Chemical Composition of Royal Jelly and Effects of Synbiotic with Two Different Locally Isolated Probiotic Strains on Antioxidant Activities

Abstract

This study was carried out to investigate the antioxidant properties of synbiotic product, Lactobacillus acidophilus supplemented with 2.5% royal jelly in skim milk and Bifidobacterium bifidum supplemented with 7.5% royal jelly in skim milk, using DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging assay, reducing power, total antioxidant in linoleic acid system and formation of diene-conjugation assay. Results showed that the synbiotic effect of royal jelly and probiotic bacteria provided substantial antioxidant activities. Milk samples fermented by B. bifi dum supplemented with 7.5% royal jelly and L. acidophilus supplemented with 2.5% royal jelly exhibited high scavenging activity with 96.8 and 93.3%, respectively, at a concentration of 500 μg/mL. IC50 values were estimated at 226.7 μg/mL for B. bifidum supplemented with 7.5% royal jelly and at 210.2 μg/ml for L. acidophilus supplemented with 2.5% royal jelly. On the other hand, L. acidophilus supplemented with 2.5% royal jelly and B. bifidum supplemented with 7.5% royal jelly exhibited significantly high reducing power at a concentration of 1000 μg/mL. The percentages of peroxide inhibition of L. acidophilus supplemented with 2.5% royal jelly and B. bifidum with 7.5% royal jelly were 52% and 42%, respectively. Significant inhibitions were found in the formation of conjugated diene at 66.9% and 65.8% for L. acidophilus with 2.5% royal jelly and B. bifidum with 7.5% royal jelly, respectively. These results were compared with standards BHT, ascorbic acid and Trolox.

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© 2014 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Pol. J.Food Nutr. Sci., 2014, Vol. 64, No. 3, pp. 171-180
DOI: 10.2478/pjfns-2013-0015
http://journal.pan.olsztyn.pl
INTRODUCTION
At present days, remarkable changes were observed inun-
derstanding therole offoods inhuman health. Enormous in-
terest ofconsumers has been directed toward healthy foods
which help inpreventing theinitiation ofdiseases andtheir
development; hence, anew term “functional food” was sug-
gested [Hardy, 2000; Grajek etal., 2005].
Functional food isconsidered to beany food or food com-
ponent that provides health benefi ts beyond basic nutrition.
Recently, agreat deal ofinterest has been paid bythecon-
sumers towards natural bioactive compounds as functional
ingredients inthediets due to their various health benefi cial
effects [Vo & Kim, 2013]. Afunctional food may benatural or
beobtained bytheelimination or themodifi cation ofone or
more ofits basic components [Perez-Alvarez etal., 2003]. Se-
lective non-digestible oligosaccharides andpolysaccharides,
some peptides andproteins, andcertain lipids, are themost
common prebiotics [Gibson & Roberfroid, 1995]. Prebiot-
ics are known to promote theproliferation ofbifi dobacteria
andlactobacilli [Roberfroid, 2000]. Among foods that can
beconsidered as functional foods, we may include all those
originating inthebeehive: honey [Haddadin etal., 2007; Mei
* Corresponding Author: E-mail address: malik.haddadin@yahoo.com
(Prof. Malik S.Y.Haddadin)
etal., 2010], propolis [Haddadin etal., 2008], androyal jelly
[Haddadin etal., 2012; Nabas, 2012].
Royal jelly (RJ) isanatural substance considered as one
ofthemost important products ofhoneybees with high nu-
tritional, functional andbiological properties. Itissecreted
from thehypopharingeal glands ofworker bees which serve
as food for thequeen bee andto theyoung larvae [Grout,
1992]. Ingeneral, royal jelly isrelatively acidic (pH 3.1–3.9)
with ahigh buffering capacity ranges between 4 and7 [Sau-
erwald etal., 1998].
Itiscomposed ofproteins, lipids, sugars, vitamins
andamino acids [Howe etal., 1985], B complex vitamins
such as B1, B2, B6 [Moreschi & Almeida-Muradian, 2009],
andbiotin [Nandhasri etal., 1990]. Moreover, itcontains
different minerals (P, S, Ca, Mg, K, Na, Zn, Fe, Cu, Mn)
andtrace elements with biological functions such as (Al,
Ba, Sr, Bi, Cd, Hg, Pb, Sn, Te, Tl, W, Sb, Cr, Ni, Ti, V, Co,
Mo) [Stocker etal., 2005]. Themain fatty acid present inRJ
is10-hydroxy-trans-2-decenoic acid (10-HDA) [Yang etal.,
2010], itplays an important role inboosting immune system,
anticancer activity. Itcontains other components such as roy-
alisin andapisin [Watanabe etal., 1996, 1998]. Royal jelly
can beincluded directly inour food anddietary supplements,
moreover, royal jelly istraditionally known to have some
biological or medical purposes such as life-span elongating
[Inoue etal., 2003], anti-fatigue [Kamakura etal., 2001], anti-
Chemical Composition ofRoyal Jelly andEffects ofSynbiotic with Two
Different LocallyIsolated Probiotic Strains on Antioxidant Activities
Zaid Nabas1, Malik S.Y.Haddadin1*, Jamal Haddadin2, Ibrahim K.Nazer3
1Department ofNutrition andFood Technology, Faculty ofAgriculture, University ofJordan, Amman, Jordan
2Department ofFood andNutrition Sciences, King Faisal University, Al Ahsa, Kingdom ofSaudi Arabia
3Department ofPlant Protection, Faculty ofAgriculture, University ofJordan, Amman, Jordan
Key words: synbiotic, royal jelly, DPPH, reducing power, linoleic acid system
This study was carried out to investigate theantioxidant properties ofsynbiotic product, Lactobacillus acidophilus supplemented with 2.5% royal
jelly inskim milk andBifi dobacterium bifi dum supplemented with 7.5% royal jelly inskim milk, using DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical
scavenging assay, reducing power, total antioxidant inlinoleic acid system andformation ofdiene-conjugation assay. Results showed that thesynbiotic
effect ofroyal jelly andprobiotic bacteria provided substantial antioxidant activities. Milk samples fermented byB. bifi dum supplemented with 7.5%
royal jelly andL. acidophilus supplemented with 2.5% royal jelly exhibited high scavenging activity with 96.8 and93.3%, respectively, at aconcentration
of500 μg/mL.IC50 values were estimated at 226.7 μg/mL for B. bifi dum supplemented with 7.5% royal jelly andat 210.2 μg/ml for L. acidophilus supple-
mented with 2.5% royal jelly. On theother hand, L. acidophilus supplemented with 2.5% royal jelly andB. bifi dum supplemented with 7.5% royal jelly
exhibited signifi cantly high reducing power at aconcentration of1000 μg/mL.Thepercentages ofperoxide inhibition ofL. acidophilus supplemented
with 2.5% royal jelly andB. bifi dum with 7.5% royal jelly were 52% and42%, respectively. Signifi cant inhibitions were found intheformation ofconju-
gated diene at 66.9% and65.8% for L. acidophilus with 2.5% royal jelly andB. bifi dum with 7.5% royal jelly, respectively. These results were compared
with standards BHT, ascorbic acid andTrolox.
