Available on-line at www.dse.usab-tm.ro/en/bjb.html
Banat′s Journal of Biotechnology
2010, I(1),
MICROBIAL BETA –CAROTENE USE AS A FOOD ADDITIVE
Alina Buţu1, Gina Fidler1, Marian Buţu 1, 2
1
National Institute of Research and Development for Biological Sciences, 0630031, Splaiul Independentei
296, Bucharest, Romania, e-mail: [email protected]
2
Faculty of Physics, University of Bucharest, 077125, Str. Atomistilor 405, Bucharest–Magurele,
Romania, e-mail: [email protected]
Abstract. The present article presents one of the possibilities to use in the food industry a microbial bio
product type of vitamin A precursor. The microbial bioproduct has a high level of β–carotene and has been
obtained from the biomass resulted from the cultivation in optimum conditions of a yeast strain. The strain
used is from Rhodotorula spp. and it has been selected from the natural environment. We studied the
possibility to enrich with β–carotene some natural syrups, by adding the microbial product in the original
recipe. The bioproduct has been used in several recipes, in different concentrations and under several forms
of presentation. We analyzed the following: the degree of bioproduct integration in the syrup, the physico–
chemical and microbiological parameters of the syrup and the stability over time.
Keywords: microbial bioproduct, carotenoid pigments, Rhodotorula spp., β–carotene
Introduction
β–carotene, which is also called
provitamin A, reaches the body through food
intake and is converted by the liver into
vitamin A as needed[BRITTON, 1995; CHARLENE, 2009;
JORDAN, 2009; CHARLWOOD, 1991]
. Vitamin A is a fat–
soluble vitamin (retinol) and contributes to the
prevention and improvement of visual
disturbance, increases body resistance against
respiratory infections and is an important
factor in maintaining healthy skin, hair, teeth
and gums. It also participates in the
biosynthesis of immunoglobulin, stimulates
the immune system, prevents and relieves
depigmentation caused by liver disease or old
age. β–carotene protects the nasal, mouth, and
throat, laryngeal, tracheal and lung mucosa is
an effective adjunct in the treatment of
respiratory infections, emphysema and
hyperthyroidism[FOOD AND NUTRITION BOARD, 2000,
HOARE, 2004, STÉPHANIE BLANQUET–DIOT, 2009, IVONNE,
2009]
.
Vitamin A accelerates the physical
development
and
increases
bone
[EVANS, 2001, GILBERT, 1996, JANET, 2010, BRIAN,
strength
2008]
. β–carotene has a role in cancer
prevention.
Prophylactic effect of β–carotene in
malignant diseases is based on the
transformation of carcinogenic metabolites
formed in the organism or of the carcinogens
coming from external environment, in some
less harmful substances more soluble and thus
easier to remove[FSA, 2006] .
β–carotene reduces the risk of
myocardial infarction and death by arrest
48
card. For the reasons mentioned above, it is
useful an intake of β–carotene from food
supplements.
The use of food additives must serve
one or more objectives and only if these
objectives cannot be achieved by other means
from economical and technological point of
view and does not pose a risk to consumers’
health. Carotenoid pigments from yeast are
biological products produced in accordance
with the legislation in use, their use as
additives in the industry is consistent with the
requirements of the objective „to improve the
conservation and stability of a food or to
improve the organoleptic properties, provided
they do not alter nature, substance or quality
of the food in order to deceive consumers”.
The use of these pigments conditioned as
microbial biological products shall, in
particular, aid the obtaining of „cleaner“
foodstuff, taking into account the fact that
there was frequently reported illness due to
long consumption of foodstuffs containing
added synthetic.
These products containing added
synthetic produce a bombardment on internal
organs of the human body, causing
destruction of the immune system, and a large
number of malignant and benign tumors.
The presence of carotenoids in
microorganisms has been reported in a large
number of species. Coloration which they
determine (various shades of yellow, orange,
red) is an important character in identifying
the species[HOUSSAINI–IRAQUI, 2001] .
