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


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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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Pol. J.Food Nutr. Sci., 2014, Vol. 64, No. 3, pp. 171-180
DOI: 10.2478/pjfns-2013-0015
At present days, remarkable changes were observed inun-
derstanding therole offoods inhuman health. Enormous in-
terest ofconsumers has been directed toward healthy foods
which help inpreventing theinitiation ofdiseases andtheir
development; hence, anew term “functional food” was sug-
gested [Hardy, 2000; Grajek etal., 2005].
Functional food isconsidered to beany food or food com-
ponent that provides health bene ts beyond basic nutrition.
Recently, agreat deal ofinterest has been paid bythecon-
sumers towards natural bioactive compounds as functional
ingredients inthediets due to their various health bene cial
effects [Vo & Kim, 2013]. Afunctional food may benatural or
beobtained bytheelimination or themodi cation ofone or
more ofits basic components [Perez-Alvarez etal., 2003]. Se-
lective non-digestible oligosaccharides andpolysaccharides,
some peptides andproteins, andcertain lipids, are themost
common prebiotics [Gibson & Roberfroid, 1995]. Prebiot-
ics are known to promote theproliferation ofbi dobacteria
andlactobacilli [Roberfroid, 2000]. Among foods that can
beconsidered as functional foods, we may include all those
originating inthebeehive: honey [Haddadin etal., 2007; Mei
* Corresponding Author: E-mail address:
(Prof. Malik S.Y.Haddadin)
etal., 2010], propolis [Haddadin etal., 2008], androyal jelly
[Haddadin etal., 2012; Nabas, 2012].
Royal jelly (RJ) isanatural substance considered as one
ofthemost important products ofhoneybees with high nu-
tritional, functional andbiological properties. Itissecreted
from thehypopharingeal glands ofworker bees which serve
as food for thequeen bee andto theyoung larvae [Grout,
1992]. Ingeneral, royal jelly isrelatively acidic (pH 3.1–3.9)
with ahigh buffering capacity ranges between 4 and7 [Sau-
erwald etal., 1998].
Itiscomposed ofproteins, lipids, sugars, vitamins
andamino acids [Howe etal., 1985], B complex vitamins
such as B1, B2, B6 [Moreschi & Almeida-Muradian, 2009],
andbiotin [Nandhasri etal., 1990]. Moreover, itcontains
different minerals (P, S, Ca, Mg, K, Na, Zn, Fe, Cu, Mn)
andtrace 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 etal., 2005]. Themain fatty acid present inRJ
is10-hydroxy-trans-2-decenoic acid (10-HDA) [Yang etal.,
2010], itplays an important role inboosting immune system,
anticancer activity. Itcontains other components such as roy-
alisin andapisin [Watanabe etal., 1996, 1998]. Royal jelly
can beincluded directly inour food anddietary supplements,
moreover, royal jelly istraditionally known to have some
biological or medical purposes such as life-span elongating
[Inoue etal., 2003], anti-fatigue [Kamakura etal., 2001], anti-
Chemical Composition ofRoyal Jelly andEffects ofSynbiotic with Two
Different LocallyIsolated Probiotic Strains on Antioxidant Activities
Zaid Nabas1, Malik S.Y.Haddadin1*, Jamal Haddadin2, Ibrahim K.Nazer3
1Department ofNutrition andFood Technology, Faculty ofAgriculture, University ofJordan, Amman, Jordan
2Department ofFood andNutrition Sciences, King Faisal University, Al Ahsa, Kingdom ofSaudi Arabia
3Department ofPlant Protection, Faculty ofAgriculture, University ofJordan, Amman, Jordan
Key words: synbiotic, royal jelly, DPPH, reducing power, linoleic acid system
This study was carried out to investigate theantioxidant properties ofsynbiotic product, Lactobacillus acidophilus supplemented with 2.5% royal
jelly inskim milk andBi dobacterium bi dum supplemented with 7.5% royal jelly inskim milk, using DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical
scavenging assay, reducing power, total antioxidant inlinoleic acid system andformation ofdiene-conjugation assay. Results showed that thesynbiotic
effect ofroyal jelly andprobiotic bacteria provided substantial antioxidant activities. Milk samples fermented byB. bi dum supplemented with 7.5%
royal jelly andL. acidophilus supplemented with 2.5% royal jelly exhibited high scavenging activity with 96.8 and93.3%, respectively, at aconcentration
of500 μg/mL.IC50 values were estimated at 226.7 μg/mL for B. bi dum supplemented with 7.5% royal jelly andat 210.2 μg/ml for L. acidophilus supple-
mented with 2.5% royal jelly. On theother hand, L. acidophilus supplemented with 2.5% royal jelly andB. bi dum supplemented with 7.5% royal jelly
exhibited signi cantly high reducing power at aconcentration of1000 μg/mL.Thepercentages ofperoxide inhibition ofL. acidophilus supplemented
with 2.5% royal jelly andB. bi dum with 7.5% royal jelly were 52% and42%, respectively. Signi cant inhibitions were found intheformation ofconju-
gated diene at 66.9% and65.8% for L. acidophilus with 2.5% royal jelly andB. bi dum with 7.5% royal jelly, respectively. These results were compared
with standards BHT, ascorbic acid andTrolox.
Original article
Section: Food Chemistry
172 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
allergic andimmunoregulatory effect [Okamoto etal., 2003],
andanti-in ammatory [Kohno etal., 2004], anti-bacterial,
andantioxidant activity [Nagai & Inoue, 2004; El-Nekeety
etal., 2007; Eshraghi & Seifollahi, 2003; Barnutiu etal., 2011;
Guo etal., 2005; Liu etal., 2008].
Aprobiotic isabene cial living microbial food ingredient
whose growth andactivity are stimulated byprebiotics [Sal-
minen etal., 1998]. According to Chandan [1999], themost
widely used probiotics infoods, especially indairy products,
are themembers ofthegenera Enterococcus, Lactobacillus,
andBi dobacterium. Probiotic bacteria proved to have de-
sirable clinical bene ts, therapeutic actions andprotective
effects against oxidative damages [Amaretti, etal., 2013;
Bernardeau etal., 2008; Osuntoki & Korie, 2010; Pan etal.,
2009]. Recently they are being recognized for their remarkable
role inregulating thehost metabolic processes, weight gain
andobesity [Thomas & Versalovic, 2010].
Inpractice, combined mixtures ofprobiotics andprebiot-
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 bene cial effects on thehost byenhancing sur-
vival andimplantation oflive microbial dietary supplements
inthegastrointestinal microbiota, byselectively stimulating
thegrowth or activating thecatabolism ofone or alimited
number ofhealth-promoting bacteria inthegastrointestinal
tract, andbyimproving theintestinal tract’s microbial balance
[Wollowski etal., 2001]. Moreover, probiotic andprebiotic ef-
fects might beadditive or even synergistic [Roberfroid, 2000].
Theobjective ofthis study was to evaluate theantioxidant
properties ofthesynbiotic effect ofroyal jelly with two differ-
ent locally isolated strains Lactobacillus acidophilus andBi -
dobacterium bi dum using different invitro assays.
Probiotic bacterial strains
L. acidophilus andB. bi dum isolates used inthis research
were previously isolated from new born breast fed infants
stool [Haddadin, 2004], at Queen Alia hospital. One gram
ofeach freeze-dried powders ofthese isolates was transferred
aseptically into 50mL sterile MRS broth supplemented with
5 g/L L(+) cysteine–HCL (99.6% purity, Sigma, USA), then
incubated at 37°C for 20 h inan anaerobic jar (Oxoid, UK).
