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Probiotics as potential antioxidants: A Systematic Review Vijendra Mishra, Chandni Shah, Narendra Mokashe, Rupesh Chavan, Hariom Yadav, and Jashbhai Prajapati J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506326t • Publication Date (Web): 26 Mar 2015 Downloaded from http://pubs.acs.org on March 29, 2015
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Journal of Agricultural and Food Chemistry
Probiotics as potential antioxidants: A Systematic Review
Vijendra Mishra*1, Chandani Shah2, Narendra Mokashe1, Rupesh Chavan1, HariomYadav3, JashbhaiPrajapati2
1
National Institute of Food Technology Entrepreneurship and Management Kundli, Sonipat (India)
2
Dairy Microbiology Department, Anand Agricultural University, Anand, Gujarat (India) 3
National Agri Biotechnology Institute, Mohali (India)
*Corresponding Author Phone: +91-0130-2281167, Email: [email protected]
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ABSTRACT
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Probiotics are known for their health beneficial effects and have established as dietary
3
adjuncts. Probiotics have been known for many beneficial health effects. In this view, there is
4
interest to find the potential probiotic strains that can exhibit antioxidant properties along
5
with health benefits. In vitro and in vivo study provides that probiotics exhibit antioxidant
6
potential. In this view, consumption of probiotics alone or foods supplemented with
7
probiotics may reduce oxidative damage, free radical scavenging rate and modification in
8
activity of crucial antioxidative enzymes in human cells. Incorporation of probiotics in foods
9
can provide a good strategy to supply dietary anti-oxidants, but more studies are needed to
10
standardize methods and evaluate antioxidant properties of probiotics, before they can be
11
recommended for antioxidant potential. In this paper, literature related to known antioxidant
12
potential of probiotics and proposed future perspective to conduct such studies has been
13
reviewed.
14
Keywords: Antioxidants, Oxidative stress, DPPH activity, Lactobacillus, Bifidobacteria,
15
Probiotics
16
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Introduction
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Oxidative stress is the causeof various chronic human diseases. Oxidative stress is caused by
19
increased activity of reactive oxidative species (ROS) through oxidation process. Oxidation is
20
a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation
21
reactions can produce free radicals having unpaired electron and are capable of carrying out a
22
rapid chain reaction, thus destabilize other molecules and generate further free
23
radicals. Examples of free radicals include the superoxide anion, hydroxyl radical, transition
24
metals such as iron and copper, nitric acid and ozone. Oxygen containing ROS is the most
25
biologically significant free radicals. In oxidative stress conditions, cellular mitochondria
26
produce more ROS
27
peroxide) than enzymatic antioxidants(Table 1) (e.g., superoxide dismutase (SOD),
28
glutathione peroxidase (GPX), peroxidase, Glutathione-S-transferase and catalase) and non-
29
enzymatic antioxidants e.g. ascorbic acid (vitamin C), tocopherol (vitamin E), glutathione,
30
carotenoids and flavonoids present/ supplied to the cells. ROS mediated oxidative stress are
31
known to play vital role in the development of chronic diseases (Fig. 1) such as cancer,
32
diabetes, heart disease, stroke, Alzheimer's disease, rheumatoid arthritis, cataract and aging1-
33
3
34
terminate the chain reaction before damage is done to the vital molecules. All these
35
molecules carry out diverse physiological role in the body by acting as an inhibitor of the
36
process of oxidation. Antioxidants terminate the chain reactions by removing free radical
37
intermediates and inhibit other oxidation reactions by neutralizing free radicals. But in the
38
process, antioxidants are themselves oxidized. Thus there is a constant need to replenish
39
antioxidant resources as one antioxidant molecule can only react with a single free radical4.
40
Consequently, it is essential to search and develop natural nontoxic antioxidants to protect
41
human body from free radicals and slowdown the progress of many chronic diseases. Due to
(e.g., superoxide anion radicals, hydroxyl radicals and hydrogen
. Antioxidants are molecules which interact with free radicals generated in cells and
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changes in consumer perception towards foods as sources of therapeutic value, there is a
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spurt in the market of food based ingredients and supplements which provide antioxidants.
44
The projected market value of antioxidants to grow to$238.5 million by 2018, at the CAGR
45
of 4.5%5. There is renewed interest in the search of new sources of antioxidants, which can
46
be safely used in food. Out of various sources, probiotics have been considered as an
47
emerging source of effective antioxidants. Due to their long tradition of safe use, along with
48
potential therapeutic benefits, role of probiotics as an antioxidant is being keenly
49
investigated.
50
Mode of action of food derived antioxidants
51
According to the mode of action, two main groups of antioxidants can be distinguished. The
52
first comprises the chemical constituents which interrupt the propagation of the free radical
53
chain by hydrogen donation to radicals or stabilization of relocated radical electrons. Such
54
mode of action is demonstrated by tocopherols, gallusans and hydrochinons6. The second
55
group is characterized by a synergistic mode of action. It includes oxygen scavengers and
56
chelators that bind to ions involved in free radical formation. Their activity consists of
57
hydrogen delivery to phenoxy radicals that leads to the reconstitution of the primary function
58
of antioxidants. This role is played by substances binding to metal ions, e.g. citric acid and by
59
secondary antioxidants, such as amino acids, flavonoids, β-carotene and selenium7. The
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main source of these substances is plant material like garlic, broccoli, green tea, soybean,
61
tomato, carrot, brussels sprouts, kale, cabbage, onions, cauliflower, red beets, cranberries,
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cocoa, blackberry, blueberry, red grapes, prunes and citrus fruits. With a huge market for
63
functional foods comprising antioxidative attributes, there is renewed interest in search of
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new diverse sources of antioxidants that can be safely used. Dairy products have also been
65
reported to possess antioxidant properties (Table 2) and among various dairy supplements,
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probiotics have been recently known for potential sources of antioxidants
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Probiotics
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Probiotics are defined as “live microorganisms which when administered in adequate
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amounts, confer health benefits to the host”8. Probiotics have established their efficacy as
70
dietary factors which can regulate gastrointestinal functions thereby imparting health benefits
71
to consumers. Alleviation of lactose intolerance, prevention of different forms of diarrhoea
72
and urogenital infections, cholesterol reduction, reduction of atopic diseases and modulation
73
of the immune system are some of the functions attributed to probiotics9-10. Other benefits
74
include prevention of cancer, particularly colon carcinoma and food allergy11-12. The
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competitive exclusion of pathogens and reduction in number as well as metabolic activities of
76
harmful organisms by probiotics has been demonstrated in vitro13. Lactobacilli are the major
77
source of probiotics and are usually explained as Gram positive bacteria, devoid of
78
cytochromes and preferring anaerobic conditions, but are aero tolerant, fastidious, strictly
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fermentative and produce lactic acid as main product14. Various species and strains of
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lactobacilli i.e. L. acidophilus, L. casei, L. rhamnosus and L. helveticus are considered as
81
successful probiotics and different variants have been available in market for human
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consumption. Bifidobacterium, even though not grouped with lactic acid bacteria, is another
83
genus with probiotic functions. B. lactis (B. animalis ssp. lactis) is a very commonly used
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probiotic, although it is not a normal inhabitant of the human gastrointestinal tract. B. longum
85
ssp. longum (and ssp. infantis) and B. breve is mainly used in supplements15. Although,
86
various strains of lactobacilli and bifidobacteria have been known for various health
87
beneficial effects, but their role as anti-oxidant have been understudied.
88
Application of probiotics in food
89
During last 20 years, consumer’s perception towards functional foods including probiotics
90
has changed. The presence of probiotics in commercial food products has been very well
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accepted and has been known for number of health benefits. In this direction, industries
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worldwide are concentrating on applications of probiotics in food products as well as
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constructing a new era of “probiotic health” foods. Foods are considered as better delivery
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vehicles for probiotic organisms than others due to the role played by food matrix in
95
maintaining the viability of organisms during the transition through the gastrointestinal tract.
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Dairy foods are considered natural carriers for lactobacilli and bifidobacteria due to their
97
ability to utilize lactose and produce lactic acid.
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Methods for antioxidant activity screening of probiotics
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Various methodologies have been adopted to determine the antioxidative potential for natural
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compounds; however, it is important to use consistent and rapid methods. Each antioxidative
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activity assay has specific target within the well-defined matrix. Each method has its own
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merits and demerits. In the absence of a universal method which can give unambiguous
103
results, the best way out is to use the different methods instead of one. Some of the
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procedures involve use of synthetic antioxidants or free radicals, few are specific for lipid
105
peroxidation and require animal and plant cells, some have a broader scope, some require
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minimum preparation and information, and few methods are speedy to produce results16. The
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antioxidative activity of food products including probiotic products has been studied by using
108
different in vitro and in vivo methods.
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In vitro antioxidative methods. The in vitro assays are based on determination of
110
scavenging capacity against ROS such as superoxide anion scavenging assay, hydrogen
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peroxide scavenging assay and scavenging activity against stable non biological radicals.
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Several assays have been used to assess the total antioxidant content in foods such as 6-
113
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent antioxidant
114
capacity (TEAC) assay, the ferric-reducing antioxidant power assay (FRAP) and the oxygen
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radical absorbance capacity assay (ORAC) assay. However, it has been found that the most
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common and reliable methods are the 2,2-Diphenyl-1-Picrylhydrazyl(DPPH) and 2,2’-azino-
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bis-3-ethylbezothiazoline-6-sulfonic acid(ABTS) methods; these methods are simple, rapid,
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sensitive and reproducible and have been modified and improved as per research
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requirements17. A brief description of some antioxidant potential screening methods used for
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probiotics is presented here and summarized in Table 3.