Original article
Section: Food Chemistry

172 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
allergic andimmunoregulatory effect [Okamoto etal., 2003],
andanti-infl ammatory [Kohno etal., 2004], anti-bacterial,
andantioxidant activity [Nagai & Inoue, 2004; El-Nekeety
etal., 2007; Eshraghi & Seifollahi, 2003; Barnutiu etal., 2011;
Guo etal., 2005; Liu etal., 2008].
Aprobiotic isabenefi cial living microbial food ingredient
whose growth andactivity are stimulated byprebiotics [Sal-
minen etal., 1998]. According to Chandan [1999], themost
widely used probiotics infoods, especially indairy products,
are themembers ofthegenera Enterococcus, Lactobacillus,
andBifi dobacterium. Probiotic bacteria proved to have de-
sirable clinical benefi ts, therapeutic actions andprotective
effects against oxidative damages [Amaretti, etal., 2013;
Bernardeau etal., 2008; Osuntoki & Korie, 2010; Pan etal.,
2009]. Recently they are being recognized for their remarkable
role inregulating thehost metabolic processes, weight gain
andobesity [Thomas & Versalovic, 2010].
Inpractice, combined mixtures ofprobiotics andprebiot-
ics are often used because their synergic effects are conferred
onto food products. For this reason, such mixtures are called
synbiotics [Gibson & Roberfroid, 1995]. Synbiotic products
may have benefi cial effects on thehost byenhancing sur-
vival andimplantation oflive microbial dietary supplements
inthegastrointestinal microbiota, byselectively stimulating
thegrowth or activating thecatabolism ofone or alimited
number ofhealth-promoting bacteria inthegastrointestinal
tract, andbyimproving theintestinal tract’s microbial balance
[Wollowski etal., 2001]. Moreover, probiotic andprebiotic ef-
fects might beadditive or even synergistic [Roberfroid, 2000].
Theobjective ofthis study was to evaluate theantioxidant
properties ofthesynbiotic effect ofroyal jelly with two differ-
ent locally isolated strains Lactobacillus acidophilus andBifi -
dobacterium bifi dum using different invitro assays.
MATERIALS ANDMETHODS
Probiotic bacterial strains
L. acidophilus andB. bifi dum isolates used inthis research
were previously isolated from new born breast fed infants
stool [Haddadin, 2004], at Queen Alia hospital. One gram
ofeach freeze-dried powders ofthese isolates was transferred
aseptically into 50mL sterile MRS broth supplemented with
5 g/L L(+) cysteine–HCL (99.6% purity, Sigma, USA), then
incubated at 37°C for 20 h inan anaerobic jar (Oxoid, UK).
Repeated streaking onto MRS agar plates were used for
purifi cation ofboth isolates, theisolates ofL. acidophilus
andB. bifi dum were identifi ed physiologically andbiochemi-
cally according to Bergey’s Manual [Kandler & Weiss, 1986]
andProkaryotes [Hammes etal., 1992]. Theisolates were ac-
tivated making sub-culturing twice inMRS broth containing
0.5% L(+) cysteine–HCL, using 1% inoculum and18–20h
ofincubation at 37°C.Each isolate was sub-cultured three
times prior to every test [Walker & Gilliland, 1993].
Royal jelly
Royal jelly samples were collected during thespring
andsummer oftheyear 2011 from Langstroth hives with
colonies ofthemost common honeybee species Apis mellifera
located at theUniversity ofJordan Apiary – Faculty ofAgri-
culture, using theartifi cial cups method according to Grout
[1992]. Samples ofroyal jelly were diluted with distilled
water andfi ltered under avacuum, using, Grade No.1 fi lter
paper, andGrade No. 40 fi lter paper (Whatman membrane,
England), and0.45 μm nylon membrane. Cold sterilization
was performed via micro-fi ltration unit using 0.20 μm sterile
cellulose-ester membranes (Advantec MFS, Japan) [Hadda-
din etal., 2007]. A9% skimmed milk was reconstituted indis-
tilled water andsterilized at 115ºC for 10 min. After cooling
to 37ºC, fi ve different concentrations ofsterilized royal jelly
(0, 2.5, 5, 7.5, 10% m/v) were added to reconstituted milk
in100mL Duran bottles. Acontrol sample was used with-
out theaddition ofroyal jelly [Haddadin etal., 2012; Nabas,
2012]. Royal jelly/milk samples with highest counts ofeach
probiotic bacteria were chosen to examine theantioxidant
activities. ThepH ofRJ was measured using adigital pH me-
ter (Hanna instrument model HI 8519, Italy). ThepH was
directly measured after adding 2 g ofRJ to 4mL ofdistilled
water (pH 7.00).