From all the carotenoid–producing
Banat’s University of Agricultural Sciences and Veterinary Medicine from
Timisoara,
Department of Exact Sciences,
Contact: web: www.dse.usab-tm.ro, e-mail: [email protected]
microorganisms, the yeast strain Rhodotorula
spp. is the one that synthesizes large amounts
of beta–carotene[BUZZINI, 1999] .
Carotenoid pigments are sensible to
light, heat, oxygen, acids and alkaline bases.
Due to this sensitivity the biological material
containing carotenoids and their solutions
should be protected from the action of
light[MACÍAS–SÁNCEZ, 2005] .
By exposure to light, especially in
direct sunlight or to UV light, cis–trans
photoizomerization is made, which may lead
to their photodestruction.
The presence of carotenoids in
bacteria chromoplasts, yeast, fungi or algae
gives them protection from harmful influence
that light or oxygen can have.
Materials and methods
Materials and experimental variants
Yeasts of the genus Rhodotorula are
some of the biggest producers of beta–
carotene and are widespread in nature, being
generally responsible for the degradation of
foodstuffs. To isolate such species we used
food in various stages of degradation.
The biological material was the
culture of 4–7 days of RD yeast grown on
average 1.5–2% agarized Sabouraud. The RD
yeast strain is a strain having high potential in
producing beta–carotene, being part of
Rhodotorula species and was isolated in our
laboratory on grapes. Yeast strain was
cultivated on liquid medium (5% peptone, 3%
yeast extract, 3% malt extract and 10%
dextrose) at 20–220C, initial pH 5.0 to 5.5 and
agitation 175 –190 rmp for 48 hours. Biomass
obtained was inactivated and centrifuged 20
min. at 4000 rpm. Bioproduct microbial
deposits resulted from the spin.
Bioproduct
variants
used
in
experiments were as follows:
– B1 – deposit as such;
– B2 – deposit powder;
– B3 – dry storage at room temperature;
– B4 – dry storage at 370C.
Five variants of natural syrups were
considered:
– S1 – propolis elder flowers;
– S2 – seabuckthorn propolis;
– S3 – rosehip propolis;
– S4 – pine propolis;
– S5 – fir shoots with Echinacea and propolis;
In each of the five variants S1–S5 were
added the B1–B4 variants in different
concentrations.
Methods
For culture medium the initial pH was
measured as well as β–carotene in the final
biomass. Over cultivation the optical density
in the culture medium was monitored.
For the resulted deposit after
centrifugation of yeast biomass was
determined the dry matter and β–carotene.
Final results after adding the microbian
bioproducts
were
analyzed
from
organoleptical,
physico–chemical
and
microbiological point of view. The analysis
carried out for the microbian bioproduct were:
soluble dry matter, acid, benzoic acid, total
no. of mezofili aerobic plate, coliform
bacteria, salmonella, no. yeasts and molds,
sensory examination (appearance, color, taste,
smell), heavy metals, carotenoids;
Analysis and verification methods
used were the following:
– sample preparation – STAS 1631/1979;
– organoleptic examination – STAS 1631/
1979;
– soluble dry matter (refractometry degrees) –
STAS 5956/1971;
– acidity – STAS 5952/1971;
– benzoic acid – STAS 11488/1981;
– carotenoids – spectrophotometry method;
– heavy metals:
− lead – STAS 5955/1986;
− copper – STAS 5454/2/1977; and
− arsenic – STAS 7118/1979.
– total number of mesophyll aerobic plate
count – STAS 6349/3/1980;
– coliforms–STAS 6349/4/1980.
– Salmonella – STAS 6348/11/1983
– yeasts and molds – STAS 184/1977
– particle size – Romanian Pharmacopoeia X
− pH – potentiometry
Results and Discussion
In the first phase it was tested the
microbial bioproduct degree of integration in
natural syrups taken into account. Thus,
variant B1 of bioproduct fits very well in all
five types of syrup, mixing is complete and
there are no particles in suspension. Variant
B2 requires prior dissolution in the
concentrated solution of citric acid (one of the
syrup recipe ingredients) and 5% is the
maximum concentration of the bioproduct in
49
Available on-line at www.dse.usab-tm.ro/en/bjb.html
Banat′s Journal of Biotechnology
2010, I(1),
this variant, which can be integrated in syrup,
to have a good homogenization.