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2,2-Diphenyl-1-Picrylhydrazyl (DPPH)assay. This widely used discoloration method was
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first described by Blois (1958)18. This assay is based on the premise that a hydrogen donor is
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an antioxidant. The antioxidants are able to reduce the free, stable and purple colored DPPH
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radical to the yellow coloured diphenyl-picrylhydrazine, which is monitored by using
125
colorimeter. DPPH is usually used as a reagent to evaluate free-radical scavenging activity of
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antioxidants19.
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2,2’-azino-bis(3-ethylbezothiazoline-6-sulfonic acid (ABTS)assay. The ABTS radical
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scavenging method was developed 20and was then modified by others21. In this assay, ABTS
129
is oxidized by oxidants to its radical cation, ABTS , which is intensely coloured and
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antioxidant capacity is measured as the ability of test compounds to decolorize the ABTS
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radical directly. ABTS
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cation forms the basis of one of the spectrophotometric methods that have been applied for
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measuring the antioxidant property of pure substances, aqueous mixtures and beverages22.
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Hydroxyl radical scavenging activity assay. The hydroxyl radical (.OH) is the neutral form
135
of the hydroxide ion (OH ). This radical is known as the most reactive species and is known
136
for lipid peroxidation and DNA damages23. In this assay, the hydroxyl radical is indirectly
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confirmed by the hydroxylation of p-hydroxybenzoic acid. However, iron plays central role
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in the hydroxyl radical formation via Fenton reaction.
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Ferric reducing antioxidant power (FRAP) assay. The FRAP assay is characterized by
140
ability of antioxidant to reduce ferric 2,4,6-tripyridyl-s-triazine complex [Fe -(TPTZ)2]
•-
•+
radicals are more reactive than DPPH while generation of its radical
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3+
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2+
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depending on the available reducing species followed by the alteration of
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[Fe -(TPTZ)2]
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color from yellow to blue in acidic condition (pH 3.6) and is analyzed through a
143
spectrophotometer24-26.This method was employed to measure reducing power in plasma, but
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the assay subsequently has also been adapted and used for the assay of antioxidants in
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different food or beverages27-32, tea and fruits. A drawback of this method is that antioxidant
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capacity of certain compounds which can react with ferrous ion (Fe ) and SH group-
147
containing antioxidants cannot bemeasured33.
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Oxygen radical absorbance capacity (ORAC) assay. The ORAC assay uses area-under-
149
curve technique and combines the inhibition time and inhibition degree of free radical action
150
by an antioxidant into a single quantity. On the other hand, similar methods use either
151
inhibition time at a fixed inhibition degree or the inhibition degree at fixed time as basis for
152
quantifying the outputs34-36. ORAC evaluates antioxidant inhibition of peroxy radical-
153
induced oxidations and thus reflects the classical radical chain-breaking antioxidant activity
154
by hydrogen atom transfer (HAT)37. The peroxy radicals are generated through thermal
155
decomposition of “2,2’-azobis(2-amidino-propane) dihydrochloride” (AAPH) in aqueous
156
buffer. The principle of this method is based on decreasing the intensity of fluorescent
157
molecule such as β-phycoerythrin or fluorescein of the target along with time under
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reproducible and constant flux of peroxy radicals.
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Antioxidative potential of probiotic microorganisms and foods
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Each characteristic of probiotic organism is first established using in vitro methods and
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subsequently in vivo experiments conducted in suitable animal models. On confirmation of
162
suitability of organism, it is incorporated in suitable food product and further studies are
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conducted using probiotic food to establish its efficacy, survival under storage conditions
164
etc8. In vitro antioxidative studies have been conducted on different probiotic organisms
165
(mainly lactobacilli and bifidobacteria) as well as food products containing probiotic
2+
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organisms have also studied. Following section reviews these studies and summary is
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presented in Table 4.
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Lactobacillus sp.
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Lactobacillus rhamnosus. Different antioxidative capacities of lactobacilli species to modify
170
the bacterial profile and prevent the oxidative stress in the Fe-overloaded mice colon were
171
studied 38. The generation of hydrogen peroxide and hydroxyl radical as well as growth of Fe-
172
dependent bacteria is encouraged by Iron (Fe). Findings of this study showed that survival
173
time of L. rhamnosus GG strain was significantly longer in the presence of H2O2 and
174
hydroxyl radical as compared to non-antioxidative strains, L. paracasei Fn032 and L.
175
plantarum Fn001, respectively. In addition, L. paracasei Fn032 and L. plantarum Fn001were
176
specific for free radical scavenging activities of their intracellular free extracts (ICFE).
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Scavenging activities increased to varying extent when bacterial strains were exposed to
178
gastric and pancreatic juice. However, Achuthan et al., (2012)39 also reported the resistance of
179
a total 39 Lactobacillus cultures to ROS. Most of the isolates showed moderate to strong
180
resistance towards 0.4 mM H2O2. Majority of cultures demonstrated high resistance to
181
hydroxyl radicals and isolate L.plantarum21 (Lp21) was most resistant with log count
182
reduction of 0.20 fold only. Out of 39 isolates, Lactobacillus spp. S3 showed highest total
183
antioxidative activity of 77.85 followed by 56.1 of isolate L.plantarum55(Lp55) in terms of
184
percent inhibition of linolenic acid oxidation. Moreover, isolate L. plantarum 9 (Lp9) up
185
regulated the expression of SOD (superoxide dismutase) gene 2 in HT-29 cell at the
186
concentrations of 0.1mM (1.997 folds) and 1.0 mM H2O2 (2.058 folds). In addition to this,
187
cultures L.plantarum9 (Lp9), L.plantarum91 (Lp91) Lp91 and L.plantarum55 (Lp55) showed
188
significant upregulation in the expression of glutathione peroxidase-I gene to the level of
189
5.451, 8.706, and 10.083 folds respectively when HT-29 cells were challenged with 0.1mM
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H2O2. On the other hand, catalase gene expression was also significantly regulated by all the
191
isolates used in this study in presence of 0.1 mM H2O2.
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Grompone et al., (2012)40 developed a new, fast, predictive and convenient in vitro method
193
for screening of antioxidative potential of new probiotic strains using the nematode
194
Caenorhabditis elegans as host (model organism). In this study, total 78 strains of lactic acid
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bacteria (Lactobacillus and Bifidobacterium) were examined. This collection is composed by
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62 Lactobacillus strains belonging to L. acidophilus, L. bulgaricus, L. casei, L. paracasei, L.
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plantarum and L. rhamnosus species, 9 Streptococcus thermophilus isolates and 6
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Bifidobacterium strains belonging to the B. animalis, B. breve and B. longum species. The
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LAB stains used in this study are fed to C. elegans and survival rate of C. elegans was
200
examined upon exposure to H2O2-induced stress by comparing with the protective effect
201
exerted by E. coli OP50.As a result of this screening, a Lactobacillus rhamnosus strain (Lb.
202
rhamnosus CNCM I- 3690) demonstrated a high antioxidant capacity.
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Kapila et al., (2006)41examined the antioxidative activity of ICFE of 13 strains of
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Lactobacillus sp. by using Microsome-Thiobarbituric acid(MS-TBA) and linoleic acid
205
peroxidation method. Maximum antioxidant capacity in terms of percent inhibition of
206
oxidation was expressed in L. casei ssp. casei 19 (76.82%) followed by L. acidophilus 14
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(62.21%), Lactobacillus sp. L13 (59.25%), L. casei ssp. casei 63 (53.30%), L. helveticus 6
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(52.70%) and L. delbreuckii ssp. bulgaricus 4 (52.64%) while remaining strains showed less
209
than 50% activity. In addition to this, strain L. casei ssp. casei 19 showed 72.04% linoleic
210
acid peroxidation followed by L. acidophilus 14 (51.74%), Lactobacillus sp. L13 (51.38%)
211
and rest of all strains exhibited < 50% activity.
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Similarly, otherauthors42reported the antioxidative potential of ICFE of 19 strains of lactic
213
acid bacteria (16 lactobacilli, 2 streptococci, 2 lactococci) out of 570 strains using
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Microsome-Thiobarbituric acid method (MS-TBA). However, highest inhibitory activity of 10 ACS Paragon Plus Environment
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oxidation was observed in Lactobacillus sp. SBT-2028 (91%) followed by L, casei ssp.
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pseudoplantarum SBT-0624 (77%). The antioxidative potential of ICFE of 19 strains of LAB
217
including L. acidophilus B, E, N1, 4356, LA-1 and Farr;L. bulgaricus12, 278, 448, 449, Lb,
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1006, and 11 842; Streptococcus thermophilus821, MC,573, 3641, and 19 987; and B.
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longum B6 and 15 708 was studied by using inhibition of ascorbate auto-oxidation, ferrous
220
and copper ion chelating assay, hydroxyl and hydrogen peroxide radical scavenging activity,
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reducing power assay, SOD activity and SOD induction assay43. All strains exhibited the
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inhibition of ascorbate auto-oxidation in the range of 7-12%. The strain L. acidophilus E
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showed highest (33.1 mM) and S. thermophilus 3641 exhibits the lowest (0.5 mM) hydroxyl
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radical scavenging capacity that is equivalent to uric acid. Furthermore, the reducing activity
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of B. longum B6 was found to be higher, equivalent to 225.3 µM of cysteine and 50 fold
226
higher than that of S. thermophilus 19 987, which had the reducing activity equivalent to 4.3
227
µM of cysteine while strains of L. acidophilus B, E, N1 and 4356 also showed lowest
228
reducing activity. Among the 19 strains of lactic acid bacteria tested, L. bulgaricus strains,
229
demonstrated higher metal ion Fe chelating ability (ranging 5.6-52.9 ppm) than L.