Repeated streaking onto MRS agar plates were used for
puri cation ofboth isolates, theisolates ofL. acidophilus
andB. bi dum were identi ed physiologically andbiochemi-
cally according to Bergey’s Manual [Kandler & Weiss, 1986]
andProkaryotes [Hammes etal., 1992]. Theisolates were ac-
tivated making sub-culturing twice inMRS broth containing
0.5% L(+) cysteine–HCL, using 1% inoculum and18–20h
ofincubation 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 thespring
andsummer oftheyear 2011 from Langstroth hives with
colonies ofthemost common honeybee species Apis mellifera
located at theUniversity ofJordan Apiary – Faculty ofAgri-
culture, using thearti cial cups method according to Grout
[1992]. Samples ofroyal jelly were diluted with distilled
water and ltered under avacuum, using, Grade No.1 lter
paper, andGrade No. 40 lter paper (Whatman membrane,
England), and0.45 μm nylon membrane. Cold sterilization
was performed via micro- ltration unit using 0.20 μm sterile
cellulose-ester membranes (Advantec MFS, Japan) [Hadda-
din etal., 2007]. A9% skimmed milk was reconstituted indis-
tilled water andsterilized at 115ºC for 10 min. After cooling
to 37ºC, ve different concentrations ofsterilized royal jelly
(0, 2.5, 5, 7.5, 10% m/v) were added to reconstituted milk
in100mL Duran bottles. Acontrol sample was used with-
out theaddition ofroyal jelly [Haddadin etal., 2012; Nabas,
2012]. Royal jelly/milk samples with highest counts ofeach
probiotic bacteria were chosen to examine theantioxidant
activities. ThepH ofRJ was measured using adigital pH me-
ter (Hanna instrument model HI 8519, Italy). ThepH was
directly measured after adding 2 g ofRJ to 4mL ofdistilled
water (pH 7.00).
Evaluation ofbacterial growth
One percent % (v/v) ofL. acidophilus or B. bi dum was
added to theprepared milk/royal jelly samples. Thecultures
were then incubated at 37ºC inanaerobic jars for 24 h. After
incubation, serial dilutions (10–1 – 10–7) were realized us-
ing sterile 0.1% peptone broth andplated on MRS agar sup-
plied with cysteine-HCl (5 g/L) andincubated at 37°C for
48h under anaerobic condition. Theresults were recorded as
CFU/mL ofculture [Harrigan & McCane, 1996].
Determination oftotal avonoids ofroyal jelly
Thetotal avonoid content ofRJ was determined accord-
ing to Ja etal. [1999]. Five grams ofRJ were added to 50mL
ofdistilled water and ltered using Grade No. 1 Filter Paper
(Whatman membranes, England). Then, 0.5mL ofthesolu-
tion was mixed with 0.3mL ofsodium nitrite solution (5g/L).
After 5 min, 0.3mL ofaluminum chloride (1 g/L) was added.
After another 5 min, 2mL of1 mol/L ofsodium hydroxide
was added to themixture. Total volume was made up to
10mL with distilled water andthesample was sonicated im-
mediately after preparation. Theabsorbance was measured
at 510nm against water blank using UV/Visible Spectropho-
tometer (Jasco-V-530, Japan). Calibration curve was plotted
bypreparing asolution ofrutin (0–200 μg/mL). Concentra-
tions are expressed as (μg rutin equivalent /mg RJ).
Determination oftotal phenolic content ofroyal jelly
Total phenolic content ofRJ was determined byFo-
lin–Ciocalteu method according to Singleton etal. [1999].
To this end, 0.5mL from theprevious solution ofRJ used
in avonoid assay was mixed with 2.5mL of0.2N Folin-Ci-
ocalteu reagents (Sigma –Aldrich, USA) for 5 min andthen
2mL of7.5% sodium carbonate were added. Theabsor-
bance was measured at 760nm after 2 h ofincubation at
room temperature against methanol blank using UV/Visible
spectrophotometer (Jasco-V-530, Japan). Theconcentration
between 0.01–0.05 mg/mL ofgallic acid was used as stan-
dard for thecalibration curve. Thetotal phenolic content was
expressed as μg gallic acid equivalent mg RJ.
Z.Nabas etal. 173
Moisture content ofroyal jelly
For thedetermination ofmoisture content, 2.5g ofroyal
jelly was placed inacrucible anddried intheoven at 105°C
for three hours, covered andthen cooled inadesiccator.
Theprocess was repeated at aone hour drying intervals until
aconstant weight was obtained [Adebiyi etal., 2004].
Ash content ofroyal jelly
For thedetermination ofash, 2.5 g ofroyal jelly was
placed inacrucible anddried intheoven at 105°C for three
hours, after cooling thesample was ashed inamuf e furnace
(carbolite model ELF 11/14) at 550°C for 8 h; then cooled
andweighed [Adebiyi etal., 2004].
Total nitrogen content ofroyal jelly
To determine thetotal nitrogen content, theKjeldahl
method for thedetermination oforganic nitrogen was used.
One gram ofroyal jelly was mixed with 15mL sulphuric acid
andplaced inadigestion tube, followed bydistilling andti-
trating. Thenitrogen content (N % w/w) was then calculated
using 6.38 as thefactor for converting nitrogen to protein
[AOAC, 1980].
Mineral content ofroyal jelly
Calcium (Ca), iron (Fe), andmanganese (Mn) were de-
termined directly intheash solution byatomic absorption
spectrometer (Perkin ElmerInstrument). Five grams ofroy-
al jelly were ashed inafurnace at 550°C until theappear-
ance ofwhite-grey ash. Theash was dissolved innitric acid
andboiled, andtheresidue was dissolved indistilled water
with 1mL compensating solution to prevent any possible in-
terference with measurement ofdifferent metals. Theconcen-
tration ofeach metal was extrapolated from standard curves
[Vinas etal., 1997].
Total lipid content ofroyal jelly
Lipid content was determined bySoxhlet procedure us-
ing diethylether as asolvent according to astandard AOAC
procedure [1980].
Total carbohydrates content ofroyal jelly
Total carbohydrate was obtained bydifference [AOAC,
1980], using theformula: Total carbohydrate (CHO) = (100
– moisture%+ protein%+ lipids% + ash%).
Antioxidant activities
Thehighest count ofB. bi dum was 9.89 log10 CFU/mL
when 7.5% (w/v) royal jelly was added to skimmed milk, while
thehighest count ofL. acidophilus was 8.93 log10 CFU/mL
when 2.5% (w/v) ofroyal jelly was added [Nabas, 2012]. Fer-
mented milk samples were centrifuged at 3000×g for 15 min.
Supernatants were used inall experiments.
DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging
Thefree radical scavenging activity offermented milk
was measured according to Sanchez-Moreno etal. [1998].
A0.5mL dose ofdifferent concentrations offermented milk
(50, 100, 200, 300, 400, 500 μg/mL) was mixed with 0.5mL
of0.1 mmol/L DPPH (Sigma-Aldrich, USA), and2mL
ofmethanol, shaken vigorously byrotomixer (Velp-Scienti -
ca, Italy) andincubated indark for 30 min at room tempera-
ture. Absorbance was measured using UV/VIS spectropho-
tometer at 517nm against ablank. Thesample concentration
that caused adecrease intheinitial DPPH concentration
by50% was calculated from thegraph ofscavenging effect
percentage against thesample concentration. TheDPPH re-
duction (GR) in30 min was calculated using thefollowing
GR(%) = [(1Af /A0)×100]
where: Af: Absorbance ofsample after 30 min; A0: Absor-
bance ofsample at themoment ofsolution preparation.
Theinhibition activity was calculated using thefollowing
DPPH radical scavenging activity (%) = [(1A1/A0) ×100]
where: A0: Absorbance ofcontrol; A1: Absorbance ofsample.
