Proceeding of the International Conference BIOATLAS 2010 Transilvania University of Brasov, Romania

THE EFFECT OF HONEY, POLLEN AND INULIN ON THE PROBIOTIC COMPONENT OF A SYNBIOTIC ECOLOGIC PRODUCT
A.VAMANU* D.PELINESCU ** I.SRBU** E.VAMANU*

Abstract: This study presents the multiplication of Lactobacillus fermentum BS2,

Lactobacillus plantarum BS1, Lactobacillus paracasei BS6, Lactobacillus plantarum BS3, Bifidobacterium bifidum BS4 and Bifidobacterium bifidum BS5 probiotic strains on a natural and ecological medium containing pollen and honey that is supplemented with inulin. At each 20 g polyflower pollen, 3 g polyflower honey, 10 ml sterile distilled water and 1% inulin were added, the necessary quantities for the obtaining of a homogenous medium. Two variants of culture media were used, one containing ground pollen and the other, unground pollen. The inoculation was made with 2,5 g freeze-dried biomass of Lactobacillus fermentum BS2, Lactobacillus plantarum BS1, Lactobacillus paracasei BS6, Lactobacillus plantarum BS3, Bifidobacterium bifidum BS4 and Bifidobacterium bifidum BS5, in equal parts. The researches were performed in tightly closed recipients and samples were taken every 24 hours. The following parameters were determined: viability, total glucides consumption and lactic acid production. The conclusion is that pollen grinding has a direct effect on the viability and lactic acid production, represented by the increasing of their values compared to the culture medium containing unground pollen. The supplementation with inulin of the samples containing ground/unground pollen, honey and biomass stimulated especially the multiplication of lactic bacteria.

Keywords: Lactobacillus, Bifidobacterium, freeze-dried, biomass

1. Introduction Traditional biotechnological processes are used in recent decades to obtain natural foods. Functional products are obtained in relatively small quantities. If speaking about natural biotechnological products, the quantity, the appearance and the price highly depend on the source of raw material. Another important aspect is the equipment used in their manufacturing, the production method and the final appearance of the product. In the main, traditional products make use of convenient techniques able to determine a natural appearance and aroma. An example of such product is the one obtained from pollen, honey and probiotic strains of lactic bacteria and bifidobacteria. [1, 2] The study is aimed at obtaining a product similar to the bee bread in beehives which proves to be a powerful nutraceutical synbiotic food. Thus, the formation of the product we intend to obtain is relatively similar to that of bee bread formation. Pollen is mixed with honey by the bees and, under the influence of microorganisms of anaerobic lactic bacteria, of humidity and a1 temperature of about 35C, it becomes bee bread. In its formation, the fermentative action of microorganisms in bee products is highly important. The output, called bee bread, has high nutritional value. Bee bread is rich in vitamins, minerals, amino acids and simple carbohydrates easy to assimilate. It also has a high antioxidant activity. It is much better tolerated by human body as compared with traditional pollen. It can be stored for long periods due to the lactic acid contained. The only clear restraint refers to the people suffering from diabetes because of the carbohydrate content easy to assimilate. Likewise, people allergic to bee products should consume it in moderation. [3]

* 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

Figure 1. The accumulation of lactic acid on B1 and B2 media

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.

Figure 2. Glucides consumption on B1 and B2 media

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 (%)

Figure 3. The number of microorganisms on B1 medium

Figure 4. The number of microorganisms on B2 medium

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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/).

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Popa, O., Ni, S., Bbeanu, N.: The obtaining by biotechnological methods of an ecological product based on pollen, honey and probiotic biomass of lactic bacteria, Journal of EcoAgriTourism, 4(1 2), 2008, p. 280 284.

2. Vamanu, E., Vamanu, A., Popa, O.,

Cmpeanu, Gh., Albulescu, M., Drugulescu, M.: Biotechnological researches concerning the multiplication of a Lactobacillus plantarum strain on media with pollen for the obtaining of a probiotic product, Roum. Biotechnol. Letters, Bucharest, 11(2), 2006, p. 2627 2635. 3. Vamanu, E., Vamanu, A., Popa, O., Bbeanu, N.: Obtaining of symbiotic products from apicultural products and probiotic biomass of Bifidobacterium bifidum, Scientific bulletin Series F XIII Biotechnology, 2006, p.24 31. 4. Vamanu, A., Vamanu, E., Popa, O., Drugulescu, M., Cmpeanu, Gh.: Identification of a lactic bacterium strain used for obtaining a pollen based probiotic product, Turkish Journal of Biology, 30(2), 2006, p. 75-80.

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