230
acidophilus. On the other hand, S. thermophilus strains showed a very wide range of Fe
231
chelating ability ranging 0.5-72.7 ppm. S. thermophilus 821 and 19 987 demonstrated the
232
highest and lowest Fe
233
high chelating activity for Fe
234
Moreover, for copper ion chelation, L. acidophilus and S. thermophilus strains showed a wide
235
range of chelation ability ranging from 0 to 34.8 ppm and from 2.4 to 51.0 ppm, respectively.
236
Six L. bulgaricus strains tested had high Cu chelating ability ranging from 13.2 to 48.3
237
ppm. Both B. longum B6 and 15 708 had not only high Fe chelating ability but also high
2+
2+
2+
chelating ability, respectively while, B. longum B6 and 15708 had 2+
at 40.7 and 26.6 ppm, respectively.
2+
2+
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Cu chelating ability (18.5 and 63 ppm, respectively). All the strains were unable to inhibit
239
the SOD activity while SOD activity also not induced when growth medium was
240
supplemented with metal ions Mn , Fe
241
Saide and Gilliland (2005)44, demonstrated the antioxidative activity by evaluating the
242
oxygen radical absorbance capacity of whole cells and ICFE of selected strains of lactobacilli
243
species which was expressed as trolox equivalents per 10 cells. Cell free extract of L.
244
delbrueckii ssp. lactis RM5-4, L. acidophilus NCFM, L. delbrueckii ssp. bulgaricus 18,10442
245
and Y-23 and L. casei 9018 exhibited higher oxygen radical absorbance capacity (total
246
antioxidative capacity) than that of the intact cells.
247
Ou et al. (2009)45reported the comparative antioxidative potential of intact cells and ICFE of
248
B. longum and L. delbrueckii ssp. bulgaricus. In this direction, intact cells of both strains
249
showed 71.3% and 70.4% and ICFE of both strains 72.6% and 75.1%, respectively DPPH
250
radical scavenging activity inhibited the liposome peroxidation by 25-31%. However, there
251
was no significant difference between the antioxidative potential of intact cells and the
252
intracellular extracts; and also significantly decreased the malondialdehyde production in
253
intestine 407 cells.
254
L. fermentum ME-3. L. fermentum ME-3 strain has been comprehensively studied at
255
University of Turku, Finland. Itis a unique strain of Lactobacillus species, having at the same
256
time the antimicrobial and physiologically effective antioxidative properties and expressing
257
health-promoting characteristics45. Research on L. fermentum ME-3 has established that the
258
particular strain has double functional properties: antimicrobial activity against intestinal
259
pathogens, high total antioxidative activity (TAA) and total antioxidative status (TAS) of
260
intact cells and lysates. L. fermentum ME-3 is characterized by a complete glutathione
261
system: synthesis, uptake and redox turnover. The functional efficacy of the antimicrobial
2+
2+
2+
2+
or Cu andZn .
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and antioxidative property has been proved by the eradication of Salmonella and the
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reduction of liver and spleen granulomas in S. typhimurium-infected mice treated with the
264
combination of ofloxacin and L. fermentumME-346-47. It also revealed the antioxidative
265
potential of probiotic, L. fermentum ME-3 in soft cheese spreads containing different fatty
266
acids. Moreover, L. fermentumE-3 and E-18 contained a remarkable level of glutathione and
267
expressed Mn-SOD, which is of vital importance for prevention of the lipid peroxidation and
268
secrete hydrogen peroxide48. Few researchers49worked on L. fermentumFTL2311 and L.
269
fermentum FTL10BR strains isolated from miang, traditional fermented tea leaves, which
270
liberate certain substances that possess antioxidative activity expressed in terms of as the
271
trolox equivalent antioxidant capacity(TEAC) and equivalent concentration (EC) values for
272
free radical scavenging and reducing mechanisms, respectively. The culture supernatant of L.
273
fermentum FTL2311revealed TEAC and EC values of 22.54±0.12 and 20.63±0.17 µM/mg
274
respectively, whereas that of L. fermentum FTL10BR yielded TEAC and EC values of
275
24.09±0.12 and21.26±0.17µM/mg respectively. These two strains isolated from miang
276
present high potential as promising health-promoting probiotics50.On the other hand, L.
277
fermentum demonstrated higher free radical scavenging activities of against hydroxyl,
278
superoxide and DPPH radicals. Scavenging activity at a population of 10 and 10 CFU/mL
279
for hydroxyl radical, superoxide radical and DPPH was 7.35%, 12.86%, 64.26% and 91.84%,
280
80.56%, 87.89% respectively51.
281
Lactobacillus acidophilus. Antioxidative potential of intestinal lactic acid bacteria L.
282
acidophilus ATCC 4356 was reported52. In this study, both intact cells and ICFE showed
283
28.1% and 45.3% inhibition of linoleic acid peroxidation while 43.2% and 20.8% DPPH
284
radical scavenging activity respectively. Cytotoxicity of 4-nitroquinoline-N- oxide (4NQO) to
285
intestine 407 cells was reduced by intact cells up to 49% while no inhibition was observed for
286
ICFE. Findings of this study suggest that ICFE showed better antioxidative activity than
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intact cells.
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Lactobacillus plantarum. Studies by others53, exhibited the antioxidative attributes of the
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intact cells of L.plantarumAS1 isolated from South Indian fermented food Kallappam. The
290
strain AS1showed 50.96% inhibition of linoleic acid peroxidation while DPPH radical
291
scavenging activity was found to be 29.15%. This study concluded that significant
292
antioxidant effect might be a reason for the reduction of cancerous tumors and related
293
symptoms in rats (also discussed in vivo studies).
294
Antioxidative property of L. rhamnosus in combination with B. longum containing fruit juice
295
permeates such as lemon juice, mango and guava pulps was studied54. The superoxide anion
296
radical scavenging capacity of L. rhamnosus GG was higher than several other bacteria like
297
L. rhamnosus Lc 705, L. acidophilus LA, L. paracasei YEC, BifidobacteriumBB12 and
298
Escherichia coli55.
299
Lactobacillus casei. Both intact cells and ICFE of L. casei KCTC 3260 demonstrated high
300
antioxidative activity and inhibited lipid peroxidation by 46.2% and 72.9%, respectively. The
301
culture had higher level of chelating activity for both Fe
302
21.8 ppm, respectively, which suggested that the antioxidative capacity of L. casei KCTC
303
3260 may be caused by chelating metal ions instead of SOD activation56. Moreover, Yoon
304
and Byun (2004)57 accounted that L. casei HY 2782 contained the highest level of
305
(glutathione sulfhydryl (GSH) among different probiotic strains tested. GSH levels in L. casei
306
HY 2782 reached maximum values after 24 h of cultivation while decreased for 72 h. Effect
307
of culture media on GSH activity was also studied and it was found that GSH levels were
308
significantly higher during cultivation in de Man Rogosa and Sharpe (MRS) broth than in
309
tryptone phytone yeast extract broth or bromcresol purple dextrose broth. Significant positive
310
correlation between antioxidative activity and cellular GSH contents was observed.
311
Lactobacillus gasseri. Kim et al., (2006)58investigated the protective effect of the selected L.
+2
and Cu
14 ACS Paragon Plus Environment
+2
ions at 10.6 ppm and
Page 15 of 48
Journal of Agricultural and Food Chemistry
312
gasseri NLRI 312 against oxidative damage to cellular membrane lipid and DNA in Jurkat
313
cell line. L. gasseri NLRI-312 had protective effect on the Jurkat cell lines in terms of
314
oxidative damage though supplementation had no effect on malondialdehyde (MDA)
315
production. There was 50% reduction in DNA damage after probiotic treatment of cell lines.
316
Many peptides are identified in fermented milk that contributes to antioxidant potential of
317
products. A κ-casein derived peptide with DPPH radical scavenging activity has been found
318
in milk fermented with L. delbrueckii ssp. bulgaricus59.
319
Lactobacillus helveticus. L. helveticus CD6 producing a folic acid derivative 5-methyl
320
tetrahydrofolate (5-MeTHF) was studied for antioxidative activity. The ICFE of L.
321
helveticusCD6 demonstrated antioxidative activity with the inhibition rate of ascorbate auto-
322
oxidation in the array of 27.5% which showed highest metal ion chelation ability for Fe
323
(0.26±0.06 ppm) as compared to Cu . The DPPH and hydroxyl radical scavenging activity
324
of intact cells found to be 24.7% and 20.8% proved its antioxidative potential. Moreover, it
325
demonstrated 14.89% inhibition of epinephrine autoxidation, 20.9±1.8 µg Cysteine
326
equivalent reducing activity and 20.8% hydroxyl radical scavenging effect. This study also
327
reported the detection of Superoxide Dismutase (SOD) by using 10% non-denaturing
328
polyacrylamide gel electrophoresis. It was observed that SOD activity was absent in the ICFE
329
of L. helveticus CD660.
330
Two strains each of lactobacilli (L. rhamnosus GD 11 and L. plantarum LA3) and
331
bifidobacteria (B. breve A28 and B. breveA10) were studied for exopolysachharide (EPS)
332
production, antioxidative characteristics and their role in gingival fibroblast. Among four
333
cultures, B. breve A28 showed high EPS production at 122 mg/L. It also showed 72% DPPH
334
free radical scavenging activity 88% iron ion chelation activity. The strain also showed 71%
335
inhibition of lipid peroxidation61.