Determination ofreducing power
Thereducing power offermented milk supplied with
royal jelly was determined according to Oyaizu [1986]. Two
andone-half mL ofdifferent concentrations offermented
milk samples (50, 100, 200, 500 and1000 μg/mL) were
mixed with 2.5mL ofsodium phosphate buffer (0.2 mol/L,
pH 6.6) and2.5mL ofpotassium ferricyanide (1%) solu-
tion. Themixture was incubated at 50°C for 20 min followed
bytheaddition of2.5mL oftrichloroacetic acid (10%)
andcentrifuged at 1400×g for 10 min. Next, 1mL ofthesu-
pernatant was mixed with 1mL distilled water and(0.1%)
ferric chloride. Theabsorbance was measured at 700nm
using UV/VIS spectrophotometer (Elico, SL 150, India).
Ascorbic acid was used as astandard andphosphate buffer
as blank.
Total antioxidant activity inlinoleic acid system
Thetotal antioxidant activity offermented milk supple-
mented with royal jelly was measured bytheuse ofalinoleic
acid system [Mitsuda etal., 1996]. Thelinoleic acid emulsion
was prepared bymixing 0.28 g oflinoleic acid (Sigma-Aldrich,
USA), 0.28 g ofTween-20 emulsi er (Sigma-Aldrich, USA),
and50mL ofphosphate buffer (0.2 mol/L, pH 7.0). Themix-
ture was then homogenized (Janike@Kunkel, IKA® homog-
enizer, Germany). A0.5mL of100 μg/mL offermented milk
or standard sample (inmethanol) was mixed with linoleic
acid emulsion (2.5 mL, 0.2 mol/L, at pH 7.0) andphosphate
buffer (2 mL, 0.2 mol/LM, pH 7.0). Thereaction mixture was
incubated at 37ºC inthedark to accelerate theperoxidation
process. Thelevels ofperoxidation were determined accord-
ing to thethiocyanate method bysequentially adding etha-
nol (5 mL, 75% v/v), ammonium thiocyanate (0.1 mL, 30%
v/v), sample solution (0.1 mL), andferrous chloride (0.1 mL,
20mmol/L in3.5% HCl). After mixing for 3 min, theperoxide
values were determined byreading theabsorbance at 500nm,
using UV/Vis spectrophotometer. 6-Hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) (Sigma-Aldrich,
174 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
USA) was used as positive control. Thepercentage inhibition
ofperoxide value was calculated using thefollowing formula:
Inhibition (%) = [(1A1/A0) ×100]
where: A0: Absorbance ofcontrol; A1: Absorbance ofsample.
Diene-conjugation formation method
Theeffects offermented milk supplied with royal jelly
on theformation ofconjugated diene hydroperoxides were
determined according to themethod ofTee etal. [2002]. Lin-
oleic emulsion system was prepared as described inthepre-
vious section. Fermented milk with themost effective con-
centrations ofroyal jelly (50, 100, and200 μg/mL), BHT
(100μg/mL) and100 μg/mL ofTrolox (Sigma - Aldrich,
USA) were added to each system, respectively. Theemulsion
was homogenized for 30 sec andincubated at 40˚C.Thefor-
mation ofconjugated diene was monitored daily for seven
days bysolubilizing 0.2mL ofthelinoleic acid emulsion
in5mL ofabsolute methanol. Theabsorbance was mea-
sured at 234nm using UV/Vis spectrophotometer. Theper-
centage inhibition ofconjugated diene value was calculated
using thefollowing formula:
Inhibition (%) = [(1A1/A0) ×100]
where: A0: Absorbance ofcontrol; A1: Absorbance ofsample.
Statistical analysis
Thegeneral linear model (GLM), produced bytheSta-
tistical Analysis System (SAS) version 7 [SAS® system for
Microsoft® Windows®. 2001], was used to analyse thedata.
All thedata ofthis research were designed to apply two repli-
cates ofeach level for every experiment. Differences between
themeans oftreatments were tested using theleast signi -
cant difference (LSD) test at (p<0.05). Repeated measures
analysis ofvariance (ANOVA) was used to analyse thedata
within thesame 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 andComplete Randomized Design for reduc-
ing power andDPPH scavenging activity. Results are shown
as themean values±standard deviation.
Royal jelly isaviscous jelly substance, often not homog-
enous due to thepresence ofundissolved granules ofvarying
size. Itispartially soluble inwater with adensity of1.1 g/mL.
Its colour iswhitish to yellow, theyellow colour increasing
upon storage. Its odour issour andpungent, thetaste being
sour andsweet [Lercker, 2003].
Theamounts ofconstituents inroyal jelly determined
inthis study are reported inTable 1. Thecomposition
ofthemain constituents ofRJ, carbohydrates, proteins
andlipids isreported intheliterature [Pourtallier etal., 1990;
Lercker, 2003; Garcia-Amoedo & Almeida-Muradian, 2007].
Thedifferences observed are probably due to thedifferent
number ofsamples taken indifferent places andat differ-
ent times ofproduction. Besides to that different methods
ofsampling andanalysis were used. Moreover, royal jelly
isanaturally heterogeneous product.
Thetotal lipid fraction inthetested royal jelly islike-
wise present inreasonably modest concentration, 10.18%,
(Table1), but indeed represents thethird most important
component ofRJ after carbohydrate andproteins. Thelipid
portion infact consists primarily oforganic acids (80–90%),
most ofwhich free, with arather unusual structure rarely en-
countered innature: they are infact mono- anddihydroxy ac-
ids anddicarboxylic acids with 8 and10 carbon atoms, which
show acharacteristic arrangement [Lercker etal., 1993].
Hydroxy acids with 10 carbon atoms (10 hydroxydecenoic
and10-hydroxy-2-decenoic acid) considered themain factor
for antimicrobial activity [Fujiwara etal., 1990]. Eshraghi &
Seifollahi [2003] examined theantibacterial effect ofRJ on
Escherichia coli (ATCC 29532), Staphylococcus aureus (ATCC
14776), Streptomyces griseus (ATCC 11746), andthree dif-
ferent Streptomyces sp. (S.46) (S.F8) and(S.66). They found
that theapplication ofether soluble fraction ofRJ was more
effective than pure RJ.On theother hand, itwas found that
theinhibitory effect of30 mg/mL ether soluble fraction
ofRJ was stronger than thesame concentration ofether
non soluble fraction which has no effect even at 300mg/mL
ofRJ.Theidenti cation ofthis fraction andspecially thear-
rangement andquantitative ratios offree organic acids,
isbelieved to represent thecriteria ofchoice for recognizing
theauthenticity ofRJ andfor con rming andquantifying
theclaimed existence ofroyal jelly inother products. Besides
thefree fatty acids, thelipid fraction consist ofsome neutral
lipids, sterols (including cholesterol) andan unsaponi able
fraction ofhydrocarbons similar to beeswax extracts [Lercker
etal., 1981, 1982, 1984, 1993].
Thetotal carbohydrate content ofroyal jelly was 14.07%
(Table 1) andmostly consisted offructose andglucose. Fruc-
tose isprevalent. Inmany cases fructose andglucose together
account for 90% ofthetotal sugars. Sucrose isalways present
but often inhighly variable concentrations. Itisalso possible
to detect other oligosaccharides such as trehalose, maltose,
gentiobiose, isomaltose, raf nose, erlose, melezitose [Garcia-
-Amoedo & Almeida-Muradian, 2007]; though present invery
TABLE 1. General chemical composition ofRoyal jelly produced inJordan.
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 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 ofduplicates determinations ± standard deviation.
Z.Nabas etal. 175
small quantities they are useful for identifying acharacteristic
pattern, which iscomparable to that ofhoney andinsome
cases indicative ofthegenuineness oftheproduct [Sabatini
etal., 2009].
Thevalues ofcrude protein content inroyal jelly are
presented in(Table 1). Theobtained data are infull compli-
ance with ndings ofother authors [Krell, 1996; Wongchai &
Ratanavalachai, 2002; Nagai & Inoue, 2004; Garcia-Amoedo
& Almeida-Muradian, 2007], who reported therange from
12 to 15%.