Variants B3 and B4 have a good
integration up to the concentration of 15%,
between 15% and 25% is required a prior
dissolution of the bioproduct in concentrated
solution of citric acid and over 25% there are
particles that remain in suspension and
homogenization is not complete.
After
establishing
maximum
concentration that can be used depending on
the variant taken into account, experimental
versions have been realized in order to study
the stability in time. The concentrations used
were as follows: B1 – 5%, 10%, 25%, B2 –
2%, 3%, 5%, B3 and B4 – 5%, 10%, 15%.
Concentrations were chosen based on β–
carotene intake on syrups and the bioproducts’
degree of integration in the natural syrups.
The analysis on the samples of natural
syrup with added beta–carotene monitored if
organoleptic,
physicochemical
and
microbiological properties of the samples are
within the limits of admissibility of the
technical specifications of basic products. In
all variants the witness was the natural syrup
that was not added microbial bioproduct.
Results of tests on samples from all
experimental
variants
considered
are
described below. Organoleptic properties:
− appearance: is preserved, viscous–syrupy
liquid;
− color: is changed, color is orange intensity
increases with increasing concentration of
microbial bioproduct;
– S1 – yellow–orange, slightly opalescent;
– S2 – yellow–orange, slightly opalescent;
– S3 – caramel, slightly opalescent;
– S4 – yellow–orange, slightly opalescent;
and
– S5 – yellow–orange, slightly opalescent.
– Odor: it preserves aroma; and
– Taste: is kept, it is sweet and sour.
Figure 1. Seabuckthorn syrup with propolis (S2)
with added β–carotene microbial B4 variant (from
left to right: control, with added 5%, 10%, 15%)
Physico–chemical and microbiological properties of natural syrups with added β–
carotene are presented in table 1.
Table 1.
Physicochemical and biological properties
1.
2.
3.
4.
5.
6.
7.
8.
9.
Determination
soluble dry matter (refractometric
degrees) minimum
acidity (%) minimum
benzoic acid (%) maximum
heavy metals (mg / kg) maximum
–Lead
–Copper
–Arsenic
total number of aerobic plate count/1
ml
coliforms
salmonella
yeasts and molds
pesticides / 1ml
Conditions of eligibility
Observations
62
comply in all samples analyzed
1.2
0.5
comply in all samples analyzed
comply in all samples analyzed
1.0
10.0
0.2
comply in all samples analyzed
100
comply in all samples analyzed
absent
absent
100
absent
comply in all samples analyzed
comply in all samples analyzed
comply in all samples analyzed
comply in all samples analyzed
Conclusions
Quality of food products is an
essential condition for which the producers
are directly responsible.
Obtaining of food products at top
quality parameters, and in large quantities is a
necessity that more and more producers are
aware of. Therefore, the use of microbial
products, as an alternative to synthetic
50
products, is becoming more agreeable,
because, besides their beneficial effect
through the application of provitamin A, they
lead to improved product quality, in the sense
of reducing the accumulation of toxic
substances and increasing the accumulation of
benefice substances (carbohydrates, vitamins).
The main control parameters were the
total protein and concentration of carotenoids,
Banat’s University of Agricultural Sciences and Veterinary Medicine from
Timisoara,
Department of Exact Sciences,
Contact: web: www.dse.usab-tm.ro, e-mail: [email protected]
microbial bioproduct quality indicators of
major importance which gives specific action
of pigmentation.
Complex studies performed in the
laboratory led to the establishment of the
optimal formula for integrating microbial
biopreparation with high content of β–
carotene in natural syrups. Tests performed on
the production line for obtaining the finished
product showed that function of the type of
integrated product (variant of bioproduct) we
can establish the concentration and integration
formula. B1 variant can be used in any
concentration in the final form of natural
syrup without changing technological process
of obtaining syrup; variants B2, B3, B4 are
used in concentrations up to 5% for B2 and
15% for B3 and B4 and modify the
technological
process
of
obtaining
intermediate syrup.
8.
9.
10.
11.
References
1.
2.