336
Bifidobacterium sp. Several studies have been conducted on antioxidative activity of
+2
+2
15 ACS Paragon Plus Environment
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Page 16 of 48
337
Bifidobacerium sp. Lin and Chang (2000)52 investigated the antioxidative potential of the
338
intestinal lactic acid bacteria B. longum ATCC 15708. Both intact cells and ICFE,
339
respectively showed 32 and 48% inhibition of linoleic acid peroxidation and 52 and 42%
340
DPPH radical scavenging activity. The cytotoxicity of 4-nitroquinoline-N-oxide (4NQO) to
341
intestine 407 cells was reduced by intact cells up to 89% while no inhibition was observed for
342
intracellular extract. Furthermore, intact cells and ICFE showed 16.2% and 34.3% inhibition
343
rates of plasma lipid peroxidation for 10 cells of B. longum ATCC 15708. Another
344
study62reported that Bifidobacterium involved in the transformation of lignans glucosides. In
345
this
346
(Secoisolariciresinoldiglucoside),
347
monoglucoside with yields < 25% out of twenty eight strains. B. pseudocatenulatum WC 01
348
gave highest SDG conversion to SECO, which exhibited 75% yield in cellobiose based
349
medium after 48h. Antioxidative activity of B. animalis 01 culture supernatant, intact cells
350
and intracellular cell- free extracts exhibited inhibitory effect on linoleic acid peroxidation.
351
The inhibitory effect was 30.48%, 41.12% and 71.02% for culture supernatant, intact cells,
352
and ICFE, respectively. Culture supernatant of B. animalis 01 demonstrated highest DPPH
353
scavenging effect (73.11%),which was significantly (P < 0.05) higher than MRS broth while
354
relatively lowest DPPH free radicals scavenging effect was found in intact cells among the
355
tested groups. In addition, culture supernatant showed 78.32% hydroxyl radical and 86.39%
356
superoxide anion radical scavenging activity which was also significantly higher than that of
357
MRS broth whereas intact cell exhibit weak scavenging effect63. Work by others64, reported
358
the in vitro antioxidative properties of thirty four strains of Bifidobacterium, Lactobacillus
359
and Lactococcus against ascorbic and linolenic acid oxidation (TAAAA and TAALA), trolox-
360
equivalent antioxidant capacity (TEAC),intracellular glutathione (TGSH), and superoxide
361
dismutase (SOD). Out of these cultures B. animalis subsp.lactisDSMZ 23032, L. acidophilus
9
view,
only
ten
Bifidobacterium giving
both
cultures
partially
Secoisolariciresinol
16 ACS Paragon Plus Environment
hydrolyzed (SECO)
and
SDG the
Page 17 of 48
Journal of Agricultural and Food Chemistry
362
DSMZ 23033, and L. brevisDSMZ23034 exhibited among the highest TAAAA, TAALA,
363
TEAC, and TGSH values.
364
Probiotic products with antioxidant activity. Fermented milk was prepared from cow, goat
365
and camel milk supplemented with probiotic bacteria Pediococcus pentosus and studied for
366
antioxidative property and fatty acidprofile65. The finding showed that probiotic fermented
367
milk obtained from goat milk has highest DPPH radical scavenging activity (98%) followed
368
by product from camel milk (86%) and cow milk (79%). Moreover, it has also been reported
369
that different protein peptides present in fermented milk are responsible for increased radical
370
scavenging activity66.
371
Recently the antioxidative potential of two synbiotic dairy products viz. symbiotic lassi with
372
honey and whey based synbiotic drink with inulin and orange juice containing L. helveticus
373
MTCC 5463 as probiotic strain was evaluated67. Both products showed antioxidative activity
374
in terms of hydroxyl radical scavenging activity, radical scavenging activity and anti- radical
375
scavenging activity. The storage of products at 4±1°C had adverse effect on these activities as
376
for whey based drink, the activity reduced for all parameters. The synbiotic yogurt samples
377
containing L. plantarum and L. fermentum, expressed the radical scavenging in the form of
378
the DPPH radical inhibition to be 85% and 82%, respectively, at day 1 and the values
379
increased as the storage time increased68. In this study, fructooligosaccharide was used as
380
prebiotic for preparation of synbiotic yogurt. To measure the antioxidant activity of probiotic
381
strain L. plantarum NBIMCC 2415, which was used as starter culture for production of dried
382
fermented products69four methods for cell disintegration of strain L. plantarum NBIMCC
383
2415 were investigated such as temperature, alkaline, enzymatic and ultrasound. Alkaline cell
384
lysis method was found to be appropriate and with this method L. plantarum NBIMCC 2415
385
showed high antioxidant activity (5.57 µmol TE/logN). The study concluded that meat starter
386
culture, L. plantarum NBIMCC 2415 not only involved in fermentation process, but also 17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
387
preserved the colour, formed the flavor and increased the meat products’ shelf-life as well.
388
Antioxidative activity in soymilk fermented with LAB and bifidobacteria simultaneously was
389
reported to be significantly higher than the fermented with either of the cultures individually.
390
Unfermented soymilk showed hydrogen peroxide-scavenging effect. Freeze-dried product
391
had significantly lesser reduction in the antioxidative activity of soymilk than spray-dried.
392
The antioxidative activity of fermented soymilk reduced after drying70.
393
Some researchers worked on the effect of prebiotics (inulin, lactulose and raffinose) on
394
multiplication of some probiotic strains of Lactobacillus and Bifidobacterium, used in a
395
pollen and honey medium for obtaining a bee bread-like product. The prebiotics were tested
396
and compared to pollen and honey medium for the viability and total antioxidative activity. In
397
this view, medium B2 supplemented with ground pollen and honey (inoculated with studied
398
probiotic strain) showed ~45% DPPH radical scavenging activity after 7 days of incubation71.
399
In vivo studies in animal model
400
Effect of oral administration of live B. breve strain Yakult (BBY) on skin barrier perturbation
401
caused by UV and reactive oxygen species was studied in hairless mice72. The strain BBY
402
prevented the UV induced increase in transepidermal waterloss (TEWL). It also significantly
403
suppressed the hydrogen peroxide level, oxidation of proteins and lipids and xanthine oxidase
404
activity, induced by UV in skin.
405
Wang et al., (2009)51 reported that L. fermentum could keep pigs growing healthy by
406
increasing superoxide dismutase and glutathione peroxidase activities and decreasing
407
malondialdehyde (MDA)levels in serum. Similarly, increased activity of hepatic catalase was
408
also observed with decreased MDA levels., The effect of supplemented diets on generation of
409
intestinal nitric oxide (NOx) and antioxidant activities with two strains of L. delbruckii subsp.
410
bulgaricus B3 and A13 which produced highest (211 mg/L) and lowest (27 gm/L) amount of
411
exopolysaccharide was studied73. Result showed that the small intestinal glutathione (GSH)
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Page 19 of 48
Journal of Agricultural and Food Chemistry
412
and thiobarbituric acid-reactive substances (TBARS) was increased by administration of the
413
probiotic organisms, but amino-acid levels did not changed in either control or experimental
414
groups. Nitric oxide (NOx) also increased in both samples. Excessive amount of NOx may
415
contribute to lipid peroxidation because of increased ROS and reactive nitrogen species.
416
Antioxidative capacity of small intestine was ameliorated by administration of two strains of
417
L. delbruckii subsp.bulgaricus B3 and A13.Probiotic Dahi was prepared by co-culturing
418
selected strains of L. acidophilus and B. bifidum and Dahi culture in standardized buffalo
419
milk and evaluated its antioxidative effects on rats74. The activities of superoxide dismutase
420
(SOD) and catalase were determined in red blood cells (RBC) at monthly interval and in liver
421
and colorectal tissue at conclusion of experiment. The SOD activity in RBC increased with
422
probiotic Dahi but not with Indian Dahi. In addition, the catalase activity in RBC increased
423
with probiotic Dahi as well as normal Dahi and the former was more efficacious. In liver, the
424
SOD activity was stimulated by probiotic Dahi, while in colorectal tissue, both normal and
425
probiotic Dahi effectively stimulated SOD activity. Milk fermented with L. acidophilus and
426
B. bifidum when fed to rats, enhanced activity of SOD and decreased TBARS accumulation
427
in liver and colorectal tissue.
428
Probiotic Dahi containing L. acidophilusLaVK2 (La-Dahi) and B. bifidumBbVK3 (LaBb-
429
Dahi) alleviated age-inflicted accumulation of oxidation products, antioxidant enzymes and
430
improved expression of biomarkers of ageing in mice75.In this direction, La-Dahi or LaBb-
431
Dahi increased catalase (CAT) activity and glutathione peroxidase (GPX) activity in RBCs,
432
hepatic tissue and a significant decline in TBARS in plasma, kidney, hepatic tissues and
433
protein carbonyls in plasma of mice. Probiotic Dahi also reversed age related decline in
434
expression of biomarkers of ageing, peroxisome proliferators activated receptor-α,
435
senescence marker protein-30 (SMP-30) and Klotho in hepatic and kidney tissue. The
436
findings of this study suggested that probiotic Dahi containing selected strains of lactic acid
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
437
bacteria can be used as potential nutraceutical intervention to combat oxidative stress and
438
molecular alterations associated with ageing. On the other hand, in vivo antioxidant activities
439
of selenium/zinc enriched probiotics preparation were studied using indigenous dogs (30 days
440
trial)76. In this study, two probiotic strains particularly L. acidophilus and Candida utilis,
441
highly tolerant to high concentration of inorganic Selenium (Se), Zinc (Zn), bile salts and low
442
pH conditions were used. Results of this study indicated that the SOD (P
Review
Probiotics as potential antioxidants: A Systematic Review Vijendra Mishra, Chandni Shah, Narendra Mokashe, Rupesh Chavan, Hariom Yadav, and Jashbhai Prajapati J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506326t • Publication Date (Web): 26 Mar 2015 Downloaded from http://pubs.acs.org on March 29, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 48
Journal of Agricultural and Food Chemistry
Probiotics as potential antioxidants: A Systematic Review
Vijendra Mishra*1, Chandani Shah2, Narendra Mokashe1, Rupesh Chavan1, HariomYadav3, JashbhaiPrajapati2
1
National Institute of Food Technology Entrepreneurship and Management Kundli, Sonipat (India)
2
Dairy Microbiology Department, Anand Agricultural University, Anand, Gujarat (India) 3
National Agri Biotechnology Institute, Mohali (India)
*Corresponding Author Phone: +91-0130-2281167, Email: [email protected]
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
Probiotics are known for their health beneficial effects and have established as dietary
3
adjuncts. Probiotics have been known for many beneficial health effects. In this view, there is
4
interest to find the potential probiotic strains that can exhibit antioxidant properties along
5
with health benefits. In vitro and in vivo study provides that probiotics exhibit antioxidant
6
potential. In this view, consumption of probiotics alone or foods supplemented with
7
probiotics may reduce oxidative damage, free radical scavenging rate and modification in
8
activity of crucial antioxidative enzymes in human cells. Incorporation of probiotics in foods
9
can provide a good strategy to supply dietary anti-oxidants, but more studies are needed to
10
standardize methods and evaluate antioxidant properties of probiotics, before they can be
11
recommended for antioxidant potential. In this paper, literature related to known antioxidant
12
potential of probiotics and proposed future perspective to conduct such studies has been
13
reviewed.