Ingeneral, water content inroyal jelly isfairly uniform
andmakes up about two third offresh royal jelly. Table1
shows that royal jelly consists of61.5% ofwater which
agreed with therange of57–70% reported byKrell [1996]
and60–70% byGarcia-Amoedo & Almeida-Muradian
[2007]. Theconstancy ofthewater content isbasically as-
sured, inside thehive, bythecontinuous provision offresh
supplies ofthis substance bynurse bees, bythenatural hygro-
scopicity ofroyal jelly andtheentire colony’s efforts to main-
tain alevel ofambient humidity; moreover thenon-solubility
ofsome compounds can explain thevariations inmoisture
content [Sabatini etal., 2009].
Thetotal amounts ofphenolic andtotal avonoids
contents inthetested royal jelly are presented inTable 1.
Thephenolic andtotal avonoids content oftheRJ were as
follows: 23.3 μg/mg ofRJ and1.28 μg/mg ofRJ, respectively.
These quantities ofphenolic compounds were very similar
to that found byNagai & Inoue [2004] which is21.2 μg/mg
ofRJ.Thetotal phenolic contents of219.2 μg/g, were found
inroyal jelly obtained from Taiwan collected 24 h after graft-
ing, while thetotal phenolic contents ofroyal jelly collected
after 48 and72 h was 194.4 μg/g and131.7 μg/g, respectively
[Liu etal., 2008].
Liu etal. [2008] indicated that regardless ofinitial lar-
val age, RJ collected 24 h after thelarval transfer contained
higher total polyphenolic contents than theRJ collected 48 or
72 h after thetransfer.
These compounds have antimicrobial activity andexplain
theantimicrobial capacity ofroyal jelly. Moreover, ithas
been reported that phenolics are ef cient antioxidant, im-
munomodulator andanti-in amatory agents [Nagai & In-
oue, 2004]. Thevariation ofthetotal phenolic contents isat-
tributed to several factors such as climate and oral source.
Thephenolic compounds ofroyal jelly could originate from
plants where they are widely distributed in nature [Bravo,
1998; Wongchai & Ratanavalachai, 2002; Liu etal., 2008].
Themain groups ofphenolic compounds present inplants,
whether infree form or as glucosides, are derivatives ofcin-
namic acid, coumarins, and avonoids [Manthey & Grohm-
ann, 2001]. Inroyal jelly, most ofthephenolic compounds
are intheform of avonoids whose concentration depends
on various factors, including plant species used bythebees,
health oftheplant, season, environmental factors, andso on
[Kucuk etal., 2007].
ThepH value ofRJ was 3.42, which agreed with thepH
value reported byKrell [1996]. Percentage ofash inroyal jelly
sample was 1.10%, this value iscomparable to themean ash
content ofThailand royal jelly with 1.14%. On theother hand,
itwas reported that theash content offresh royal jelly reached
0.8–3.0% [Wongchai & Ratanavalachai 2002; Garcia-Amoe-
do & Almeida-Muradian, 2007].
Concentrations ofminerals detected inthis research varied,
calcium was themost abundant element compared with other
determined minerals with 64.5 ppm followed byFe (0.63 ppm)
andvery low concentration ofMn (0.03 ppm). These results
agree with those reported byStocker etal. [2005], who detected
different trace andmineral elements inroyal jelly that could
beattributed to an external factor such as environment, differ-
ent botanical andgeological origins andto some extent internal
factors such as biological factors related to thebees [Stocker
etal., 2005; Garcia-Amoedo & Almeida-Muradian, 2007].
Theproton radical scavenging has been reported to
beamajor mechanism for antioxidation. Theassay for theas-
sessment ofproton radical-scavenging activity with DPPH
isrelatively simple andenough reproducible; this compound
has been reported to bestoichiometrically decolorized byan-
tioxidants. Thereduction intheDPPH would beobserved
as its absorbance at acharacteristic wavelength isdecreased
when itistreated with proton radical scavengers. TheDPPH
isastable radical with amaximum absorption at 517nm
that can readily undergo scavenging byan antioxidant [Lu &
Foo, 2001]. Ithas been widely used to test theability ofcom-
pounds as free-radical scavengers or hydrogen donors andto
evaluate theantioxidative activity ofplant extracts andfoods
[daPorto etal., 2000].
Theantioxidant capacity offermented milk (L. acidophi-
lus supplemented with 2.5% royal jelly andB. bi dum sup-
plemented with 7.5% royal jelly) was evaluated indifferent
invitro tests. Thepercent ofDPPH radical scavenging activity
offermented milk samples containing royal jelly, with ascor-
bic acid andbutylated hydroxytoluene (BHT) as reference
samples are listed inFigures 1 and2. Thescavenging effects
offermented milk with B. bi dum andstandards on DPPH
radicals were 8.6, 17.1, 18.9 and48.5% at aconcentration of
50 μg/mL for B. bi dum, B. bi dum supplemented with 7.5%
RJ, BHT andascorbic acid, respectively (Figure 1). Asigni -
cant DPPH radical scavenging activity was observed when
fermented milk with B. bi dum supplemented with 7.5% RJ
was used at 500μg/mL with an effect of96.8% (Figure1).
Moreover, thevalue oftheantioxidant capacity offerment-
ed milk with L. acidophilus supplemented with 2.5% RJ was
12.6% at aconcentration of50 μg/mL.This value was in-
creased signi cantly to 93.2% at aconcentration of500 μg/mL
(Figure2). As presented inFigures 1 and2, thescavenging
abilities ofdifferent samples were clearly demonstrated dose-
dependent antioxidant activity against DPPH.These ndings
agree with observations indicated byother researchers [Nagai
& Inoue, 2004; Sowndhararajan & Kang, 2013; Zhang etal.,
2011]. Nagai & Inoue [2004] demonstrated that theradicals
scavenging activities ofroyal jelly samples tended to decrease
with adecreasing concentration ofthesample.
Themaximum GR percentage for fermented milk was ob-
tained at aconcentration of500 μg/mL with values of28.4,
92.0, 91.8 and80.5% for B. bi dum, B. bi dum supplemented
with 7.5% RJ, ascorbic acid andBHT, respectively. On theother
hand, themaximum GR percentage for fermented milk was ob-
tained also at aconcentration of500 μg/mL with values of15.4,
93.2, 91.8 and80.5% for L. acidophilus, L. acidophilus supple-
176 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
Ascorbic acid
DPPH Scavenging Activity (%)
Concentration ( g/mL)µ
50 100 200 300 400 500
B. bifidum + 7.5% royal jelly
B. bifidum
FIGURE 1. Scavenging activity ofdifferent concentrations ofascorbic
acid, BHT, B. bi dum supplemented with 7.5% royal jelly andB. bi dum.
Ascorbic acid
DPPH Scavenging Activity (%)
Concentration ( g/mL)µ
50 100 200 300 400 500
L. acidophilus + 2.5% royal
L. acidophilus
FIGURE 2. Scavenging activity ofdifferent concentrations ofascorbic acid,
BHT, L. acidophilus supplemented with 2.5% royal jelly andL. acidophilus.
TABLE 2. Thereduction ofDPPH (GR) ofascorbic acid, BHT, B. bi dum andB. bi dum supplemented with 7.5% RJ, L. acidophilus andL. acidophilus
supplemented with 2.5% RJ.