Evaluation ofbacterial growth
One percent % (v/v) ofL. acidophilus or B. bifi dum was
added to theprepared milk/royal jelly samples. Thecultures
were then incubated at 37ºC inanaerobic jars for 24 h. After
incubation, serial dilutions (10–1 – 10–7) were realized us-
ing sterile 0.1% peptone broth andplated on MRS agar sup-
plied with cysteine-HCl (5 g/L) andincubated at 37°C for
48h under anaerobic condition. Theresults were recorded as
CFU/mL ofculture [Harrigan & McCane, 1996].
Determination oftotal fl avonoids ofroyal jelly
Thetotal fl avonoid content ofRJ was determined accord-
ing to Ja etal. [1999]. Five grams ofRJ were added to 50mL
ofdistilled water andfi ltered using Grade No. 1 Filter Paper
(Whatman membranes, England). Then, 0.5mL ofthesolu-
tion was mixed with 0.3mL ofsodium nitrite solution (5g/L).
After 5 min, 0.3mL ofaluminum chloride (1 g/L) was added.
After another 5 min, 2mL of1 mol/L ofsodium hydroxide
was added to themixture. Total volume was made up to
10mL with distilled water andthesample was sonicated im-
mediately after preparation. Theabsorbance was measured
at 510nm against water blank using UV/Visible Spectropho-
tometer (Jasco-V-530, Japan). Calibration curve was plotted
bypreparing asolution ofrutin (0–200 μg/mL). Concentra-
tions are expressed as (μg rutin equivalent /mg RJ).
Determination oftotal phenolic content ofroyal jelly
Total phenolic content ofRJ was determined byFo-
lin–Ciocalteu method according to Singleton etal. [1999].
To this end, 0.5mL from theprevious solution ofRJ used
infl avonoid assay was mixed with 2.5mL of0.2N Folin-Ci-
ocalteu reagents (Sigma –Aldrich, USA) for 5 min andthen
2mL of7.5% sodium carbonate were added. Theabsor-
bance was measured at 760nm after 2 h ofincubation at
room temperature against methanol blank using UV/Visible
spectrophotometer (Jasco-V-530, Japan). Theconcentration
between 0.01–0.05 mg/mL ofgallic acid was used as stan-
dard for thecalibration curve. Thetotal phenolic content was
expressed as μg gallic acid equivalent mg RJ.

Z.Nabas etal. 173
Moisture content ofroyal jelly
For thedetermination ofmoisture content, 2.5g ofroyal
jelly was placed inacrucible anddried intheoven at 105°C
for three hours, covered andthen cooled inadesiccator.
Theprocess was repeated at aone hour drying intervals until
aconstant weight was obtained [Adebiyi etal., 2004].
Ash content ofroyal jelly
For thedetermination ofash, 2.5 g ofroyal jelly was
placed inacrucible anddried intheoven at 105°C for three
hours, after cooling thesample was ashed inamuffl e furnace
(carbolite model ELF 11/14) at 550°C for 8 h; then cooled
andweighed [Adebiyi etal., 2004].
Total nitrogen content ofroyal jelly
To determine thetotal nitrogen content, theKjeldahl
method for thedetermination oforganic nitrogen was used.
One gram ofroyal jelly was mixed with 15mL sulphuric acid
andplaced inadigestion tube, followed bydistilling andti-
trating. Thenitrogen content (N % w/w) was then calculated
using 6.38 as thefactor for converting nitrogen to protein
[AOAC, 1980].
Mineral content ofroyal jelly
Calcium (Ca), iron (Fe), andmanganese (Mn) were de-
termined directly intheash solution byatomic absorption
spectrometer (Perkin ElmerInstrument). Five grams ofroy-
al jelly were ashed inafurnace at 550°C until theappear-
ance ofwhite-grey ash. Theash was dissolved innitric acid
andboiled, andtheresidue was dissolved indistilled water
with 1mL compensating solution to prevent any possible in-
terference with measurement ofdifferent metals. Theconcen-
tration ofeach metal was extrapolated from standard curves
[Vinas etal., 1997].
Total lipid content ofroyal jelly
Lipid content was determined bySoxhlet procedure us-
ing diethylether as asolvent according to astandard AOAC
procedure [1980].
Total carbohydrates content ofroyal jelly
Total carbohydrate was obtained bydifference [AOAC,
1980], using theformula: Total carbohydrate (CHO) = (100
– moisture%+ protein%+ lipids% + ash%).
Antioxidant activities
Thehighest count ofB. bifi dum was 9.89 log10 CFU/mL
when 7.5% (w/v) royal jelly was added to skimmed milk, while
thehighest count ofL. acidophilus was 8.93 log10 CFU/mL
when 2.5% (w/v) ofroyal jelly was added [Nabas, 2012]. Fer-
mented milk samples were centrifuged at 3000×g for 15 min.
Supernatants were used inall experiments.
DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging
activity
Thefree radical scavenging activity offermented milk
was measured according to Sanchez-Moreno etal. [1998].
A0.5mL dose ofdifferent concentrations offermented milk
(50, 100, 200, 300, 400, 500 μg/mL) was mixed with 0.5mL
of0.1 mmol/L DPPH (Sigma-Aldrich, USA), and2mL
ofmethanol, shaken vigorously byrotomixer (Velp-Scientifi -
ca, Italy) andincubated indark for 30 min at room tempera-
ture. Absorbance was measured using UV/VIS spectropho-
tometer at 517nm against ablank. Thesample concentration
that caused adecrease intheinitial DPPH concentration
by50% was calculated from thegraph ofscavenging effect
percentage against thesample concentration. TheDPPH re-
duction (GR) in30 min was calculated using thefollowing
equation:
GR(%) = [(1 – Af /A0)×100]
where: Af: Absorbance ofsample after 30 min; A0: Absor-
bance ofsample at themoment ofsolution preparation.