14
Keywords: Antioxidants, Oxidative stress, DPPH activity, Lactobacillus, Bifidobacteria,
15
Probiotics
16
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Page 2 of 48
Page 3 of 48
Journal of Agricultural and Food Chemistry
17
Introduction
18
Oxidative stress is the causeof various chronic human diseases. Oxidative stress is caused by
19
increased activity of reactive oxidative species (ROS) through oxidation process. Oxidation is
20
a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation
21
reactions can produce free radicals having unpaired electron and are capable of carrying out a
22
rapid chain reaction, thus destabilize other molecules and generate further free
23
radicals. Examples of free radicals include the superoxide anion, hydroxyl radical, transition
24
metals such as iron and copper, nitric acid and ozone. Oxygen containing ROS is the most
25
biologically significant free radicals. In oxidative stress conditions, cellular mitochondria
26
produce more ROS
27
peroxide) than enzymatic antioxidants(Table 1) (e.g., superoxide dismutase (SOD),
28
glutathione peroxidase (GPX), peroxidase, Glutathione-S-transferase and catalase) and non-
29
enzymatic antioxidants e.g. ascorbic acid (vitamin C), tocopherol (vitamin E), glutathione,
30
carotenoids and flavonoids present/ supplied to the cells. ROS mediated oxidative stress are
31
known to play vital role in the development of chronic diseases (Fig. 1) such as cancer,
32
diabetes, heart disease, stroke, Alzheimer's disease, rheumatoid arthritis, cataract and aging1-
33
3
34
terminate the chain reaction before damage is done to the vital molecules. All these
35
molecules carry out diverse physiological role in the body by acting as an inhibitor of the
36
process of oxidation. Antioxidants terminate the chain reactions by removing free radical
37
intermediates and inhibit other oxidation reactions by neutralizing free radicals. But in the
38
process, antioxidants are themselves oxidized. Thus there is a constant need to replenish
39
antioxidant resources as one antioxidant molecule can only react with a single free radical4.
40
Consequently, it is essential to search and develop natural nontoxic antioxidants to protect
41
human body from free radicals and slowdown the progress of many chronic diseases. Due to
(e.g., superoxide anion radicals, hydroxyl radicals and hydrogen
. Antioxidants are molecules which interact with free radicals generated in cells and
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
42
changes in consumer perception towards foods as sources of therapeutic value, there is a
43
spurt in the market of food based ingredients and supplements which provide antioxidants.
44
The projected market value of antioxidants to grow to$238.5 million by 2018, at the CAGR
45
of 4.5%5. There is renewed interest in the search of new sources of antioxidants, which can
46
be safely used in food. Out of various sources, probiotics have been considered as an
47
emerging source of effective antioxidants. Due to their long tradition of safe use, along with
48
potential therapeutic benefits, role of probiotics as an antioxidant is being keenly
49
investigated.
50
Mode of action of food derived antioxidants
51
According to the mode of action, two main groups of antioxidants can be distinguished. The
52
first comprises the chemical constituents which interrupt the propagation of the free radical
53
chain by hydrogen donation to radicals or stabilization of relocated radical electrons. Such
54
mode of action is demonstrated by tocopherols, gallusans and hydrochinons6. The second
55
group is characterized by a synergistic mode of action. It includes oxygen scavengers and
56
chelators that bind to ions involved in free radical formation. Their activity consists of
57
hydrogen delivery to phenoxy radicals that leads to the reconstitution of the primary function
58
of antioxidants. This role is played by substances binding to metal ions, e.g. citric acid and by
59
secondary antioxidants, such as amino acids, flavonoids, β-carotene and selenium7. The
60
main source of these substances is plant material like garlic, broccoli, green tea, soybean,
61
tomato, carrot, brussels sprouts, kale, cabbage, onions, cauliflower, red beets, cranberries,
62
cocoa, blackberry, blueberry, red grapes, prunes and citrus fruits. With a huge market for
63
functional foods comprising antioxidative attributes, there is renewed interest in search of
64
new diverse sources of antioxidants that can be safely used. Dairy products have also been
65
reported to possess antioxidant properties (Table 2) and among various dairy supplements,
66
probiotics have been recently known for potential sources of antioxidants
4 ACS Paragon Plus Environment
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Page 5 of 48
Journal of Agricultural and Food Chemistry
67
Probiotics
68
Probiotics are defined as “live microorganisms which when administered in adequate
69
amounts, confer health benefits to the host”8. Probiotics have established their efficacy as
70
dietary factors which can regulate gastrointestinal functions thereby imparting health benefits
71
to consumers. Alleviation of lactose intolerance, prevention of different forms of diarrhoea
72
and urogenital infections, cholesterol reduction, reduction of atopic diseases and modulation
73
of the immune system are some of the functions attributed to probiotics9-10. Other benefits
74
include prevention of cancer, particularly colon carcinoma and food allergy11-12. The
75
competitive exclusion of pathogens and reduction in number as well as metabolic activities of
76
harmful organisms by probiotics has been demonstrated in vitro13. Lactobacilli are the major
77
source of probiotics and are usually explained as Gram positive bacteria, devoid of
78
cytochromes and preferring anaerobic conditions, but are aero tolerant, fastidious, strictly
79
fermentative and produce lactic acid as main product14. Various species and strains of
80
lactobacilli i.e. L. acidophilus, L. casei, L. rhamnosus and L. helveticus are considered as
81
successful probiotics and different variants have been available in market for human
82
consumption. Bifidobacterium, even though not grouped with lactic acid bacteria, is another
83
genus with probiotic functions. B. lactis (B. animalis ssp. lactis) is a very commonly used
84
probiotic, although it is not a normal inhabitant of the human gastrointestinal tract. B. longum
85
ssp. longum (and ssp. infantis) and B. breve is mainly used in supplements15. Although,
86
various strains of lactobacilli and bifidobacteria have been known for various health
87
beneficial effects, but their role as anti-oxidant have been understudied.
88
Application of probiotics in food
89
During last 20 years, consumer’s perception towards functional foods including probiotics
90
has changed. The presence of probiotics in commercial food products has been very well
91
accepted and has been known for number of health benefits. In this direction, industries
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
92
worldwide are concentrating on applications of probiotics in food products as well as
93
constructing a new era of “probiotic health” foods. Foods are considered as better delivery
94
vehicles for probiotic organisms than others due to the role played by food matrix in
95
maintaining the viability of organisms during the transition through the gastrointestinal tract.
96
Dairy foods are considered natural carriers for lactobacilli and bifidobacteria due to their
97
ability to utilize lactose and produce lactic acid.
98
Methods for antioxidant activity screening of probiotics
99
Various methodologies have been adopted to determine the antioxidative potential for natural
100
compounds; however, it is important to use consistent and rapid methods. Each antioxidative
101
activity assay has specific target within the well-defined matrix. Each method has its own
102
merits and demerits. In the absence of a universal method which can give unambiguous
103
results, the best way out is to use the different methods instead of one. Some of the
104
procedures involve use of synthetic antioxidants or free radicals, few are specific for lipid
105
peroxidation and require animal and plant cells, some have a broader scope, some require
106
minimum preparation and information, and few methods are speedy to produce results16. The
107
antioxidative activity of food products including probiotic products has been studied by using
108
different in vitro and in vivo methods.
109
In vitro antioxidative methods. The in vitro assays are based on determination of
110
scavenging capacity against ROS such as superoxide anion scavenging assay, hydrogen
111
peroxide scavenging assay and scavenging activity against stable non biological radicals.
112
Several assays have been used to assess the total antioxidant content in foods such as 6-
113
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent antioxidant
114
capacity (TEAC) assay, the ferric-reducing antioxidant power assay (FRAP) and the oxygen
115
radical absorbance capacity assay (ORAC) assay. However, it has been found that the most
116
common and reliable methods are the 2,2-Diphenyl-1-Picrylhydrazyl(DPPH) and 2,2’-azino-
6 ACS Paragon Plus Environment
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Page 7 of 48
Journal of Agricultural and Food Chemistry
117
bis-3-ethylbezothiazoline-6-sulfonic acid(ABTS) methods; these methods are simple, rapid,
118
sensitive and reproducible and have been modified and improved as per research
119
requirements17. A brief description of some antioxidant potential screening methods used for
120
probiotics is presented here and summarized in Table 3.