3.
4.
5.
6.
7.
Britton G., Structure and properties of
carotenoids in relation to function, Journal
of the Federation of American Societies for
Experimental Biology, 1995, 9, 1551–
1558.
Charlene C. Ho, Fabiana F. de Moura,
Seung–Hyun Kim, Betty J. Burri, Andrew
J. Clifford, A Minute Dose of 14C–β–
Carotene Is Absorbed and Converted to
Retinoids in Humans, J. Nutr., 2009,
139,1480 – 1486.
Jordan P. Mills, Gaston A. Tumuhimbise,
Kazi M. Jamil, Sagar K. Thakkar, Mark L.
Failla, Sherry A. Tanumihardjo, Sweet
Potato
β–Carotene
Bioefficacy
Is
Enhanced by Dietary Fat and Not Reduced
by Soluble Fiber Intake in Mongolian
Gerbils, J. Nutr.,2009,139,44–50.
Charlwood B.V., Banthorpe D.V., Methods
in plant biochemistry, Academic Press,
1991, v.7, pp. 473–519.
Food and Nutrition Board IoM. Dietary
reference intakes for vitamin C, vitamin E,
selenium, and carotenoids. Washington,
DC: National Academy Press, 2000.
Hoare J, Henderson L, Bates CJ, et al., The
national diet and nutrition survey: adults
aged. London:The Stationery Office, 2004,
pp.19-64.
Stéphanie Blanquet–Diot, Maha Soufi,
Mathieu
Rambeau,
Edmond
Rock,
Monique Alric, Digestive Stability of
12.
13.
14.
15.
16.
Xanthophylls Exceeds That of Carotenes
As Studied in a Dynamic in Vitro
Gastrointestinal System, J. Nutr., 2009,
139, 876 – 883.
Ivonne Sluijs, Joline W. J. Beulens,
Diederick E. Grobbee, Yvonne T. van der
Schouw, Dietary Carotenoid Intake Is
Associated with Lower Prevalence of
Metabolic Syndrome in Middle–Aged and
Elderly Men, J. Nutr., 2009, 139, 987 –
992.
Evans P, Halliwell B. Micronutrients:
oxidant/antioxidant status. Br, J Nutr,
2001, 85 (suppl 2), pp. 67–74.
Gilbert S. Omenn, et all, Effects of a
Combination of Beta Carotene and Vitamin
A on Lung Cancer and Cardiovascular
Disease, The New England Jourmal of
Medicine, 1996, 334, pp.1150–1155.
Janet A. Novotny, Dawn J. Harrison,
Robert Pawlosky, Vincent P. Flanagan,
Earl H. Harrison, Anne C. Kurilich, β–
Carotene Conversion to Vitamin A
Decreases As the Dietary Dose Increases
in Humans, J. Nutr., 2010, 140, 915 – 918.
Brian Buijsse, Edith J. M. Feskens,
Lemogang Kwape, Frans J. Kok, Daan
Kromhout, Both – and β–Carotene, but Not
Tocopherols and Vitamin C, Are Inversely
Related to 15–Year Cardiovascular
Mortality in Dutch Elderly Men, J. Nutr.,
2008, 138, 344 – 350.
Food Standards Agency. Eat well, be well.
London: FSA, 2006.
Houssaini–Iraqui M., Khamlichi N.,
Yamani J.E., Rastogi N., Response of
Escherichia coli containing mycobacterial
carotene genes to UV radiation, J. of
Biomedicine and Biotechnology, 2001, 1–
3, pp.79–84.
Buzzini, P., A. Martini. Production of
carotenoids by strains of Rhodotorula
glutinis cultured in raw materials of agro–
industrial origin, Biores. Technol. 1999,
71, pp.41–44.
Macías–Sáncez
M.D.,
Mantell
C.,
Rodríguez M., Martínez De La Ossa E.,
Lubián L., Montero O., Supercritical fluid
extraction of carotenoids and chlorophyll a
from Nannochloropsis gaditana, Journal of
Food Engineering, 2005, 66 (2), pp. 245–251.
Received: March 11, 2010
Accepted: May 5, 2010
51