GR (%)
Ascorbic acid BHT B. bi dum
B. bi dum
with 7.5% RJ
L. acidophilus
L. acidophilus
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 thesame row are signi cantly different (p<0.05) according to LSD.Signi cant differences between various
treatments within each experiment are indicated bysuperscript letters (a, b, c andd). Results are mean values ofduplicates determinations ± standard
mented with 2.5% RJ, ascorbic acid andBHT, respectively
(Table 2). Also, theinvitro antioxidant activity was expressed
interms ofIC50 value, theIC50 value isde ned as theconcen-
tration ofsubstrate that causes 50% loss oftheDPPH activity
(colour). TheIC50 was determined bylinear regression equa-
tion for fermented milk with B. bi dum supplemented with
7.5% RJ andfor fermented milk with L. acidophilus supple-
mented with 2.5% RJ.TheIC50 value offermented milk with dum supplemented with 7.5% RJ reached at aconcentra-
tion of226.7μg/mL (y = 0.188x + 7.38 R² = 0.968), while
thevalue of IC50 ofL. acidophilus supplemented with 2.5% RJ
reached 210.2 μg/mL (y = 0.181x + 11.95 R² = 0.979).
Reducing power indicates theability ofenzymes (catalase,
NADH oxidase, andNADH peroxidase) or non-enzymatic
compounds (ascorbate, tocopherol, andglutathione) to re-
duce oxygen radicals or iron that then become unavailable for
oxidative reactions [Warriner & Morris, 1995; Liu etal., 2008].
Several researches have reported that thereducing power
ofacompound may beused as asigni cant indicator ofits
potential antioxidant activity [Meir etal., 1995]. Inreducing
power assay, fermented milk containing L. acidophilus supple-
mented with 2.5% royal jelly andB. bi dum supplemented
with 7.5% royal jelly reduced Fe3+ to Fe2+ byreducing oxygen
radical that isusually considered as promotive to oxidative
reactions. However, theamount ofFe2+ complex can bethen
monitored bymeasuring theformation ofPerl’s Prussian
blue at 700nm [Dorman etal., 2003; Ara & Nur, 2009]. Fer-
mented milk containing B. bi dum supplemented with 7.5%RJ
exhibited signi cantly higher reducing power than ascorbic
acid or fermented milk containing B. bi dum alone with val-
ues of1.253, 0.759 and0.330, respectively at aconcentration
of500 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 and0.441, respectively, at 1000 μg/mL as dose
dependent manner (Figure 4). Based on theresults, inorder to
get theeffect ofreducing power ofthemixture (RJ andprobi-
otic) thedose should not beless than 200 μg/mL.This could
berelated to asuf cient amount ofreductive substances, with
thecapacity to react with free radicals to stabilize andtermi-
nate theradical reactions. Wang etal. [2009] reported that
lactic acid bacteria exhibited astrong reducing power ability.
Thedevelopment oflipid peroxidation offermented milk
containing royal jelly over afermentation period of24 h
isshown inFigures 5 and6. Thetotal antioxidant capacity was
determined bythiocyanate method. This method measures
thetotal amount ofperoxide produced during theinitial stages
ofoxidation. The% inhibition ofB. bi dum supplemented with
7.5% royal jelly andL. acidophilus was 42% and52%, respec-
tively. Theprocess oflipid peroxidation isinitiated byan at-
tack on afatty acid or fatty acyl side chain byany chemical
species featuring suf cient reactivity to take ahydrogen atom
away from amethylene carbon intheside chain. Theresulting
lipid radicals then undergo molecular rearrangement, followed
byreacting with oxygen to produce peroxyl radicals, which are
Z.Nabas etal. 177
capable ofabstracting hydrogen from adjacent fatty acid side
chains andso propagating achain reaction oflipid peroxida-
tion [Halliwell & Gutteridge, 1986; Prakash & Gupta, 2009].
Lipid peroxidation isnot only associated with food deteriora-
tion andlowers thenutritional quality offood, but itexerts ad-
verse effects on human health. Liu etal. [2008] demonstrated
that royal jelly exhibited an inhibitory effect on linoleic acid
peroxidation at 27.9%. Another study byGuo etal. [2005] ex-
amined theantioxidant capacity ofroyal jelly peptides andwa-
ter-soluble protein inlinoleic acid peroxidation, where royal
jelly peptides had greater effect with 97.4% than water-soluble
royal jelly protein with 6.8% inhibition.
Thedevelopment ofdiene conjugated compounds inlin-
oleic acid system treated with 50, 100, 200 μg/mL ofboth dif-
ferent fermented milk and100 μg/mL ofTrolox andBHT are
listed inTables 3 and4. Fermented milk with B. bi dum supple-
(ass absorbance at 700 nm)
50 100 200 500 10000
Concentration ( g/mL)µ
Ascorbic acid
B. bifidum
+ 7.5 % royal jelly
B. bifidum
FIGURE 3. Reducing power ofdifferent concentrations ofascorbic acid,
B. bi dum supplemented with 7.5% royal jelly andB. bi dum.
B. bifidium
B. bifidium
+ 7.5% RJ
Absorbance at 500 nm)
Time of incubation (Day)
FIGURE 5. Antioxidant activity ofTrolox, B. bi dum andB. bi dum sup-
plemented with 7.5% royal jelly measured bythiocyanate method during
different time intervals (day).
Reducing Power
(ass absorbance at 700 nm)
50 100 200 500 10000
Concentration ( g/mL)µ
Ascorbic acid
L. acidophilus
+ 2.5 % royal jelly
L. acidophilus
FIGURE 4. Reducing power ofdifferent concentrations ofascorbic acid,
L. acidophilus supplemented with 2.5% royal jelly andL. acidophilus.
Absorbance at 500 nm
Time of incubation (Day)
1.0 Control
L. acidophilus
L. acidophilus
+ 2.5% RJ
FIGURE 6. Antioxidant activity ofTrolox, L. acidophilus andL. acidophi-
lus supplemented with 2.5% royal jelly measured bythiocyanate method
during different time intervals (day).
TABLE 3. Effect ofBHT, Trolox, B. bifdidum andB. bifdidum supplemented with 7.5% royal jelly on theformation ofdiene-conjugation (expressed as
inhibition %) using different intervals day inlinoleic acid model system.
Treatment (μg/mL) Inhibition ofdiene 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
Bi dobacterium bi 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 thesame column are signi cantly different (p<0.05) according to LSD.Signi cant differences between vari-
ous treatments within each experiment are indicated bysuperscript letters (a, b, c, d, e andf ). Results are mean values ofduplicates determinations ±
standard deviation.
178 Synbiotic Effects ofRoyal Jelly with Two Different Probiotic Strains on Antioxidant Activities
mented with 7.5% royal jelly andL. acidophilus supplemented
with 2.5% royal jelly inhibited theformation ofconjugated di-
ene inlinoleic acid system. Theinhibition effects were noticed
throughout theinduction period compared to control. Ingen-
eral, results show that as theconcentration ofboth fermented
milk with B. bi dum andL. acidophilus increased, theformation
ofoxidation products decreased andinsome concentrations
showed close absorbance values andsometimes competitive
with Trolox andBHT.Theformation oftheoxidation products
increased gradually inthecontrol system during theinduction
period andtheproducts started to decompose after thefourth
day. Thepercentage of inhibition for fermented milk with
B. bi dum were 20.3, 57.6, 65.8, 12.2, 13.3, 19.3, 67.7 and57.3
percent for 50, 100, and200μg/mL ofB. bi dum supplemented
with 7.5% royal jelly; for 50, 100, and 200 μg/mL ofB. bi dum;
100 μg/mL ofTrolox and 100μg/mL ofBHT, respectively (Ta-
ble 3). While, thepercentage ofinhibition for fermented milk
with L. acidophilus were 47.4, 60.3, 66.9, 16.4, 22.0, 24.5, 67.6
and57.3 percent for 50, 100, and200 μg/mL ofL. acidophilus
supplemented with 2.5% royal jelly; for 50, 100, and 200μg/mL
ofL. acidophilus; 100μg/mL ofTrolox and 100μg/mL ofBHT,
respectively (Table4). Tee etal. [2002] made thesame obser-
vations concerning thedecreasing ofthepercentage inhibition
ofdiene-conjugation compounds after four days ofincubation.