Theinhibition activity was calculated using thefollowing
equation:
DPPH radical scavenging activity (%) = [(1 – A1/A0) ×100]
where: A0: Absorbance ofcontrol; A1: Absorbance ofsample.
Determination ofreducing power
Thereducing power offermented milk supplied with
royal jelly was determined according to Oyaizu [1986]. Two
andone-half mL ofdifferent concentrations offermented
milk samples (50, 100, 200, 500 and1000 μg/mL) were
mixed with 2.5mL ofsodium phosphate buffer (0.2 mol/L,
pH 6.6) and2.5mL ofpotassium ferricyanide (1%) solu-
tion. Themixture was incubated at 50°C for 20 min followed
bytheaddition of2.5mL oftrichloroacetic acid (10%)
andcentrifuged at 1400×g for 10 min. Next, 1mL ofthesu-
pernatant was mixed with 1mL distilled water and(0.1%)
ferric chloride. Theabsorbance was measured at 700nm
using UV/VIS spectrophotometer (Elico, SL 150, India).
Ascorbic acid was used as astandard andphosphate buffer
as blank.
Total antioxidant activity inlinoleic acid system
Thetotal antioxidant activity offermented milk supple-
mented with royal jelly was measured bytheuse ofalinoleic
acid system [Mitsuda etal., 1996]. Thelinoleic acid emulsion
was prepared bymixing 0.28 g oflinoleic acid (Sigma-Aldrich,
USA), 0.28 g ofTween-20 emulsifi er (Sigma-Aldrich, USA),
and50mL ofphosphate buffer (0.2 mol/L, pH 7.0). Themix-
ture was then homogenized (Janike@Kunkel, IKA® homog-
enizer, Germany). A0.5mL of100 μg/mL offermented milk
or standard sample (inmethanol) was mixed with linoleic
acid emulsion (2.5 mL, 0.2 mol/L, at pH 7.0) andphosphate
buffer (2 mL, 0.2 mol/LM, pH 7.0). Thereaction mixture was
incubated at 37ºC inthedark to accelerate theperoxidation
process. Thelevels ofperoxidation were determined accord-
ing to thethiocyanate method bysequentially adding etha-
nol (5 mL, 75% v/v), ammonium thiocyanate (0.1 mL, 30%
v/v), sample solution (0.1 mL), andferrous chloride (0.1 mL,
20mmol/L in3.5% HCl). After mixing for 3 min, theperoxide
values were determined byreading theabsorbance at 500nm,
using UV/Vis spectrophotometer. 6-Hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich,

174 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
USA) was used as positive control. Thepercentage inhibition
ofperoxide value was calculated using thefollowing formula:
Inhibition (%) = [(1 – A1/A0) ×100]
where: A0: Absorbance ofcontrol; A1: Absorbance ofsample.
Diene-conjugation formation method
Theeffects offermented milk supplied with royal jelly
on theformation ofconjugated diene hydroperoxides were
determined according to themethod ofTee etal. [2002]. Lin-
oleic emulsion system was prepared as described inthepre-
vious section. Fermented milk with themost effective con-
centrations ofroyal jelly (50, 100, and200 μg/mL), BHT
(100μg/mL) and100 μg/mL ofTrolox (Sigma - Aldrich,
USA) were added to each system, respectively. Theemulsion
was homogenized for 30 sec andincubated at 40˚C.Thefor-
mation ofconjugated diene was monitored daily for seven
days bysolubilizing 0.2mL ofthelinoleic acid emulsion
in5mL ofabsolute methanol. Theabsorbance was mea-
sured at 234nm using UV/Vis spectrophotometer. Theper-
centage inhibition ofconjugated diene value was calculated
using thefollowing formula:
Inhibition (%) = [(1 – A1/A0) ×100]
where: A0: Absorbance ofcontrol; A1: Absorbance ofsample.
Statistical analysis
Thegeneral linear model (GLM), produced bytheSta-
tistical Analysis System (SAS) version 7 [SAS® system for
Microsoft® Windows®. 2001], was used to analyse thedata.
All thedata ofthis research were designed to apply two repli-
cates ofeach level for every experiment. Differences between
themeans oftreatments were tested using theleast signifi -
cant difference (LSD) test at (p<0.05). Repeated measures
analysis ofvariance (ANOVA) was used to analyse thedata
within thesame experiment. Split – Spli-Plot, CRD, with re-
peated measurement design was used for conjugated diene
formation test, CRD with repeated measurement design for
peroxide values andComplete Randomized Design for reduc-
ing power andDPPH scavenging activity. Results are shown
as themean values±standard deviation.
RESULTS ANDDISCUSSION
Royal jelly isaviscous jelly substance, often not homog-
enous due to thepresence ofundissolved granules ofvarying
size. Itispartially soluble inwater with adensity of1.1 g/mL.
Its colour iswhitish to yellow, theyellow colour increasing
upon storage. Its odour issour andpungent, thetaste being
sour andsweet [Lercker, 2003].
Theamounts ofconstituents inroyal jelly determined
inthis study are reported inTable 1. Thecomposition
ofthemain constituents ofRJ, carbohydrates, proteins
andlipids isreported intheliterature [Pourtallier etal., 1990;
Lercker, 2003; Garcia-Amoedo & Almeida-Muradian, 2007].
Thedifferences observed are probably due to thedifferent
number ofsamples taken indifferent places andat differ-
ent times ofproduction. Besides to that different methods
ofsampling andanalysis were used. Moreover, royal jelly
isanaturally heterogeneous product.
Thetotal lipid fraction inthetested royal jelly islike-
wise present inreasonably modest concentration, 10.18%,
(Table1), but indeed represents thethird most important
component ofRJ after carbohydrate andproteins. Thelipid
portion infact consists primarily oforganic acids (80–90%),
most ofwhich free, with arather unusual structure rarely en-
countered innature: they are infact mono- anddihydroxy ac-
ids anddicarboxylic acids with 8 and10 carbon atoms, which
show acharacteristic arrangement [Lercker etal., 1993].
