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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. ...
... The major fatty acid in RJ is 10-HDA, which has not been reported in other natural products. Thus, the existence of 10-HDA can be considered a marker to differentiate RJ from other bee products, and its content can be used as a parameter for RJ quality (Nabas et al., 2014). The qualitative and quantitative analysis of 10-HDA was performed using HPLC photodiode array detection (HPLC-DAD) (Flanjak et al., 2017). ...
... The sugar content of RJ is usually determined through GC or HPLC methods (B arnuţiu et al., 2011;Nabas et al., 2014). The GC methods require various purification and derivatization steps and the use of an internal standard. ...
... The increase in chromosomal aberrations under RJ treatment was directly proportional to the increase in MI, Treatment with RJ in combination with salinity stress led to a significant reduction in chromosomal aberrations in comparison with nontreated stressed seeds (Table 2). Royal jelly was considered a scavenger of free radicals due to some of its components such as flavonoids and phenolic ingredients with strong antioxidant properties of hydroxyl radical scavenging (Nabas et al., 2014;Kocot et al., 2018). ...
... The moisture content of the fresh royal jelly and prepared RJP were measured as follows: 1 g of the sample was placed in the oven (UF55Memmert) adjusted at 100 ± 5°C for 3 h, cooled in a desiccator, and then weighed. This procedure was repeated at 1 h drying intervals until the sample weight became constant (Nabas et al., 2014). ...
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Royal jelly, due to its unique bioactive components, has special biological activities, but a great extent of its nutritional value is lost during processing and storage. Lyophilization, an effective preservation technique, can feasibly preserve the main bioactive compounds present in royal jelly. In this study, fresh royal jelly was subjected to the freeze‐drying process at a pressure and temperature of 100 Pa and − 70°C, respectively, for 40 h. The results obtained indicated that the pH, turbidity, total phenol content, and antioxidant activity of the royal jelly powder (RJP), during 3 months of storage at ambient temperature (30°C), were constant with values of 4.30, 1.634 (%A.U.), 0.617 (g/L), and 28.7 (%), respectively. Moisture content of the prepared RJP was less than 1%, while that of the fresh royal jelly was 70%. Furthermore, for the fresh royal jelly, the mentioned parameters were significantly (p < .05) decreased after 2 months of storage at freezer temperature (−20°C). GC–MS analysis indicated that the amount of 10‐hydroxy‐2‐decanoic acid (10H2DA) in RJP was 3.85 times more than that of fresh royal jelly. The obtained results also indicated that prepared RJP had a high bactericidal effect toward Escherichia coli and Staphylococcus aureus, with clear zone diameters of 12 and 15 mm, respectively. The present study provides a foundation for research on the potential application of prepared RJP and the development of dietary supplements and functional foods.
... It is known that DBL have antioxidant properties due to their polyphenol content [29]. According to the previous studies, flavonoids-phenolic compounds and 10-HDA which are important components of royal jelly, are responsible for antioxidant activity [34,35]. ...
Full-text available
Therapeutic properties of products such as propolis, pollen and bee venom have been investigated before, but studies about the biological properties of drone brood (DBL) and queen bee larvae (QBL) are very limited. In addition, there are many factors that affect the biological activity power of royal jelly (RJ). This study was carried out to evaluate the antibacterial and antioxidant activity, total phenolic (TPC) and protein content and 10-hydroxy-2-decenoic acid (10-HDA) amount of Anatolian RJ, DBL and QBL. As a result of Folin–Ciocalteu method, there were significant differences between the samples, and the highest value was obtained from the QBL. Bradford Coomassie Brilliant Blue method results showed that QBL had most abundant total protein content while RJ had the lowest amount. According to the HPLC analysis RJ showed the highest 10-HDA amounts, while DBL had the lowest. When the antioxidant activity values were evaluated together, it was understood that the antioxidant capacity of DBL and QBL is significantly higher than RJ. When the data were evaluated statistically, both differences and negative and positive correlations were obtained between the parameters. As a result of MIC experiment Mycobacterium smegmatis was the most susceptible bacterium. DBL and QBL samples did not show any antimicrobial activity against selected microorganisms. This is the first study that focuses on the biological properties of QBL. As a result, Anadolu RJ is promising candidates for the treatment of some infectious diseases, and DBL and QBL are promising candidates for the development of products that can be used as food supplements.
Royal jelly (RJ) is a bee product produced by young adult worker bees, composed of water, proteins, carbohydrates and lipids, rich in bioactive components with therapeutic properties, such as free fatty acids, mainly 10-hydroxy-trans-2-decenoic acid (10-H2DA) and 10-hydroxydecanoic acid (10-HDA), and major royal jelly proteins (MRJPs), as well as flavonoids, most flavones and flavonols, hormones, vitamins and minerals. In vitro, non-clinical and clinical studies have confirmed its vital role as an antioxidant and anti-inflammatory. This narrative review discusses the possible effects of royal jelly on preventing common complications of non-communicable diseases (NCDs), such as inflammation, oxidative stress and intestinal dysbiosis, from the viewpoint of predictive, preventive and personalised medicine (PPPM/3PM). It is concluded that RJ, predictively, can be used as a non-pharmacological therapy to prevent and mitigate complications related to NCDs, and the treatment must be personalised.
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Royal jelly is one of the most important bee products. The biological activities of royal jelly can be influenced by various factors such as geographic origin, climatic conditions, vegetation. This study was carried out to evaluate the antibacterial and antioxidant activity of royal jelly samples collected from beehives from different geographical regions including mountain, coastal and plain regions in northern Iran. Antibacterial activity of royal jelly samples against ten bacteria was determined using agar well diffusion method. The MIC and MBC of royal jelly samples were determined by the broth microdilution method. Folin-Ciocâlteu reagent and reaction with DPPH were used to determine the total phenolic content and antioxidant potential of royal jelly samples, respectively. The MIC of samples ranged from 0.78 to 12.5% and MBC - from 3.12 to 50%. Samples collected from mountain regions showed the highest antibacterial activity with MIC for Gram-positive bacteria from 0.78 to 1.56% and for Gram-negative bacteria: from 1.56 to 3.12%. The total phenolic content and DPPH radical scavenging activity in royal jelly samples of the mountain region was significantly higher than those from the two regions with other climates. The results of this study indicated that the climate of the geographic region of sampling location had an effect on the antibacterial and antioxidant activity of royal jelly which may be due to differences in plant vegetation and the origin of the flowers of bees.
Larval feeds for different castes of honey bees include exclusively royal jelly from 4–9 days of development for the queen, and for worker larvae, royal jelly and worker jelly for 4-6 and 6–9 days respectively, whereas for drone larvae, royal jelly and a blended composite mixture of honey and pollen grain for 4-6 and 6–9 days respectively. For the queen, worker, and drone larvae, larval feeds include royal jelly and worker jelly for 4-6 and 6–9 days respectively. Royal jelly is a thick, creamy substance that is produced by the hypopharyngeal and mandibular glands of worker honey bees. Its primary components include water, hydrocarbons, proteins, lipids, minerals, vitamins, and a small amount of various types of polyphenols. Because the queen eats different larvae than the worker bees, this triggers a chain reaction of biochemical reactions, which ultimately leads to a high concentration of juvenile and ecdysone hormones being released. These hormones, in turn, regulate the expression of different genes in a sequential manner. Queen larvae have a variant proteomic that promotes the healthy development of the female reproductive system, which in turn leads to profound fertility and immune protection, as well as a longer life span for the queen.
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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.