Royal jelly isanatural substance considered as one
ofthemost important products ofhoneybees with high nutri-
tional, functional andbiological properties. Therefore, itcould
beconcluded from theobtained results that thesynbiotic effect
ofprobiotic bacteria L. acidophilus andB. bi dum with theroyal
jelly can beused inthedairy industry. This mixture, which ispo-
tentially bene cial to enhance human health andprotect against
oxidative damage andwould beconsidered astrong competi-
tor for thesynthetic antioxidants. Synthetic antioxidants such
as butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT) andn-propyl gallate (PG) exhibit strong antioxidant
activity against several oxidation systems. However, synthetic
antioxidants pose potential risks invivo; their use infood isre-
stricted or prohibited insome countries. Antioxidants from
natural sources are likely to bemore desirable than those chemi-
cally produced because some synthetic antioxidants have been
reported to beside effects [Osuntoki & Korie, 2010].
This research was supported by the University of Jordan,
Amman, Jordan.
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TABLE 4. Effect ofBHT, Trolox, L. acidophilus andL. acidophilus supplemented with 2.5% royal jelly on theformation ofdiene-conjugation (expressed
as inhibition %) using different intervals day inlinoleic acid model system.
Treatment (μg/mL) Inhibition ofdiene conjugation (%) during different intervals (day)
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... The amount of sugar present in RJ is relatively similar to the previous report by Nabas, et al. [20], which was also within the limits proposed by Sabatini, et al. [8]. The total sugar content was mostly occupied by glucose and fructose [21]. ...
... The total protein content of RJ was estimated to be 12.5 mg/ml. The present results of phenolics content in RJ agreed with the previous studies of Nabas, et al. from A. mellifera species [20]. They reported that RJ contains 23.3±.92 µg/mg of phenolic compounds. ...
... The flavonoid content of RJ was similar to the value, ±0.09 RE µg/mg of flavonoid which was obtained by Nabas, et al. from A. mellifera species [20]. Both phenolics and flavonoid compounds present in RJ provide antioxidant activity to RJ. ...
... and strong buffering capability (ranging from 4-7). The taste and odor of RJ are slightly bitter and partially water-soluble, having a 1.1 g/mL density [143]. ...
... The lipid components are the unique feature of RJ composition. About 90% of lipids are composed of fatty acids, and the remaining components include waxes (5-6%), phenols (4-10%), steroids (3-4%), and phospholipids (0.4-0.8%) [143,149]. Several vitamins (water-soluble) have been identified in RJ composition. Vitamin B5 (52.80 mg/100 g) [150] is the chief vitamin component of the composition. ...
... The mineral elements include Ca, Cu, Fe, K, Mn, Mg, Na, and Zn. Some trace elements have also been identified, including Cd, Al, Cr, Ba, Co, Bi, Mo, Ni, Sr, Sb, Sn, Hg, V, Pb, Te, Ti, and Tl [143]. Some minor compounds have also been identified, including heterocyclic components, neopterin, and biopterin. ...
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Bee products have been extensively employed in traditional therapeutic practices to treat several diseases and microbial infections. Numerous bioactive components of bee products have exhibited several antibacterial, antifungal, antiviral, anticancer, antiprotozoal, hepatoprotective, and immunomodulatory properties. Apitherapy is a form of alternative medicine that uses the bioactive properties of bee products to prevent and/or treat different diseases. This review aims to provide an elaborated vision of the antiviral activities of bee products with recent advances in research. Since ancient times, bee products have been well known for their several medicinal properties. The antiviral and immunomodulatory effects of bee products and their bioactive components are emerging as a promising alternative therapy against several viral infections. Numerous studies have been performed, but many clinical trials should be conducted to evaluate the potential of apitherapy against pathogenic viruses. In that direction, here, we review and highlight the potential roles of bee products as apitherapeutics in combating numerous viral infections. Available studies validate the effectiveness of bee products in virus inhibition. With such significant antiviral potential, bee products and their bioactive components/extracts can be effectively employed as an alternative strategy to improve human health from individual to communal levels as well.
... El porcentaje de cenizas se encontró dentro de los parámetros establecidos por la Norma Mexicana y por la Comisión Internacional de Miel (IHC) . Los porcentajes fueron similares a los reportados en Brasil con valores que van de 0.83% (Sereia y Arnaut de Toledo, 2013) a 1.06% (Garcia-Amoedo y De Almeida-Muradian, 2007), Rumania con porcentajes de 0.95 a 1.12 , y Jordania con un total de 1.01% (Nabas, Haddadin, Haddadin y Nazer, 2014). Al no existir diferencias entre los tres tipos de jaleas producidas en el mismo ambiente y periodo de tiempo se puede asumir que el tipo de dieta no influyó directamente en la concentración de cenizas presentes en la JR. ...
... Un exceso en los valores de P puede ser un indicador de contaminación por pesticidas (Brodschneider y Crailsheim, 2010); sin embargo, se ha reportado que los suelos de 59 Yucatán poseen una concentración alta de P (Borges-Gómez, et al., 2014). La concentración de Ca en las jaleas fue mayor a lo reportado por otros autores (6.45 mg/100 g) (Nabas et al., 2014), este se encuentra en altas concentraciones en la miel, por lo que existe la posibilidad de que la miel que se utilizó para las dietas y el néctar que pecorearon las abejas poseyera altas cantidades del mineral, además de que se ha reportado que el suelo de Yucatán posee niveles altos de Ca (Borges-Gómez, et al., 2014). El valor de Fe fue similar a lo reportado en JR de China (2.36 mg/100 gr) pero es mayor a lo obtenido para JR de Jordania (0.063 mg/100 gr). ...
... La JRM fue la que presentó el valor más alto (11.02 ± 0.19 μg de ácido gálico/mL) y la JRP el valor menor (6.83 ± 0.86 μg de ácido gálico/mL). Diversos autores han medido la concentración de fenoles totales en la JR, utilizando condiciones diferentes de extracción; por ejemplo, Nabas et al. (2014), reportaron un valor de 23.3 ±0.92 μg de ácido gálico/mg para un extracto acuoso de JR y Nagai e Inoue (2004) reportaron valores de 21.2 μg/mg de polvo para el extracto acuoso y de 22.8 μg/mg para el extracto alcalino. La mayor concentración reportada por los autores se puede deber a que trabajaron con extractos acuosos y los fenoles al ser solubles en agua se pudieron concentrar en el extracto (Pugalenthi et al., 2005); además de que la presencia de los compuestos fenólicos en la JR depende de factores como el tipo de planta donde pecorearon las abejas, la estación del año y factores ambientales (Viuda-Martos, et al., 2008), por lo que los valores pueden variar de una región a otra. ...
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La Jalea Real (JR) es el alimento de las larvas de las abejas melíferas (Apis mellifera L.) y de la reina durante toda su vida, esta les proporciona una nutrición óptima para su crecimiento y su desarrollo, para que las nodrizas puedan producir JR es necesario que las glándulas hipofaríngeas se encuentren completamente desarrolladas, esto se consigue cuando las obreras durante sus primeros días de vida consumen Polen con alto contenido de proteína. El uso de suplementos proteicos es necesario dentro de los sistemas de producción; sin embargo, existe poca información sobre como influyen diferentes fuentes de proteína utilizadas en la alimentación de las abejas en la composición fisicoquímica y en la presencia de los componentes bioactivos en la JR. La fuente proteica utilizada para alimentar a las abejas que producen JR influye de manera significativa en mejorar la composición químico-proximal y las propiedades bioactivas de tipo antioxidante y antihipertensiva que tiene el alimento. El uso de M. pruriens como proteína mejora algunas características fisicoquímicas de la JR, al igual que algunas respuestas en la actividad biológica; sin embargo, la dieta de Polen también otorgó beneficios a la JR, por lo que no hay una dieta que sea mejor que otra. El incremento en la bioactividad de la JR proveniente de los tratamientos de suplementación proteica en comparación a la JRC, se puede deber a la mayor concentración de proteína, ácidos grasos y compuestos fenólicos que presentaron la JRM y JRP respectivamente. No es posible indicar de manera general si alimentar con una u otra fuente proteica es mejor, todo dependerá de la actividad biológica que se evalue; no obstante, se puede asegurar que es mejor alimentar a las colonias de abejas de manera constante con una proteína de calidad para incrementar sus componentes bioactivos y de esta forma obtener en mayor beneficio al consumo. La información generada en este trabajo puede ser utilizada para el desarrollo de dietas apícolas encaminadas a obtener una JR con mayor actividad biológica.
