease were
reduced during initiation of sprouting in clove. Anthocyanin content was increased
after harvesting and then reduced during sprouting (Mashayekhi et al. 2016).

3.1.2.21 Grapes (Vitis vinifera L.)

Three grape cultivars of Vitis vinifera L., namely, Rubi, Borbon, and Benitaka, were
studied for the evaluation of activities of enzymes, peroxidase (POD), and PPO. The
effect of temperature on the activity was also studied, and the data showed that Rubi
and Benitaka cultivars were more thermostable than Borbon cultivar for both
enzymes (Troiani et al. 2003).

3.1.2.22 Mustard (Brassica juncea) and Cabbage (Brassica oleracea)

The research study on flavor enzymes from cabbage was undertaken. It was shown
that thioglucosidases are present as a constituent of enzyme system in cabbage and
mustard and their assay can be done easily by action in the release of glucose from
sinalbin and sinigrin. Sensory panel data, nevertheless, revealed that the enzyme
extract from cabbage and mustard sources could be differentiated by their effects on
flavor development in the reconstituted dehydrated cabbage. It was also indicated
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 57

that a mechanism of enzyme activity would be more clear by estimating the released
isothiocyanates in mustard oil (Mackay and Hewitt 1959).

3.1.3 Food Enzymes in Pharmaceutical Industry

The enzymes used in digestion have the tendency to decompose the complex
nutrients into simpler ones. The significance of these enzymes can be gaged from
the idea that the simpler products of digestion enter the bloodstream for either
catabolism to release energy or for anabolic purposes. In case of any digestive
disorder, the whole process is disturbed leading to severe effects. Therefore, the
digestive enzyme supplements might be very useful alternative in such cases, e.g.,
lactose intolerance and cystic fibrosis. Keeping this in mind, many products
containing these enzymes are accessible in the marketplace. The difference in
these formulations lies in the enzyme content, its source, and the dose. But still,
the research in this regard is not sufficient as the scope in this regard is infinite in
pharmaceutical industry. In the present scenario, the sources of the enzymes include
microbes, animals, and plants. However, plant-derived enzymes offer much more
potential as a source of digestive enzymes for many obvious reasons. But, the
research in this area, mechanism-based approaches, patenting, and commercializa-
tion are the need of the hour (Fig. 3.3).
In case of the disorders of pancreas, like pancreatitis, cancer, cystic fibrosis, or
diabetes (Borowitz et al. 2011; Domínguez-Muñoz 2007; Imrie et al. 2010; Olesen
et al. 2013; Wiera and Kuhnb 2011; Zubarik and Ganguly 2011) diseases related to
lactose metabolism (Kanabar et al. 2001), celiac disease (Mitea et al. 2008), enzyme
therapy has been used. Till April, 2010, the approval of the FDA was not required for
any pancreatic therapy, but after that FDA made the clinical trials and investigational
new drug application submission mandatory for the authorization of pancreatic
enzyme formulations in the United States, which led to the withdrawal of earlier
stuffs from the market (Wiera and Kuhnb 2011). The formulations which received
approval from the FDA in United States include Creon and Zenpep (2009),
Pancreaze (2010), and Ultresa, Viokace, and Pertzye (2002) (United States Pharma-
copeia 2002). In addition to this, lactase products including Lactogest, soft gel
capsule; Lactaid, caplet; and DairyEase, chewable tablet were used on patients of
lactose intolerance and showed positive results (Lin et al. 1993).

3.2 Conclusion

There is a requirement to include more and more raw, uncooked, and unprocessed
food in our diet. The popularity of vegetable juice lies in the fact that they are living
raw food which leads to marked improvements in our energy status and health
issues. The exposure of enzymes to heat renders them devoid of the function for
which they were made. Cooking of foods destroys the enzyme component and leads
to disruption of assisted food digestion which might result into diseases related to
58 I. Z. Ahmad et al.

Fig. 3.3 A working model of drug-enzyme nutrient interactions

digestion. The digestion of cooked food utilizes valuable metabolic enzymes for
food digestion and requires more energy as compared to the digestion of raw food.
The digestion of raw food is quite rapid and less time-consuming than the cooked
food. The study of food enzymes must be taken simultaneously with food minerals
and vitamins as a part of our nutrition in order to bring out its significance to the
society. The enzymes are being produced by our digestive system in the body, yet
food enzymes play significant role in maintaining optimum health, and they are
constituent of uncooked foods such as fresh fruits and vegetables, raw sprouted
grains, and unpasteurized dairy products.
A healthy body is strong and less prone to diseases, and it has the ability to
maintain its normal weight, metabolism, and self-healing with better immune sys-
tem. In case of digestive diseases including malfunction in the digestive organs and
glands, malabsorption, and inborn errors of metabolism, the food enzyme supple-
ment from plant sources will prove to be very useful in the treatment. But, one has to
take care of the availability of sufficient research data, proper clinical trials, and
approval from the FDA before commercializing these products. Moreover, the
ethical, safety, and toxicity issues must also be considered. The future of dietary
enzymes seems to be very bright and very promising area demanding much more
literature and studies.
3 Food Enzymes in Pharmaceutical Industry: Perspectives and Limitations 59

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