Hydroxy acids with 10 carbon atoms (10 hydroxydecenoic
and10-hydroxy-2-decenoic acid) considered themain factor
for antimicrobial activity [Fujiwara etal., 1990]. Eshraghi &
Seifollahi [2003] examined theantibacterial effect ofRJ on
Escherichia coli (ATCC 29532), Staphylococcus aureus (ATCC
14776), Streptomyces griseus (ATCC 11746), andthree dif-
ferent Streptomyces sp. (S.46) (S.F8) and(S.66). They found
that theapplication ofether soluble fraction ofRJ was more
effective than pure RJ.On theother hand, itwas found that
theinhibitory effect of30 mg/mL ether soluble fraction
ofRJ was stronger than thesame concentration ofether
non soluble fraction which has no effect even at 300mg/mL
ofRJ.Theidentifi cation ofthis fraction andspecially thear-
rangement andquantitative ratios offree organic acids,
isbelieved to represent thecriteria ofchoice for recognizing
theauthenticity ofRJ andfor confi rming andquantifying
theclaimed existence ofroyal jelly inother products. Besides
thefree fatty acids, thelipid fraction consist ofsome neutral
lipids, sterols (including cholesterol) andan unsaponifi able
fraction ofhydrocarbons similar to beeswax extracts [Lercker
etal., 1981, 1982, 1984, 1993].
Thetotal carbohydrate content ofroyal jelly was 14.07%
(Table 1) andmostly consisted offructose andglucose. Fruc-
tose isprevalent. Inmany cases fructose andglucose together
account for 90% ofthetotal sugars. Sucrose isalways present
but often inhighly variable concentrations. Itisalso possible
to detect other oligosaccharides such as trehalose, maltose,
gentiobiose, isomaltose, raffi nose, erlose, melezitose [Garcia-
-Amoedo & Almeida-Muradian, 2007]; though present invery
TABLE 1. General chemical composition ofRoyal jelly produced inJordan.
Chemical analysis Content
pH 3.42±0.02
Moisture (%) 61.5±0.25
Ash content (%) 1.10±0.05
Protein content (%) 13.15±0.60
Fat (%) 10.18±3.48
CHO (%) 14.07±0.0
Total phenolics (μg galic acid/mg RJ ) 23.3±0.92
Total fl avonoids (μg rutin/mg RJ) 1.28±0.09
Ca (ppm) 64.5±6.50
Fe (ppm) 0.63±0.10
Mn (ppm) 0.03±0.01
Results are mean values ofduplicates determinations ± standard deviation.

Z.Nabas etal. 175
small quantities they are useful for identifying acharacteristic
pattern, which iscomparable to that ofhoney andinsome
cases indicative ofthegenuineness oftheproduct [Sabatini
etal., 2009].
Thevalues ofcrude protein content inroyal jelly are
presented in(Table 1). Theobtained data are infull compli-
ance with fi ndings ofother authors [Krell, 1996; Wongchai &
Ratanavalachai, 2002; Nagai & Inoue, 2004; Garcia-Amoedo
& Almeida-Muradian, 2007], who reported therange from
12 to 15%.
Ingeneral, water content inroyal jelly isfairly uniform
andmakes up about two third offresh royal jelly. Table1
shows that royal jelly consists of61.5% ofwater which
agreed with therange of57–70% reported byKrell [1996]
and60–70% byGarcia-Amoedo & Almeida-Muradian
[2007]. Theconstancy ofthewater content isbasically as-
sured, inside thehive, bythecontinuous provision offresh
supplies ofthis substance bynurse bees, bythenatural hygro-
scopicity ofroyal jelly andtheentire colony’s efforts to main-
tain alevel ofambient humidity; moreover thenon-solubility
ofsome compounds can explain thevariations inmoisture
content [Sabatini etal., 2009].
Thetotal amounts ofphenolic andtotal fl avonoids
contents inthetested royal jelly are presented inTable 1.
Thephenolic andtotal fl avonoids content oftheRJ were as
follows: 23.3 μg/mg ofRJ and1.28 μg/mg ofRJ, respectively.
These quantities ofphenolic compounds were very similar
to that found byNagai & Inoue [2004] which is21.2 μg/mg
ofRJ.Thetotal phenolic contents of219.2 μg/g, were found
inroyal jelly obtained from Taiwan collected 24 h after graft-
ing, while thetotal phenolic contents ofroyal jelly collected
after 48 and72 h was 194.4 μg/g and131.7 μg/g, respectively
[Liu etal., 2008].
Liu etal. [2008] indicated that regardless ofinitial lar-
val age, RJ collected 24 h after thelarval transfer contained
higher total polyphenolic contents than theRJ collected 48 or
72 h after thetransfer.
These compounds have antimicrobial activity andexplain
theantimicrobial capacity ofroyal jelly. Moreover, ithas
been reported that phenolics are effi cient antioxidant, im-
munomodulator andanti-infl amatory agents [Nagai & In-
oue, 2004]. Thevariation ofthetotal phenolic contents isat-
tributed to several factors such as climate andfl oral source.
Thephenolic compounds ofroyal jelly could originate from
plants where they are widely distributed in nature [Bravo,
1998; Wongchai & Ratanavalachai, 2002; Liu etal., 2008].
Themain groups ofphenolic compounds present inplants,
whether infree form or as glucosides, are derivatives ofcin-
namic acid, coumarins, andfl avonoids [Manthey & Grohm-
ann, 2001]. Inroyal jelly, most ofthephenolic compounds
are intheform offl avonoids whose concentration depends
on various factors, including plant species used bythebees,
health oftheplant, season, environmental factors, andso on
[Kucuk etal., 2007].