... RJ is a milky substance comprised mainly of water, proteins, sugars, lipids (Bărnuţiu et al., 2011;Zheng et al., 2011;Xu and Gao, 2013;Maghsoudlou et al., 2019) and other minor components, e.g. vitamins, phenols (Bărnuţiu et al., 2011;Nabas et al., 2014;Wang et al., 2016;Kazemi et al., 2019). ...
... The chemical composition of RJ has been studied by several authors (Boselli et al., 2003;Sabatini et al., 2009;Zheng et al., 2011;Balkanska et al., 2013;Nabas et al., 2014;Wang et al., 2016;Mărgăoan et al., 2017;Ab Hamid et al., 2018;Kazemi et al., 2019). For example, RJ shows an average of 60 -70% moisture, 9 -18% crude protein, 7 -18% carbohydrates, 3 -13% fructose, 4 -8% glucose, 3 -8% lipids, and 0.8 -3% ash (Sabatini et al., 2009), these are considered quality standards. ...
... For example, RJ shows an average of 60 -70% moisture, 9 -18% crude protein, 7 -18% carbohydrates, 3 -13% fructose, 4 -8% glucose, 3 -8% lipids, and 0.8 -3% ash (Sabatini et al., 2009), these are considered quality standards. The nutritive and therapeutic properties of RJ have led to its involvement in formulating supplementary sources of food for human nutrition (Sabatini et al., 2009;Bărnuţiu et al., 2011;Balkanska et al., 2014;Nabas et al., 2014;Ajitha Nath et al., 2019;Maghsoudlou et al., 2019). Most of RJ producers harvest RJ after 72 hrs of grafting (Lercker et al., 1985;Taha, 2000). ...
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Royal jelly (RJ) was harvested after 24, 48, and 72 hrs of grafting to study the effect of harvest time on the yield of RJ/queen cell. The changes in the nutritional components in RJ harvested at different times post-grafting were also studied. The highest yield of RJ/queen cell was harvested after 72 hrs of grafting, while the lowest yield was harvested after 24 hrs of grafting. The lowest moisture content was found in RJ harvested after 72 hrs of grafting. The maximum concentrations of crude protein, ash, fructose, and glucose were found in RJ harvested after 72 hrs of grafting. Conversely, the highest lipid content was obtained from RJ harvested after 24 hrs of grafting. The pH value decreased and acidity increased over time after grafting. It was concluded that RJ can be harvested after 72 hrs of grafting to obtain maximum yield. The nutritional components in RJ may be changed over time after grafting, and it could differentiate between RJ harvested at different times post-grafting.
... Our finding of the moisture content for drone brood was similar to earlier reported data [28][29][30]. Royal jelly was more acidic but exhibited lower total acidity compared to DB. Obtained data agreed with the values reported by Krell [31], Ramadan and Al-Ghamdi [8], and Nabas et al. [32]. The observed differences in pH and acidity values could be a useful tool in the detection of RJ adulteration with drone brood. ...
... The obtained DB total proteins content was consistent with the literature data [35,36]. The value found for RJ samples in this study was within the range reported by several authors [8,32,36,37]. The water-soluble proteins accounted for only 25-35% of the total protein content in the drone brood and 50% in the royal jelly. ...
Drone brood is a little-known bee product which is frequently considered as a male equivalent of royal jelly and is sometimes used as its adulterant. The aim of the study was to compare the chemical composition and biological activity of both bee products originated from the same apiaries (n = 3) limiting the influence of genetic and environmental factors. Moreover, for drone brood study covered testing three stages of larval development (days 7, 11, and 14). The comparison included mineral composition (ICP-OES method), protein content and protein profile (SDS-PAGE), testosterone and estradiol content (ELISA tests). HPTLC method was used to analyze of sugar, amino acids, and polyphenolic profile of drone brood and royal jelly. Moreover, their antioxidant and enzymatic properties were compared. A lot of similarities between drone brood and royal jelly were found in terms of chemical components. However, drone brood was more abundant in iron and manganese, reducing sugars and some amino acids, especially proline, tyrosine, and leucine. It contained more testosterone (especially on the 14th day) and estradiol (on the 7th day). The greatest differences in the enzymatic activities and polyphenolic profile were found. Diastase and α-glucosidase activity were found as specific enzymes of the drone brood. Similarly, ferulic and ellagic acids were characteristic for brood and were not present in royal jelly. The study showed a lot of similar features for both tested bee products, however, some specific markers which can serve to differentiate drone brood and royal jelly were found.
... Furthermore, there are other elements that influence this parameter of polyphenols, for instance, the chemical composition, but also the interaction between these micronutrients and proteins and beyond [58]. According to numerous studies, the total polyphenol content can be determined by the Folin-Ciocalteu colorimetric method [59][60][61][62][63]. ...
Full-text available
Royal jelly (RJ) is one of the most valued natural products and is known for its health-promoting properties. Due to its therapeutic effects, it has been used in medicine since antiquity. Nowadays, several studies indicate that RJ acts as a powerful antimicrobial agent. Indeed, researchers shed light on its antioxidant and anticancer activity. RJ’s biological properties are related to its bioactive compounds, such as proteins, peptides, phenolic, and fatty acids. The aim of this review is to highlight recent findings on RJ’s main bioactive compounds correlated with its health-promoting properties. The available literature suggests that these bioactive compounds can be used as an alternative approach in order to enhance human health. Moreover, throughout this paper, we underline the prominent antibacterial effect of RJ against several target bacterial strains. In addition, we briefly discuss other therapeutic activities, such as antioxidative and anticancer effects, of this outstanding natural product.
... Furthermore, there are other elements that influence this parameter of polyphenols, for instance, the chemical composition, but also the interaction between these micronutrients and proteins and beyond [58]. According to numerous studies, the total polyphenol content can be determined by the Folin-Ciocalteu colorimetric method [59][60][61][62][63]. ...
... The pH determined using pH meter device (Orion, thermo technology). Royal jelly diluted by adding 2 g in 10 mL of distilled water (pH 7.00), then mixed for 10 minutes according to Nabas, Haddadin [23]. ...
Full-text available
Royal jelly (RJ) consumption is expanding because of its high nutritional and bioactive content. However, there is a lack of information to standardize the limit of properties of Saudi Royal Jelly for regulation. The aim of this study is to investigate the quality of Saudi royal jelly associated with the international standard. Twelve fresh royal jelly samples were analyzed for moisture, pH value, total acidity, protein, carbohydrate composition, and 10-hydroxydec-2-enoic acid (10-HAD) concentration. The result of physicochemical properties was varied from 61.70 % to76.80 % for moisture; 3.14 to 3.83 for pH; 39.4 to 45.0 mL for free acidity; 0.1 N NaOH/100 g; 571.60 to 745.80 µS/cm for electrical conductivity (EC); 6.73% to 13.27% for crude protein content. Moreover, the 10-HDA, fructose, glucose, sucrose, maltose, and lactose content were ranged from 1.68% to 6.36%, 2.51 to 5.71%, 0.00% to 2.64%, 0.00% to 2.01% and 0.00 to 0.69%, respectively. The obtained results indicated that all samples matched the international standard and Gulf Standardization Organization (GSO ISO 12824:2021) for royal jelly requirements in terms of 10-HDA concentration, one of the most critical quality parameters. The statistical analysis showed a significant positive correlation (r=0.487, P < 0.01) between the EC and the total protein content while negative correlation between EC and sucrose content (r=-0.825, P < 0.01) and 10HDA (r= -0.699, P < 0.01); fructose and glucose content were shown to be statistically significant (r=0.887). The obtained results of present study will assist to the establishment of a standards of the Saudi royal jelly.
