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THE EFFECT OF HONEY, POLLEN AND INULIN ON THE PROBIOTIC COMPONENT OF A SYNBIOTIC ECOLOGIC PRODUCT
A.VAMANU* D.PELINESCU ** I.SRBU** E.VAMANU*
* University of Agronomic Sciences and Veterinary Medicine, Faculty of Biotechnology, Bd. Mrti no. 59, district 1, Bucharest, Romania **- University of Bucharest, Faculty of Biology, Splaiul Independenei no. 91-95, district 5, zip 76201, Bucharest, Romania
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Proceeding of the International Conference BIOATLAS 2010 Transilvania University of Brasov, Romania
2. Materials and Methods Microorganisms and Culture Media. Four strains of lactic bacteria were used (Lactococcus fermentum BS2, Lactobacillus plantarum BS1, Lactobacillus plantarum BS3, Lactobacillus paracasei BS6) and two strains of bifidobacteria (Bifidobacterium bifidum BS4, Bifidobacterium bifidum BS5). The six strains in the collection of the Faculty of Biotechnologies were stored in 20% glycerol at -820C. The study made use of freeze-dried biomass of these strains, obtained in a freeze dryer Alpha 1 2D. The nutritional support used to obtain the symbiotic product was ensured by 20 g of polyfloral pollen and 3 g of polyfloral honey. Two variants were made, one with unground pollen (B1), the other with ground pollen (B2). The composition was homogenized by adding 10 ml of sterilized ultrapure water. 2.5 g of freezedried biomass were used as inoculum, all the six strains being added in equal proportions. 1% inulin was also added, as prebiotic component. The product was obtained in sealed polypropylene boxes which were stored for maximum 14 days at 370C in a LabTech cooling thermostat. [1, 4] Determination of the lactic acid quantity. The lactic acid accumulation was determined by titration with HCl 0.1N. For determination, the fact that 1 ml HCl 0.1N corresponds to 0.009008 g lactic acid is taken into account. [5] Determination of the glucose quantity by using the o-toluidine method. It was performed with the o-toluidine test, made by the National Institute of Chemical-Pharmaceutical ResearchDevelopment ICCF Bucharest. [5] Microbiological analysis of pollen, honey and biomass samples. In order to determine microbiologically the pollen and honey samples supplemented with freeze-dried biomass of the six strains of lactic bacteria, 1 g of sample was homogenized in 9 ml of NaCl 0.85% isotonic solution. Of the suspensions obtained decimal serial dilutions were made and 0.1 ml was inoculated on each plate with the following media: for the selection of the strain Lactobacillus plantarum BS 1, MRS was utilized with 2% melezitose as carbon source; for the selection of the strain Lactobacillus fermentum
BS 2, MRS was utilized with 2% xylose as carbon source; for the selection of the strain Lactobacillus plantarum BS 3, MRS was utilized with 2% rhamnose as carbon source; for the selection of the strain Bifidobacterium bifidum BS4, MRS was utilized with 2% turanose as carbon source; for the selection of the strain Bifidobacterium bifidum BS 5, MRS was utilized with 2% sorbose as carbon source; for the selection of the strain Lactobacillus pracasei ssp. paracasei BS6 MRS was utilized with 2% tagatose as carbon source. From the plates with specific media colonies were replicated and examined using the API 50 CHL kit. 3. Results and Discussions The first phase of the study was aimed at determining the multiplication capacity on pollen and honey culture media utilized. The results were analyzed for the two variants of culture media, with unground pollen - B1, and with ground pollen - B2.
14 7 4 3 2 1 0 0 0,5 B2 B1
For the lactic acid synthesis (Figure 1) it can be noticed that in the first three days, B1 variant stimulates lactic acid synthesis due to the medium composed of water and honey mixed together. In this phase, pollen grains are not completely hydrated so that most of them are still intact. Together with the growth of fermentative action of bacterial strains, the homogeneity of B2 variant determines a significant increase of lactic acid synthesis, with its peak on the fourth day of fermentation. The increase and the persistence of lactic acid
Time (days)
Lactic acid (% )
1,5
2,5
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Proceeding of the International Conference BIOATLAS 2010 Transilvania University of Brasov, Romania
quantity is an important aspect for preserving the natural composition of the product, without the need to mix additives in. The consumption of reducing carbohydrates in Figure 2 shows that pollen grinding does not favor this consumption, because B2 variant has a higher initial carbohydrate content. Gradual release of a part of carbohydrates, due to the fermentative action of microorganisms, has a positive effect, in the case of B2 variant. For the microbiological analysis of samples made of a blend of ground / unground pollen, inulin and bacterial biomass of the six strains of lactic bacteria and bifidobacteria, decimal serial dilutions were made and inoculated on Petri dishes with MRS having various carbon sources. To quantify the number of microorganisms during fermentation, aseptic samples were taken each 7, 10 and 14 days.
14 7 4 3 2 1 0 0 3 6 9 12 15 B2 B1
6. At the same time, inulin addition determined increased viability, especially for two of the strains: Lactobacillus plantarum BS 3 and Lactobacillus paracasei BS 6 to the detriment of the other strains introduced. After 7 days of fermentation, within the sample based on ground pollen, 7x109cells/ml were obtained for the strain Lactobacillus plantarum BS 3, and 3x108cells/ml were quantified for Lactobacillus paracasei BS 6. For the samples based on unground pollen, it was possible to notice that the number of lactic bacteria cells was smaller: 1.6x 108cells/ ml were obtained for the strain Lactobacillus plantarum BS 3, and 2.4x107 cells/ml for Lactobacillus. paracasei BS 6.
96 colonies were replicated in liquid MRS medium with bromcresol red and with the four carbon sources for their reexamination. The indications were based on the color turning from blue to yellow following medium acidification. On turanose and sorbose media no colony was registered, indicating that the strains of Bifidobacterium bifidum BS 4 and Bifidobacterium bifidum BS5 could not be revealed following the fermentation of the product of honey and ground / unground pollen. The results confirmed the data previously obtained, according to which the medium based on pollen and honey favors the growth of four of the strains introduced in the form of bacterial biomass, namely: Lactobacillus plantarum BS 1, Lactobacillus fermentum BS 2, Lactobacillus plantarum BS 3 and Lactobacillus paracasei BS
Time (days)
Glucides (%)
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Proceeding of the International Conference BIOATLAS 2010 Transilvania University of Brasov, Romania
As it can be noticed in Figure 3 and Figure 4, for the samples composed of ground and unground pollen, respectively, the highest number of microorganisms was obtained after 7 days of fermentation. Within 14 days the viability of the strains of lactic bacteria started to decease because of the sensitivity of this type of microorganisms to accumulation of organic acids in the medium. It was noticed that within 14 days of fermentation, the pH of the samples increased to 4.2. 4. Conclusions Inulin addition to the samples based on ground / unground pollen, honey and biomass stimulated the growth of the strains Lactobacillus plantarum BS 3 and Lactobacillus paracasei BS 6, particularly. The comparative analysis of the two variants with ground and unground pollen, respectively, highlighted the fact that the use of ground pollen favors lactic bacteria growth, due to the larger contact surface with the nutritional components in pollen and honey. Acknowledgment The researches were financed through a project PNCDI II Parteneriate, Project no. 61047/2007. (http://proiectbiosin.emanuelvamanu.ro/).
References
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