ThepH value ofRJ was 3.42, which agreed with thepH
value reported byKrell [1996]. Percentage ofash inroyal jelly
sample was 1.10%, this value iscomparable to themean ash
content ofThailand royal jelly with 1.14%. On theother hand,
itwas reported that theash content offresh royal jelly reached
0.8–3.0% [Wongchai & Ratanavalachai 2002; Garcia-Amoe-
do & Almeida-Muradian, 2007].
Concentrations ofminerals detected inthis research varied,
calcium was themost abundant element compared with other
determined minerals with 64.5 ppm followed byFe (0.63 ppm)
andvery low concentration ofMn (0.03 ppm). These results
agree with those reported byStocker etal. [2005], who detected
different trace andmineral elements inroyal jelly that could
beattributed to an external factor such as environment, differ-
ent botanical andgeological origins andto some extent internal
factors such as biological factors related to thebees [Stocker
etal., 2005; Garcia-Amoedo & Almeida-Muradian, 2007].
Theproton radical scavenging has been reported to
beamajor mechanism for antioxidation. Theassay for theas-
sessment ofproton radical-scavenging activity with DPPH
isrelatively simple andenough reproducible; this compound
has been reported to bestoichiometrically decolorized byan-
tioxidants. Thereduction intheDPPH would beobserved
as its absorbance at acharacteristic wavelength isdecreased
when itistreated with proton radical scavengers. TheDPPH
isastable radical with amaximum absorption at 517nm
that can readily undergo scavenging byan antioxidant [Lu &
Foo, 2001]. Ithas been widely used to test theability ofcom-
pounds as free-radical scavengers or hydrogen donors andto
evaluate theantioxidative activity ofplant extracts andfoods
[daPorto etal., 2000].
Theantioxidant capacity offermented milk (L. acidophi-
lus supplemented with 2.5% royal jelly andB. bifi dum sup-
plemented with 7.5% royal jelly) was evaluated indifferent
invitro tests. Thepercent ofDPPH radical scavenging activity
offermented milk samples containing royal jelly, with ascor-
bic acid andbutylated hydroxytoluene (BHT) as reference
samples are listed inFigures 1 and2. Thescavenging effects
offermented milk with B. bifi dum andstandards on DPPH
radicals were 8.6, 17.1, 18.9 and48.5% at aconcentration of
50 μg/mL for B. bifi dum, B. bifi dum supplemented with 7.5%
RJ, BHT andascorbic acid, respectively (Figure 1). Asignifi -
cant DPPH radical scavenging activity was observed when
fermented milk with B. bifi dum supplemented with 7.5% RJ
was used at 500μg/mL with an effect of96.8% (Figure1).
Moreover, thevalue oftheantioxidant capacity offerment-
ed milk with L. acidophilus supplemented with 2.5% RJ was
12.6% at aconcentration of50 μg/mL.This value was in-
creased signifi cantly to 93.2% at aconcentration of500 μg/mL
(Figure2). As presented inFigures 1 and2, thescavenging
abilities ofdifferent samples were clearly demonstrated dose-
dependent antioxidant activity against DPPH.These fi ndings
agree with observations indicated byother researchers [Nagai
& Inoue, 2004; Sowndhararajan & Kang, 2013; Zhang etal.,
2011]. Nagai & Inoue [2004] demonstrated that theradicals
scavenging activities ofroyal jelly samples tended to decrease
with adecreasing concentration ofthesample.
Themaximum GR percentage for fermented milk was ob-
tained at aconcentration of500 μg/mL with values of28.4,
92.0, 91.8 and80.5% for B. bifi dum, B. bifi dum supplemented
with 7.5% RJ, ascorbic acid andBHT, respectively. On theother
hand, themaximum GR percentage for fermented milk was ob-
tained also at aconcentration of500 μg/mL with values of15.4,
93.2, 91.8 and80.5% for L. acidophilus, L. acidophilus supple-

176 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
Ascorbic acid
DPPH Scavenging Activity (%)
Concentration ( g/mL)µ
50 100 200 300 400 500
-40
-20
0
20
40
60
80
120
100
BHT
B. bifidum + 7.5% royal jelly
B. bifidum
0
FIGURE 1. Scavenging activity ofdifferent concentrations ofascorbic
acid, BHT, B. bifi dum supplemented with 7.5% royal jelly andB. bifi dum.
Ascorbic acid
DPPH Scavenging Activity (%)
Concentration ( g/mL)µ
50 100 200 300 400 500
-40
-20
0
20
40
60
80
120
100
BHT
L. acidophilus + 2.5% royal
jelly
L. acidophilus
0
FIGURE 2. Scavenging activity ofdifferent concentrations ofascorbic acid,
BHT, L. acidophilus supplemented with 2.5% royal jelly andL. acidophilus.
TABLE 2. Thereduction ofDPPH (GR) ofascorbic acid, BHT, B. bifi dum andB. bifi dum supplemented with 7.5% RJ, L. acidophilus andL. acidophilus
supplemented with 2.5% RJ.