121
2,2-Diphenyl-1-Picrylhydrazyl (DPPH)assay. This widely used discoloration method was
122
first described by Blois (1958)18. This assay is based on the premise that a hydrogen donor is
123
an antioxidant. The antioxidants are able to reduce the free, stable and purple colored DPPH
124
radical to the yellow coloured diphenyl-picrylhydrazine, which is monitored by using
125
colorimeter. DPPH is usually used as a reagent to evaluate free-radical scavenging activity of
126
antioxidants19.
127
2,2’-azino-bis(3-ethylbezothiazoline-6-sulfonic acid (ABTS)assay. The ABTS radical
128
scavenging method was developed 20and was then modified by others21. In this assay, ABTS
129
is oxidized by oxidants to its radical cation, ABTS , which is intensely coloured and
130
antioxidant capacity is measured as the ability of test compounds to decolorize the ABTS
131
radical directly. ABTS
132
cation forms the basis of one of the spectrophotometric methods that have been applied for
133
measuring the antioxidant property of pure substances, aqueous mixtures and beverages22.
134
Hydroxyl radical scavenging activity assay. The hydroxyl radical (.OH) is the neutral form
135
of the hydroxide ion (OH ). This radical is known as the most reactive species and is known
136
for lipid peroxidation and DNA damages23. In this assay, the hydroxyl radical is indirectly
137
confirmed by the hydroxylation of p-hydroxybenzoic acid. However, iron plays central role
138
in the hydroxyl radical formation via Fenton reaction.
139
Ferric reducing antioxidant power (FRAP) assay. The FRAP assay is characterized by
140
ability of antioxidant to reduce ferric 2,4,6-tripyridyl-s-triazine complex [Fe -(TPTZ)2]
•-
•+
radicals are more reactive than DPPH while generation of its radical
-
3+
7 ACS Paragon Plus Environment
3+
to
Journal of Agricultural and Food Chemistry
2+
2+
Page 8 of 48
depending on the available reducing species followed by the alteration of
141
[Fe -(TPTZ)2]
142
color from yellow to blue in acidic condition (pH 3.6) and is analyzed through a
143
spectrophotometer24-26.This method was employed to measure reducing power in plasma, but
144
the assay subsequently has also been adapted and used for the assay of antioxidants in
145
different food or beverages27-32, tea and fruits. A drawback of this method is that antioxidant
146
capacity of certain compounds which can react with ferrous ion (Fe ) and SH group-
147
containing antioxidants cannot bemeasured33.
148
Oxygen radical absorbance capacity (ORAC) assay. The ORAC assay uses area-under-
149
curve technique and combines the inhibition time and inhibition degree of free radical action
150
by an antioxidant into a single quantity. On the other hand, similar methods use either
151
inhibition time at a fixed inhibition degree or the inhibition degree at fixed time as basis for
152
quantifying the outputs34-36. ORAC evaluates antioxidant inhibition of peroxy radical-
153
induced oxidations and thus reflects the classical radical chain-breaking antioxidant activity
154
by hydrogen atom transfer (HAT)37. The peroxy radicals are generated through thermal
155
decomposition of “2,2’-azobis(2-amidino-propane) dihydrochloride” (AAPH) in aqueous
156
buffer. The principle of this method is based on decreasing the intensity of fluorescent
157
molecule such as β-phycoerythrin or fluorescein of the target along with time under
158
reproducible and constant flux of peroxy radicals.
159
Antioxidative potential of probiotic microorganisms and foods
160
Each characteristic of probiotic organism is first established using in vitro methods and
161
subsequently in vivo experiments conducted in suitable animal models. On confirmation of
162
suitability of organism, it is incorporated in suitable food product and further studies are
163
conducted using probiotic food to establish its efficacy, survival under storage conditions
164
etc8. In vitro antioxidative studies have been conducted on different probiotic organisms
165
(mainly lactobacilli and bifidobacteria) as well as food products containing probiotic
2+
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Journal of Agricultural and Food Chemistry
166
organisms have also studied. Following section reviews these studies and summary is
167
presented in Table 4.
168
Lactobacillus sp.
169
Lactobacillus rhamnosus. Different antioxidative capacities of lactobacilli species to modify
170
the bacterial profile and prevent the oxidative stress in the Fe-overloaded mice colon were
171
studied 38. The generation of hydrogen peroxide and hydroxyl radical as well as growth of Fe-
172
dependent bacteria is encouraged by Iron (Fe). Findings of this study showed that survival
173
time of L. rhamnosus GG strain was significantly longer in the presence of H2O2 and
174
hydroxyl radical as compared to non-antioxidative strains, L. paracasei Fn032 and L.
175
plantarum Fn001, respectively. In addition, L. paracasei Fn032 and L. plantarum Fn001were
176
specific for free radical scavenging activities of their intracellular free extracts (ICFE).
177
Scavenging activities increased to varying extent when bacterial strains were exposed to
178
gastric and pancreatic juice. However, Achuthan et al., (2012)39 also reported the resistance of
179
a total 39 Lactobacillus cultures to ROS. Most of the isolates showed moderate to strong
180
resistance towards 0.4 mM H2O2. Majority of cultures demonstrated high resistance to
181
hydroxyl radicals and isolate L.plantarum21 (Lp21) was most resistant with log count
182
reduction of 0.20 fold only. Out of 39 isolates, Lactobacillus spp. S3 showed highest total
183
antioxidative activity of 77.85 followed by 56.1 of isolate L.plantarum55(Lp55) in terms of
184
percent inhibition of linolenic acid oxidation. Moreover, isolate L. plantarum 9 (Lp9) up
185
regulated the expression of SOD (superoxide dismutase) gene 2 in HT-29 cell at the
186
concentrations of 0.1mM (1.997 folds) and 1.0 mM H2O2 (2.058 folds). In addition to this,
187
cultures L.plantarum9 (Lp9), L.plantarum91 (Lp91) Lp91 and L.plantarum55 (Lp55) showed
188
significant upregulation in the expression of glutathione peroxidase-I gene to the level of
189
5.451, 8.706, and 10.083 folds respectively when HT-29 cells were challenged with 0.1mM
9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
190
H2O2. On the other hand, catalase gene expression was also significantly regulated by all the
191
isolates used in this study in presence of 0.1 mM H2O2.
192
Grompone et al., (2012)40 developed a new, fast, predictive and convenient in vitro method
193
for screening of antioxidative potential of new probiotic strains using the nematode
194
Caenorhabditis elegans as host (model organism). In this study, total 78 strains of lactic acid
195
bacteria (Lactobacillus and Bifidobacterium) were examined. This collection is composed by
196
62 Lactobacillus strains belonging to L. acidophilus, L. bulgaricus, L. casei, L. paracasei, L.
197
plantarum and L. rhamnosus species, 9 Streptococcus thermophilus isolates and 6
198
Bifidobacterium strains belonging to the B. animalis, B. breve and B. longum species. The
199
LAB stains used in this study are fed to C. elegans and survival rate of C. elegans was
200
examined upon exposure to H2O2-induced stress by comparing with the protective effect
201
exerted by E. coli OP50.As a result of this screening, a Lactobacillus rhamnosus strain (Lb.
202
rhamnosus CNCM I- 3690) demonstrated a high antioxidant capacity.
203
Kapila et al., (2006)41examined the antioxidative activity of ICFE of 13 strains of
204
Lactobacillus sp. by using Microsome-Thiobarbituric acid(MS-TBA) and linoleic acid
205
peroxidation method. Maximum antioxidant capacity in terms of percent inhibition of
206
oxidation was expressed in L. casei ssp. casei 19 (76.82%) followed by L. acidophilus 14
207
(62.21%), Lactobacillus sp. L13 (59.25%), L. casei ssp. casei 63 (53.30%), L. helveticus 6
208
(52.70%) and L. delbreuckii ssp. bulgaricus 4 (52.64%) while remaining strains showed less
209
than 50% activity. In addition to this, strain L. casei ssp. casei 19 showed 72.04% linoleic
210
acid peroxidation followed by L. acidophilus 14 (51.74%), Lactobacillus sp. L13 (51.38%)
211
and rest of all strains exhibited < 50% activity.
212
Similarly, otherauthors42reported the antioxidative potential of ICFE of 19 strains of lactic
213
acid bacteria (16 lactobacilli, 2 streptococci, 2 lactococci) out of 570 strains using
214
Microsome-Thiobarbituric acid method (MS-TBA). However, highest inhibitory activity of 10 ACS Paragon Plus Environment
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215
oxidation was observed in Lactobacillus sp. SBT-2028 (91%) followed by L, casei ssp.
216
pseudoplantarum SBT-0624 (77%). The antioxidative potential of ICFE of 19 strains of LAB
217
including L. acidophilus B, E, N1, 4356, LA-1 and Farr;L. bulgaricus12, 278, 448, 449, Lb,
218
1006, and 11 842; Streptococcus thermophilus821, MC,573, 3641, and 19 987; and B.
219
longum B6 and 15 708 was studied by using inhibition of ascorbate auto-oxidation, ferrous
220
and copper ion chelating assay, hydroxyl and hydrogen peroxide radical scavenging activity,
221
reducing power assay, SOD activity and SOD induction assay43. All strains exhibited the
222
inhibition of ascorbate auto-oxidation in the range of 7-12%. The strain L. acidophilus E
223
showed highest (33.1 mM) and S. thermophilus 3641 exhibits the lowest (0.5 mM) hydroxyl
224
radical scavenging capacity that is equivalent to uric acid. Furthermore, the reducing activity
225
of B. longum B6 was found to be higher, equivalent to 225.3 µM of cysteine and 50 fold
226
higher than that of S. thermophilus 19 987, which had the reducing activity equivalent to 4.3
227
µM of cysteine while strains of L. acidophilus B, E, N1 and 4356 also showed lowest
228
reducing activity. Among the 19 strains of lactic acid bacteria tested, L. bulgaricus strains,
229
demonstrated higher metal ion Fe chelating ability (ranging 5.6-52.9 ppm) than L.