... They include trans-10hydroxy-2-decenoic acid, gluconic acid, and 10-hydroxydecanoic acid that are the main fatty acids in RJ from the quantitative point of view, followed by 9-hydroxydecanoic, 7and 8-hydroxyoctanoic, 9-hydroxy-2-decenoic, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, and 10-hydroxydecanoic acids; 2-decene-1,10-dioic and 2-octene-1,8-dioic acids have been also reported in RJ, together with the mono-and diesters of 10-hydroxy-2-trans-decenoic acid and hydroxyl-2-trans-decenoic acid 10-phosphate. 72,74,86−88 Finally, 24-methylenecholesterol and Δ 5 -avenasterol are the main sterols found in RJ. 80 With regard to micronutrients, the most abundant minerals in RJ include Ca, Cu, Fe, K, Mg, Mn, Na, and Zn, followed, in trace amounts, by aluminum, antimony, barium, bismuth, cadmium, cobalt, chrome, lead, thallium, and vanadium, among others, 75,87 while pantothenic acid is the most abundant vitamin present in RJ, followed in trace amounts by ascorbic acid, biotin, folic acid, niacin, pyridoxal, riboflavin, and thiamine. 70,89 RJ may also contain nucleotides, as both free bases, including adenosine, cytidine guanosine, uridine, and phosphates, comprising adenosine mono-, di-, and triphosphate. ...
Full-text available
Beside honey, honeybees (Apis mellifera L.) are able to produce many byproducts, including bee pollen, propolis, bee bread, royal jelly, and beeswax. Even if the medicinal properties of these byproducts have been recognized for thousands of years by the ancient civilizations, in the modern era, they have a limited use, essentially as nutritional supplements or health products. However, these natural products are excellent sources of bioactive compounds, macro- and micronutrients, that, in a synergistic way, confer multiple biological activities to these byproducts, such as, for example, antimicrobial, antioxidant, and anti-inflammatory properties. This work aims to update the chemical and phytochemical composition of bee pollen, propolis, bee bread, royal jelly, and beeswax and to summarize the main effects exerted by these byproducts on human health, from the anticancer and immune-modulatory activities to the antidiabetic, hypolipidemic, hypotensive, and anti-allergic properties.
Bee products such as honey, propolis, bee pollen, royal jelly, beeswax, and bee venom constitute important pharmaceutical and cosmetic components. Each bee product is characterized by the content of the active substance, which differentiates one bee product from another, and causes that each of them is worth using for a different skin problem. In addition, flavonoids and phenolic acids play a crucial role in influencing those products on the skin. For example, honey, propolis, and pollen are used to heal burn wounds. Moreover, bee venom called apitoxin contains active peptides and amines used in the wound’s healing process. Therefore, findings connected with wound dressing containing honey, propolis, or bee venom can be applied during wound healing therapy. Furthermore, the advantages of pharmaceuticals and cosmetics based on bee products are high effectiveness with minimal side effects. Therefore, bee products may become a new strategy in skin therapy.
Full-text available
A Study was conducted on the chemical composition of royal jelly produced by honeybe es (Apis mellifera) in Chiang Mai, Thailand. It was shown that seasonal variations had a moderating influence on the chemical composition of royal jelly, especially on carbohydrate and lipid contents, causing slight changes in protein and moisture contents but no alteration in ash content and pH value. The lipid mostly contained acidic polar compounds. The protein mostly contained a water-soluble fraction makeing up to 70To of the total protein, mainly having rather low molecular weights. As compared with other studies, royal jelly from various sources demonstrated similar chemical compositions.
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Eight Lactobacillus isolates obtained from five indigenous fermented foods (ogi, ogi baba, wara, kunnu and ugba) were investigated. Wara is a dairy-based food while the others are not dairy-based. The bacteria were isolated on MRS agar and purified by successive streaking on the same medium. The whey fraction of skimmed milk fermented with each isolate was assayed for radical scavenging effects using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. All the whey fractions showed radical scavenging activities. The five isolates with the highest activities were selected. On the basis of Gram stain reaction, cellular morphology, biochemical tests and carbohydrate utilization profiles they were identified as strains of Lactobacillus brevis, L. fermentum, L. plantarum, L. casei and L. delbrueckii. The antioxidant activities of whey fractions from 24-hour fermentations with the selected organisms were investigated using both radical scavenging effects and lipid peroxidation inhibitory activity. The radical scavenging activity was generally higher than the lipid peroxidation inhibition, except in the L. plantarum strain, which did not show any significant difference in both activities. The probiotic potential of the isolates was evaluated by pH and bile tolerance. None of the selected isolates showed any growth at pH=2.0 but L. casei and L. delbrueckii survived at this pH. Four of the five selected isolates were able to grow in 0.5 % dehydrated bile, with L. casei strain showing the highest level of growth, followed by L. delbrueckii. L. plantarum strain was not bile tolerant. The ability of L. casei and L. delbrueckii strains to survive at pH=2 and grow in the presence of bile indicates that the isolates may be able to colonize the gastrointestinal tract. The findings of this study indicate that Lactobacillus strains isolated from indigenous Nigerian fermented foods could be useful as starter cultures to provide antioxidants in food and that fermented milk may serve as a delivery vehicle for antioxidative, probiotic lactobacilli from non-dairy sources.
The kinetic behaviour of polyphenols common in fruits as free radical scavengers was studied using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH•). After addi‐tion of different standard concentrations to DPPH· (0·025 g litre⁻¹), the percentage of remaining DPPH• was determined at different times from the absorbances at 515 nm. The percentage remaining DPPH• against reaction time followed a multiplicative model equation: ln [DPPHREM•]=b ln t+ln a. The slopes of these equations may be useful parameters to define the antioxidant capacity. The steeper the slope, the lower the amount of antioxidant necessary to decrease by 50% the initial DPPH• concentration (EC50). This parameter, EC50, is widely used to measure antioxidant power, but it does not takes into account the reaction time. Time needed to reach the steady state to the concentration corresponding at EC50 (TEC50) was calculated, and antiradical efficiency (AE) was proposed as a new parameter to characterise the antioxidant compounds where AE=1/EC50TEC50. It was shown that AE is more discriminatory than EC50. AE values are more useful because they also take into account the reaction time. The results have shown that the order of the AE (×10⁻³) in the compounds tested was: ascorbic acid (11·44)>caffeic acid (2·75)⩾gallic acid (2·62)>tannic acid (0·57)⩾DL‐α‐tocopherol (0·52)>rutin (0·21)⩾quercetin (0·19)>ferulic acid (0·12)⩾3‐tert‐butyl‐4‐hydroxyanisole, BHA (0·10)>resveratrol (0·05). © 1998 SCI.
Quantitative differences were found in the chemical composition of royal jelly samples collected in spring and summer by the same producer. The main difference was in the free fatty acids, which showed a marked increase in 10-hydroxydecanoic acid in summer. The sterol and hydrocarbon fractions were also investigated. The most significant sterols were identified by gas chromatography—mass spectrometry. The hydrocarbon components, identified by their retention times, were a homologous series of straight-chain compounds, from C16 to C33, with even and odd numbers of carbon atoms. Considerable amounts of some hydrocarbons were found. Of the sterols identified, the most abundant was 24-methylene cholesterol; stigmasterol, β-sitosterol, Δ5-avenasterol, Δ7-avenasterol and cholesterol were also present.