Concentration
(μg/mL)
GR (%)
Ascorbic acid BHT B. bifi dum
B. bifi dum
supplemented
with 7.5% RJ
L. acidophilus
L. acidophilus
supplemented
with 2.5 % RJ
50 33.3±0.04a* 3.2±1.2b2.2±0.33b6.2±1.6b2.5±1.2b3.2±1.3b
100 55.7±0.64a17.6±1.4b1.7±1.53c13.3± 0.8bc 1.1±0.4d9.0±0.8c
200 80.3±1.61a43.9±0.4b6.9±0.57d28.5±2.12c3.7±1.11d17.0±1.2c
300 90.9±1.30a72.6±0.11b16.0±3.2d44.8± 1.3c7.9±0.3d48.3±1.23c
400 89.9± 0.9a76.1±1.2c27.3±1.8d83.1± 0.8b3.7± 0.7c79.4±0.9b
500 91.8± 1.1a80.5±0.8b28.4±2.2c92.0±0.73a15.4±2.4c93.2±1.4b
*Means with different superscript within thesame row are signifi cantly different (p<0.05) according to LSD.Signifi cant differences between various
treatments within each experiment are indicated bysuperscript letters (a, b, c andd). Results are mean values ofduplicates determinations ± standard
deviation.
mented with 2.5% RJ, ascorbic acid andBHT, respectively
(Table 2). Also, theinvitro antioxidant activity was expressed
interms ofIC50 value, theIC50 value isdefi ned as theconcen-
tration ofsubstrate that causes 50% loss oftheDPPH activity
(colour). TheIC50 was determined bylinear regression equa-
tion for fermented milk with B. bifi dum supplemented with
7.5% RJ andfor fermented milk with L. acidophilus supple-
mented with 2.5% RJ.TheIC50 value offermented milk with
B.bifi dum supplemented with 7.5% RJ reached at aconcentra-
tion of226.7μg/mL (y = 0.188x + 7.38 R² = 0.968), while
thevalue of IC50 ofL. acidophilus supplemented with 2.5% RJ
reached 210.2 μg/mL (y = 0.181x + 11.95 R² = 0.979).
Reducing power indicates theability ofenzymes (catalase,
NADH oxidase, andNADH peroxidase) or non-enzymatic
compounds (ascorbate, tocopherol, andglutathione) to re-
duce oxygen radicals or iron that then become unavailable for
oxidative reactions [Warriner & Morris, 1995; Liu etal., 2008].
Several researches have reported that thereducing power
ofacompound may beused as asignifi cant indicator ofits
potential antioxidant activity [Meir etal., 1995]. Inreducing
power assay, fermented milk containing L. acidophilus supple-
mented with 2.5% royal jelly andB. bifi dum supplemented
with 7.5% royal jelly reduced Fe3+ to Fe2+ byreducing oxygen
radical that isusually considered as promotive to oxidative
reactions. However, theamount ofFe2+ complex can bethen
monitored bymeasuring theformation ofPerl’s Prussian
blue at 700nm [Dorman etal., 2003; Ara & Nur, 2009]. Fer-
mented milk containing B. bifi dum supplemented with 7.5%RJ
exhibited signifi cantly higher reducing power than ascorbic
acid or fermented milk containing B. bifi dum alone with val-
ues of1.253, 0.759 and0.330, respectively at aconcentration
of500 μg/mL on dose dependent basis (Figure 3). Fermented
milk containing L. acidophilus supplemented with 2.5% RJ ex-
hibited higher reducing power than ascorbic acid or fermented
milk with L .acidophilus alone with reducing power values at
1.535, 0.943 and0.441, respectively, at 1000 μg/mL as dose
dependent manner (Figure 4). Based on theresults, inorder to
get theeffect ofreducing power ofthemixture (RJ andprobi-
otic) thedose should not beless than 200 μg/mL.This could
berelated to asuffi cient amount ofreductive substances, with
thecapacity to react with free radicals to stabilize andtermi-
nate theradical reactions. Wang etal. [2009] reported that
lactic acid bacteria exhibited astrong reducing power ability.
Thedevelopment oflipid peroxidation offermented milk
containing royal jelly over afermentation period of24 h
isshown inFigures 5 and6. Thetotal antioxidant capacity was
determined bythiocyanate method. This method measures
thetotal amount ofperoxide produced during theinitial stages
ofoxidation. The% inhibition ofB. bifi dum supplemented with
7.5% royal jelly andL. acidophilus was 42% and52%, respec-
tively. Theprocess oflipid peroxidation isinitiated byan at-
tack on afatty acid or fatty acyl side chain byany chemical
species featuring suffi cient reactivity to take ahydrogen atom
away from amethylene carbon intheside chain. Theresulting
lipid radicals then undergo molecular rearrangement, followed
byreacting with oxygen to produce peroxyl radicals, which are

Z.Nabas etal. 177
capable ofabstracting hydrogen from adjacent fatty acid side
chains andso propagating achain reaction oflipid peroxida-
tion [Halliwell & Gutteridge, 1986; Prakash & Gupta, 2009].
Lipid peroxidation isnot only associated with food deteriora-
tion andlowers thenutritional quality offood, but itexerts ad-
verse effects on human health. Liu etal. [2008] demonstrated
that royal jelly exhibited an inhibitory effect on linoleic acid
peroxidation at 27.9%. Another study byGuo etal. [2005] ex-
amined theantioxidant capacity ofroyal jelly peptides andwa-
ter-soluble protein inlinoleic acid peroxidation, where royal
jelly peptides had greater effect with 97.4% than water-soluble
royal jelly protein with 6.8% inhibition.
Thedevelopment ofdiene conjugated compounds inlin-
oleic acid system treated with 50, 100, 200 μg/mL ofboth dif-
ferent fermented milk and100 μg/mL ofTrolox andBHT are
listed inTables 3 and4. Fermented milk with B. bifi dum supple-
R
e
d
uc
i
ng
P
ower
(ass absorbance at 700 nm)
2.5
1.5
0.5
0
-0.5
50 100 200 500 10000
Concentration ( g/mL)µ
Ascorbic acid
B. bifidum
+ 7.5 % royal jelly
B. bifidum
1.0
2.0
FIGURE 3. Reducing power ofdifferent concentrations ofascorbic acid,
B. bifi dum supplemented with 7.5% royal jelly andB. bifi dum.
Control
Trolox
B. bifidium
B. bifidium
+ 7.5% RJ
0.6
0
0.2
0.4
0.8
1.2
1.4
Absorbance at 500 nm)
0123
Time of incubation (Day)
4
1.0
FIGURE 5. Antioxidant activity ofTrolox, B. bifi dum andB. bifi dum sup-
plemented with 7.5% royal jelly measured bythiocyanate method during
different time intervals (day).