230
acidophilus. On the other hand, S. thermophilus strains showed a very wide range of Fe
231
chelating ability ranging 0.5-72.7 ppm. S. thermophilus 821 and 19 987 demonstrated the
232
highest and lowest Fe
233
high chelating activity for Fe
234
Moreover, for copper ion chelation, L. acidophilus and S. thermophilus strains showed a wide
235
range of chelation ability ranging from 0 to 34.8 ppm and from 2.4 to 51.0 ppm, respectively.
236
Six L. bulgaricus strains tested had high Cu chelating ability ranging from 13.2 to 48.3
237
ppm. Both B. longum B6 and 15 708 had not only high Fe chelating ability but also high
2+
2+
2+
chelating ability, respectively while, B. longum B6 and 15708 had 2+
at 40.7 and 26.6 ppm, respectively.
2+
2+
11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
2+
238
Cu chelating ability (18.5 and 63 ppm, respectively). All the strains were unable to inhibit
239
the SOD activity while SOD activity also not induced when growth medium was
240
supplemented with metal ions Mn , Fe
241
Saide and Gilliland (2005)44, demonstrated the antioxidative activity by evaluating the
242
oxygen radical absorbance capacity of whole cells and ICFE of selected strains of lactobacilli
243
species which was expressed as trolox equivalents per 10 cells. Cell free extract of L.
244
delbrueckii ssp. lactis RM5-4, L. acidophilus NCFM, L. delbrueckii ssp. bulgaricus 18,10442
245
and Y-23 and L. casei 9018 exhibited higher oxygen radical absorbance capacity (total
246
antioxidative capacity) than that of the intact cells.
247
Ou et al. (2009)45reported the comparative antioxidative potential of intact cells and ICFE of
248
B. longum and L. delbrueckii ssp. bulgaricus. In this direction, intact cells of both strains
249
showed 71.3% and 70.4% and ICFE of both strains 72.6% and 75.1%, respectively DPPH
250
radical scavenging activity inhibited the liposome peroxidation by 25-31%. However, there
251
was no significant difference between the antioxidative potential of intact cells and the
252
intracellular extracts; and also significantly decreased the malondialdehyde production in
253
intestine 407 cells.
254
L. fermentum ME-3. L. fermentum ME-3 strain has been comprehensively studied at
255
University of Turku, Finland. Itis a unique strain of Lactobacillus species, having at the same
256
time the antimicrobial and physiologically effective antioxidative properties and expressing
257
health-promoting characteristics45. Research on L. fermentum ME-3 has established that the
258
particular strain has double functional properties: antimicrobial activity against intestinal
259
pathogens, high total antioxidative activity (TAA) and total antioxidative status (TAS) of
260
intact cells and lysates. L. fermentum ME-3 is characterized by a complete glutathione
261
system: synthesis, uptake and redox turnover. The functional efficacy of the antimicrobial
2+
2+
2+
2+
or Cu andZn .
9
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262
and antioxidative property has been proved by the eradication of Salmonella and the
263
reduction of liver and spleen granulomas in S. typhimurium-infected mice treated with the
264
combination of ofloxacin and L. fermentumME-346-47. It also revealed the antioxidative
265
potential of probiotic, L. fermentum ME-3 in soft cheese spreads containing different fatty
266
acids. Moreover, L. fermentumE-3 and E-18 contained a remarkable level of glutathione and
267
expressed Mn-SOD, which is of vital importance for prevention of the lipid peroxidation and
268
secrete hydrogen peroxide48. Few researchers49worked on L. fermentumFTL2311 and L.
269
fermentum FTL10BR strains isolated from miang, traditional fermented tea leaves, which
270
liberate certain substances that possess antioxidative activity expressed in terms of as the
271
trolox equivalent antioxidant capacity(TEAC) and equivalent concentration (EC) values for
272
free radical scavenging and reducing mechanisms, respectively. The culture supernatant of L.
273
fermentum FTL2311revealed TEAC and EC values of 22.54±0.12 and 20.63±0.17 µM/mg
274
respectively, whereas that of L. fermentum FTL10BR yielded TEAC and EC values of
275
24.09±0.12 and21.26±0.17µM/mg respectively. These two strains isolated from miang
276
present high potential as promising health-promoting probiotics50.On the other hand, L.
277
fermentum demonstrated higher free radical scavenging activities of against hydroxyl,
278
superoxide and DPPH radicals. Scavenging activity at a population of 10 and 10 CFU/mL
279
for hydroxyl radical, superoxide radical and DPPH was 7.35%, 12.86%, 64.26% and 91.84%,
280
80.56%, 87.89% respectively51.
281
Lactobacillus acidophilus. Antioxidative potential of intestinal lactic acid bacteria L.
282
acidophilus ATCC 4356 was reported52. In this study, both intact cells and ICFE showed
283
28.1% and 45.3% inhibition of linoleic acid peroxidation while 43.2% and 20.8% DPPH
284
radical scavenging activity respectively. Cytotoxicity of 4-nitroquinoline-N- oxide (4NQO) to
285
intestine 407 cells was reduced by intact cells up to 49% while no inhibition was observed for
286
ICFE. Findings of this study suggest that ICFE showed better antioxidative activity than
6
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Journal of Agricultural and Food Chemistry
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287
intact cells.
288
Lactobacillus plantarum. Studies by others53, exhibited the antioxidative attributes of the
289
intact cells of L.plantarumAS1 isolated from South Indian fermented food Kallappam. The
290
strain AS1showed 50.96% inhibition of linoleic acid peroxidation while DPPH radical
291
scavenging activity was found to be 29.15%. This study concluded that significant
292
antioxidant effect might be a reason for the reduction of cancerous tumors and related
293
symptoms in rats (also discussed in vivo studies).
294
Antioxidative property of L. rhamnosus in combination with B. longum containing fruit juice
295
permeates such as lemon juice, mango and guava pulps was studied54. The superoxide anion
296
radical scavenging capacity of L. rhamnosus GG was higher than several other bacteria like
297
L. rhamnosus Lc 705, L. acidophilus LA, L. paracasei YEC, BifidobacteriumBB12 and
298
Escherichia coli55.
299
Lactobacillus casei. Both intact cells and ICFE of L. casei KCTC 3260 demonstrated high
300
antioxidative activity and inhibited lipid peroxidation by 46.2% and 72.9%, respectively. The
301
culture had higher level of chelating activity for both Fe
302
21.8 ppm, respectively, which suggested that the antioxidative capacity of L. casei KCTC
303
3260 may be caused by chelating metal ions instead of SOD activation56. Moreover, Yoon
304
and Byun (2004)57 accounted that L. casei HY 2782 contained the highest level of
305
(glutathione sulfhydryl (GSH) among different probiotic strains tested. GSH levels in L. casei
306
HY 2782 reached maximum values after 24 h of cultivation while decreased for 72 h. Effect
307
of culture media on GSH activity was also studied and it was found that GSH levels were
308
significantly higher during cultivation in de Man Rogosa and Sharpe (MRS) broth than in
309
tryptone phytone yeast extract broth or bromcresol purple dextrose broth. Significant positive
310
correlation between antioxidative activity and cellular GSH contents was observed.
311
Lactobacillus gasseri. Kim et al., (2006)58investigated the protective effect of the selected L.
+2
and Cu
14 ACS Paragon Plus Environment
+2
ions at 10.6 ppm and
Page 15 of 48
Journal of Agricultural and Food Chemistry
312
gasseri NLRI 312 against oxidative damage to cellular membrane lipid and DNA in Jurkat
313
cell line. L. gasseri NLRI-312 had protective effect on the Jurkat cell lines in terms of
314
oxidative damage though supplementation had no effect on malondialdehyde (MDA)
315
production. There was 50% reduction in DNA damage after probiotic treatment of cell lines.
316
Many peptides are identified in fermented milk that contributes to antioxidant potential of
317
products. A κ-casein derived peptide with DPPH radical scavenging activity has been found
318
in milk fermented with L. delbrueckii ssp. bulgaricus59.
319
Lactobacillus helveticus. L. helveticus CD6 producing a folic acid derivative 5-methyl
320
tetrahydrofolate (5-MeTHF) was studied for antioxidative activity. The ICFE of L.
321
helveticusCD6 demonstrated antioxidative activity with the inhibition rate of ascorbate auto-
322
oxidation in the array of 27.5% which showed highest metal ion chelation ability for Fe
323
(0.26±0.06 ppm) as compared to Cu . The DPPH and hydroxyl radical scavenging activity
324
of intact cells found to be 24.7% and 20.8% proved its antioxidative potential. Moreover, it
325
demonstrated 14.89% inhibition of epinephrine autoxidation, 20.9±1.8 µg Cysteine
326
equivalent reducing activity and 20.8% hydroxyl radical scavenging effect. This study also
327
reported the detection of Superoxide Dismutase (SOD) by using 10% non-denaturing
328
polyacrylamide gel electrophoresis. It was observed that SOD activity was absent in the ICFE
329
of L. helveticus CD660.
330
Two strains each of lactobacilli (L. rhamnosus GD 11 and L. plantarum LA3) and
331
bifidobacteria (B. breve A28 and B. breveA10) were studied for exopolysachharide (EPS)
332
production, antioxidative characteristics and their role in gingival fibroblast. Among four
333
cultures, B. breve A28 showed high EPS production at 122 mg/L. It also showed 72% DPPH
334
free radical scavenging activity 88% iron ion chelation activity. The strain also showed 71%
335
inhibition of lipid peroxidation61.
336
Bifidobacterium sp. Several studies have been conducted on antioxidative activity of
+2
+2
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Page 16 of 48
337
Bifidobacerium sp. Lin and Chang (2000)52 investigated the antioxidative potential of the
338
intestinal lactic acid bacteria B. longum ATCC 15708. Both intact cells and ICFE,
339
respectively showed 32 and 48% inhibition of linoleic acid peroxidation and 52 and 42%
340
DPPH radical scavenging activity. The cytotoxicity of 4-nitroquinoline-N-oxide (4NQO) to
341
intestine 407 cells was reduced by intact cells up to 89% while no inhibition was observed for
342
intracellular extract. Furthermore, intact cells and ICFE showed 16.2% and 34.3% inhibition
343
rates of plasma lipid peroxidation for 10 cells of B. longum ATCC 15708. Another
344
study62reported that Bifidobacterium involved in the transformation of lignans glucosides. In
345
this
346
(Secoisolariciresinoldiglucoside),
347
monoglucoside with yields < 25% out of twenty eight strains. B. pseudocatenulatum WC 01
348
gave highest SDG conversion to SECO, which exhibited 75% yield in cellobiose based
349
medium after 48h. Antioxidative activity of B. animalis 01 culture supernatant, intact cells
350
and intracellular cell- free extracts exhibited inhibitory effect on linoleic acid peroxidation.
351
The inhibitory effect was 30.48%, 41.12% and 71.02% for culture supernatant, intact cells,
352
and ICFE, respectively. Culture supernatant of B. animalis 01 demonstrated highest DPPH
353
scavenging effect (73.11%),which was significantly (P < 0.05) higher than MRS broth while
354
relatively lowest DPPH free radicals scavenging effect was found in intact cells among the
355
tested groups. In addition, culture supernatant showed 78.32% hydroxyl radical and 86.39%
356
superoxide anion radical scavenging activity which was also significantly higher than that of
357
MRS broth whereas intact cell exhibit weak scavenging effect63. Work by others64, reported
358
the in vitro antioxidative properties of thirty four strains of Bifidobacterium, Lactobacillus
359
and Lactococcus against ascorbic and linolenic acid oxidation (TAAAA and TAALA), trolox-
360
equivalent antioxidant capacity (TEAC),intracellular glutathione (TGSH), and superoxide
361
dismutase (SOD). Out of these cultures B. animalis subsp.lactisDSMZ 23032, L. acidophilus
9
view,
only
ten
Bifidobacterium giving
both
cultures
partially
Secoisolariciresinol
16 ACS Paragon Plus Environment
hydrolyzed (SECO)
and
SDG the
Page 17 of 48
Journal of Agricultural and Food Chemistry
362
DSMZ 23033, and L. brevisDSMZ23034 exhibited among the highest TAAAA, TAALA,
363
TEAC, and TGSH values.
364
Probiotic products with antioxidant activity. Fermented milk was prepared from cow, goat
365
and camel milk supplemented with probiotic bacteria Pediococcus pentosus and studied for
366
antioxidative property and fatty acidprofile65. The finding showed that probiotic fermented
367
milk obtained from goat milk has highest DPPH radical scavenging activity (98%) followed
368
by product from camel milk (86%) and cow milk (79%). Moreover, it has also been reported
369
that different protein peptides present in fermented milk are responsible for increased radical
370
scavenging activity66.
371
Recently the antioxidative potential of two synbiotic dairy products viz. symbiotic lassi with
372
honey and whey based synbiotic drink with inulin and orange juice containing L. helveticus
373
MTCC 5463 as probiotic strain was evaluated67. Both products showed antioxidative activity
374
in terms of hydroxyl radical scavenging activity, radical scavenging activity and anti- radical
375
scavenging activity. The storage of products at 4±1°C had adverse effect on these activities as
376
for whey based drink, the activity reduced for all parameters. The synbiotic yogurt samples
377
containing L. plantarum and L. fermentum, expressed the radical scavenging in the form of
378
the DPPH radical inhibition to be 85% and 82%, respectively, at day 1 and the values
379
increased as the storage time increased68. In this study, fructooligosaccharide was used as
380
prebiotic for preparation of synbiotic yogurt. To measure the antioxidant activity of probiotic
381
strain L. plantarum NBIMCC 2415, which was used as starter culture for production of dried
382
fermented products69four methods for cell disintegration of strain L. plantarum NBIMCC
383
2415 were investigated such as temperature, alkaline, enzymatic and ultrasound. Alkaline cell
384
lysis method was found to be appropriate and with this method L. plantarum NBIMCC 2415
385
showed high antioxidant activity (5.57 µmol TE/logN). The study concluded that meat starter
386
culture, L. plantarum NBIMCC 2415 not only involved in fermentation process, but also 17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
387
preserved the colour, formed the flavor and increased the meat products’ shelf-life as well.
388
Antioxidative activity in soymilk fermented with LAB and bifidobacteria simultaneously was
389
reported to be significantly higher than the fermented with either of the cultures individually.
390
Unfermented soymilk showed hydrogen peroxide-scavenging effect. Freeze-dried product
391
had significantly lesser reduction in the antioxidative activity of soymilk than spray-dried.
392
The antioxidative activity of fermented soymilk reduced after drying70.
393
Some researchers worked on the effect of prebiotics (inulin, lactulose and raffinose) on
394
multiplication of some probiotic strains of Lactobacillus and Bifidobacterium, used in a
395
pollen and honey medium for obtaining a bee bread-like product. The prebiotics were tested
396
and compared to pollen and honey medium for the viability and total antioxidative activity. In
397
this view, medium B2 supplemented with ground pollen and honey (inoculated with studied
398
probiotic strain) showed ~45% DPPH radical scavenging activity after 7 days of incubation71.
399
In vivo studies in animal model
400
Effect of oral administration of live B. breve strain Yakult (BBY) on skin barrier perturbation
401
caused by UV and reactive oxygen species was studied in hairless mice72. The strain BBY
402
prevented the UV induced increase in transepidermal waterloss (TEWL). It also significantly
403
suppressed the hydrogen peroxide level, oxidation of proteins and lipids and xanthine oxidase
404
activity, induced by UV in skin.
405
Wang et al., (2009)51 reported that L. fermentum could keep pigs growing healthy by
406
increasing superoxide dismutase and glutathione peroxidase activities and decreasing
407
malondialdehyde (MDA)levels in serum. Similarly, increased activity of hepatic catalase was
408
also observed with decreased MDA levels., The effect of supplemented diets on generation of
409
intestinal nitric oxide (NOx) and antioxidant activities with two strains of L. delbruckii subsp.
410
bulgaricus B3 and A13 which produced highest (211 mg/L) and lowest (27 gm/L) amount of
411
exopolysaccharide was studied73. Result showed that the small intestinal glutathione (GSH)
18 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
412
and thiobarbituric acid-reactive substances (TBARS) was increased by administration of the
413
probiotic organisms, but amino-acid levels did not changed in either control or experimental
414
groups. Nitric oxide (NOx) also increased in both samples. Excessive amount of NOx may
415
contribute to lipid peroxidation because of increased ROS and reactive nitrogen species.
416
Antioxidative capacity of small intestine was ameliorated by administration of two strains of
417
L. delbruckii subsp.bulgaricus B3 and A13.Probiotic Dahi was prepared by co-culturing
418
selected strains of L. acidophilus and B. bifidum and Dahi culture in standardized buffalo
419
milk and evaluated its antioxidative effects on rats74. The activities of superoxide dismutase
420
(SOD) and catalase were determined in red blood cells (RBC) at monthly interval and in liver
421
and colorectal tissue at conclusion of experiment. The SOD activity in RBC increased with
422
probiotic Dahi but not with Indian Dahi. In addition, the catalase activity in RBC increased
423
with probiotic Dahi as well as normal Dahi and the former was more efficacious. In liver, the
424
SOD activity was stimulated by probiotic Dahi, while in colorectal tissue, both normal and
425
probiotic Dahi effectively stimulated SOD activity. Milk fermented with L. acidophilus and
426
B. bifidum when fed to rats, enhanced activity of SOD and decreased TBARS accumulation
427
in liver and colorectal tissue.
428
Probiotic Dahi containing L. acidophilusLaVK2 (La-Dahi) and B. bifidumBbVK3 (LaBb-
429
Dahi) alleviated age-inflicted accumulation of oxidation products, antioxidant enzymes and
430
improved expression of biomarkers of ageing in mice75.In this direction, La-Dahi or LaBb-
431
Dahi increased catalase (CAT) activity and glutathione peroxidase (GPX) activity in RBCs,
432
hepatic tissue and a significant decline in TBARS in plasma, kidney, hepatic tissues and
433
protein carbonyls in plasma of mice. Probiotic Dahi also reversed age related decline in
434
expression of biomarkers of ageing, peroxisome proliferators activated receptor-α,
435
senescence marker protein-30 (SMP-30) and Klotho in hepatic and kidney tissue. The
436
findings of this study suggested that probiotic Dahi containing selected strains of lactic acid
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
437
bacteria can be used as potential nutraceutical intervention to combat oxidative stress and
438
molecular alterations associated with ageing. On the other hand, in vivo antioxidant activities
439
of selenium/zinc enriched probiotics preparation were studied using indigenous dogs (30 days
440
trial)76. In this study, two probiotic strains particularly L. acidophilus and Candida utilis,
441
highly tolerant to high concentration of inorganic Selenium (Se), Zinc (Zn), bile salts and low
442
pH conditions were used. Results of this study indicated that the SOD (P