Reducing Power
(ass absorbance at 700 nm)
1.5
0.5
0
-0.5
50 100 200 500 10000
Concentration ( g/mL)µ
Ascorbic acid
L. acidophilus
+ 2.5 % royal jelly
L. acidophilus
1.0
2.0
FIGURE 4. Reducing power ofdifferent concentrations ofascorbic acid,
L. acidophilus supplemented with 2.5% royal jelly andL. acidophilus.
0.6
0
0.2
0.4
0.8
1.2
1.4
Absorbance at 500 nm
Time of incubation (Day)
01234
1.0 Control
Trolox
L. acidophilus
L. acidophilus
+ 2.5% RJ
FIGURE 6. Antioxidant activity ofTrolox, L. acidophilus andL. acidophi-
lus supplemented with 2.5% royal jelly measured bythiocyanate method
during different time intervals (day).
TABLE 3. Effect ofBHT, Trolox, B. bifdidum andB. bifdidum supplemented with 7.5% royal jelly on theformation ofdiene-conjugation (expressed as
inhibition %) using different intervals day inlinoleic acid model system.
Treatment (μg/mL) Inhibition ofdiene conjugation (%) during different intervals (day)
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
BHT (100) 42.6± 0.27c* 57.7±3.8d54.6±2.5c59.9±0.74b57.3±0.4b50.6±0.20b49.3±0.44b47.8±0.23b
Trolox (100) 61.5±1.7a74.9±0.7a68.0±2.8a68.0±1.5a67.6±0.35a61.6±0.11a59.3±1.2a58.7±0.45a
Bifi dobacterium bifi dum
0% Royal jelly (50) 1.7±0.8d14.7±2.10f6.3±2.0e8.5 ±0.6d12.2±1.52d2.8 ±0.31e1.2± 1.33d0.97±1.0d
0% Royal jelly (100) 5.3±0.23d16.9±2.01f8.2±1.98e9.3±0.32d13.2±0.83d5.7±2.6d1.4±0.66d0.83±0.73d
0% Royal jelly (200) 6.2±1.64d19.6±1.92e16.5±0.9d17.4±0.84c19.3±1.9c7.2 ±1.02cd 5.4±0.74c3.11±1.73d
7.5% Royal jelly (50 ) 3.4±0.8d15.9±1.44f17.1±0.72d18.9±0.33c20.3±0.3c8.3± 0.28c6.3±0.79c4.0 ±1.31c
7.5% Royal jelly (100) 43.2±0.47c62.1±1.70c57.4±2.53b60.9±0.4b57.6±0.77b51.7±0.28b50.2±0.54b48.6±0.2b
7.5% Royal jelly (200) 46.2±0.71b72.1±1.52b67.8±0.94a66.7±0.4a65.8±1.6a61.8±2.32a52.1±0.45b49.5±0.64b
*Means with different superscript within thesame column are signifi cantly different (p<0.05) according to LSD.Signifi cant differences between vari-
ous treatments within each experiment are indicated bysuperscript letters (a, b, c, d, e andf ). Results are mean values ofduplicates determinations ±
standard deviation.

178 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
mented with 7.5% royal jelly andL. acidophilus supplemented
with 2.5% royal jelly inhibited theformation ofconjugated di-
ene inlinoleic acid system. Theinhibition effects were noticed
throughout theinduction period compared to control. Ingen-
eral, results show that as theconcentration ofboth fermented
milk with B. bifi dum andL. acidophilus increased, theformation
ofoxidation products decreased andinsome concentrations
showed close absorbance values andsometimes competitive
with Trolox andBHT.Theformation oftheoxidation products
increased gradually inthecontrol system during theinduction
period andtheproducts started to decompose after thefourth
day. Thepercentage of inhibition for fermented milk with
B. bifi dum were 20.3, 57.6, 65.8, 12.2, 13.3, 19.3, 67.7 and57.3
percent for 50, 100, and200μg/mL ofB. bifi dum supplemented
with 7.5% royal jelly; for 50, 100, and 200 μg/mL ofB. bifi dum;
100 μg/mL ofTrolox and 100μg/mL ofBHT, respectively (Ta-
ble 3). While, thepercentage ofinhibition for fermented milk
with L. acidophilus were 47.4, 60.3, 66.9, 16.4, 22.0, 24.5, 67.6
and57.3 percent for 50, 100, and200 μg/mL ofL. acidophilus
supplemented with 2.5% royal jelly; for 50, 100, and 200μg/mL
ofL. acidophilus; 100μg/mL ofTrolox and 100μg/mL ofBHT,
respectively (Table4). Tee etal. [2002] made thesame obser-
vations concerning thedecreasing ofthepercentage inhibition
ofdiene-conjugation compounds after four days ofincubation.
CONCLUSION
Royal jelly isanatural substance considered as one
ofthemost important products ofhoneybees with high nutri-
tional, functional andbiological properties. Therefore, itcould
beconcluded from theobtained results that thesynbiotic effect
ofprobiotic bacteria L. acidophilus andB. bifi dum with theroyal
jelly can beused inthedairy industry. This mixture, which ispo-
tentially benefi cial to enhance human health andprotect against
oxidative damage andwould beconsidered astrong competi-
tor for thesynthetic antioxidants. Synthetic antioxidants such
as butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT) andn-propyl gallate (PG) exhibit strong antioxidant
activity against several oxidation systems. However, synthetic
antioxidants pose potential risks invivo; their use infood isre-
stricted or prohibited insome countries. Antioxidants from
natural sources are likely to bemore desirable than those chemi-
cally produced because some synthetic antioxidants have been
reported to beside effects [Osuntoki & Korie, 2010].
ACKNOWLEDGEMENTS
This research was supported by the University of Jordan,
Amman, Jordan.
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cepted: 10 June 2014. Published on-line: 25 July 2014.