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15
Scientific Research of the Union of Scientists in Bulgaria - Plovdiv,
series B. Natural Sciences and Humanities, Vol. XVII, ISSN 1311-9192, International Conference of Young Scientists, 11 - 13 June 2015, Plovdiv
THE EFFECTS OF PREBIOTIC ACACIA FIBREGUM INCLUSION ON SOME TECHNOLOGICAL PROPERTIES OF PROBIOTIC NATURAL - SET YOGURT
Nikola Popovski1, Dona Trombeva2, Vesna Karapetkovska Hristova3,
Meri Ilijoska4 Mentor: Prof. D-r Stevce Presilski
1,3,4 Faculty of Biotechnical Sciences, University of St. Kliment Ohridski, Partizanska bb. 7000 Bitola, Macedonia 2Clinical Hospital, Dr. Trifun Panovski, 7000 Bitola, Macedonia e-mail(1): [email protected]
Abstract
The manufacturing process and storage of probiotic natural - set yogurt enriched with prebiotic, represent a key factor for fulfilling of the therapeutic and protective effects of the probiotic bacteria. The aim of the present study was to establish the survival rate of probiotic bacteria in storage and to point out the dynamic of the total number of the bacteria Lactobacillus acidophilus, during the storage period (0, 1, 7, 14, 21, 28 days) at 4 0C. In our research the prebiotics Fibregum B (V1) and Fibregum P (V2), have been used. The sample of the natural - set yogurt without prebiotic, served as the control (K). During the first week in storage the average number of living cells of L. acidophilus was higher in the natural- set yogurt with prebiotics 100.00 - 80.33 x 10'°cfu/ml compared with the control variant (p<0.05). In general, the viability of lactobacilli at set yogurt with Fibregum P addition (variant V2), was greatest, especially during the first week of storage (106.00 - 85.00 x 10'°cfu/ml) respectively.
Key words: Acacia Fibregum, Lactobacillus acidophilus, natural - set yogurt, dynamic, technological properties
Introduction
In recent years, the implementation of new technologies for manufacturing fermented milk products with probiotic properties which poses a higher nutritional value than raw milk, has been one of the important trends in health maintenance (Kojic M., 2000)[1].
The fermentation of the milk is performed by various starter cultures that contain fermenting bacteria and probiotic bacteria and the products that are gained are listed in the list of functional foods (Wallowski, 1999) [2]. The viability of the probiotic bacteria "in vitro" and "in vivo" through the gastrointestinal tract can be improved by adding prebiotics (inulin, Fructooligosaccharides, Acacia gum, Fibregum and others) which act symbiotically (Shah, 2004) [3]. The prebiotics in their presence have an important effect on the producing of the lactic acid which inhibits the growth of the pathogenic microorganisms by lowering the pH value (Campbell et all., 1997) [4]. The lactic and acetic acids act synergistic in the inhibition of the yeasts (Moon N., 1983) [5], and Salmonella species (Rubin H. E., 1987) [6]. Also, symbiotic activity of Lactobacillus casei and the prebiotics FOS and maltodextrin, have been conducted, by reduction of the cholesterol in the
bloodstream of humans (Liongand Shah, 2005) [7].
The Fibregum is a natural, purified exudate of the tree Acacia. It contains more than 80% vegetable soluble fibres. It is composed of cellulose, hemicelluloses, oligosaccharides, pectin, waxes and lignin (Trowell, Burkitt 1986) [8]. It can be soluble or insoluble and it appears in two forms Fibregum B and Fibregum P. They are different only in that Fibregum P has left oriented activity. It is used in the food industry in the producing of bread, biscuits in the form of soluble fibres in the milk (dairy) products, and it shows no sandy texture, has no smell and no specific taste (Strect C. A. and Anderson M.D.W. 1993) [9]. Included prebiotic Fibregum, during the technological process of natural - set yogurt production, it shows the following characteristics: Better water retention capacity (lower syneresis), low viscosity, high stability on heat, pH and yeast fermentation, high stability in storage, high solubility at room temperature, low hygroscopic traits, it has a neutral taste, smell and colour (Reshetnik, E. I., E. A. Utochkina) [10].
The aim of our research was to establish the length of survival of the probiotic bacteria during storage as well as to determine in which set - yogurt sample with prebiotic inclusion will be shown the maximal viability of the probiotic culture during storage. The development of the technology of this kind probiotic yogurt represents the basis for developing new technologies of products with similar manufacturing technology.
Materials and methods
1. Preparation of probiotic natural set - yogurt with prebiotic Fibregum inclusion
The whole experiment cycle consists of a number of interrelated steps. Collected bulk cow's milk was standardized on fat content of 1 ± 0, 1 % and was homogenized at temperatures of 65°C. Into the homogenized milk with continuous mixing, a quantity of 1% of milk powder was added. After this process, the milk was pasteurized with a heat treatment of 92°C and was inoculated with a starter culture F-DVS ABT 10, (Probio-Tec-chr. Hansen, Copenhagen, Denmark) composed of Streptococcus thermophilus, Lactobacillus acidophilus h Bifidobacterium bifidus at temperature of 43°C. Later, in two variants of milk V1 and V2, prebiotics Fibregum B and Fibregum P (C.N.I. - Rovencedex.- France) were added at the rate of 1.5% (w/v) of each of the prebiotics in order to increase the growth and viability of the previously mentioned cultures. The sample of the natural -set yogurt without prebiotics, served as the control (K). Thus, enriched milk samples with prebiotics were packed in plastic tins for sales and placed in a thermostatic chamber (Instrumentaría Sutjeska, Serbia) at 43 ± 1° C until terminal coagulation. Coagulated samples after 4 hours were placed in the refrigerator at 4-6°C. All three variants V1, V2 and K, were kept in storage for 28 days and during this period the number of viable cells in the experimental variants was measured.
2. Microbiological analysis
2.1. Assessment of the growth of total bacterial cultures in experimental samples
The research was conducted on MRS agar with maltose, (Liofil chems.r.l. Bacteriology product) and prepared on Petri plates which were later sterilized in autoclave on which the starter culture F-DVS ABT 10, (Probio-Tec-chr. Hansen, Denmark) were seeded. At the end the development of the already grown colonies was read with a colony reader (pbi international F4). These measurements were conducted by the method "Hardy Diagnostics Lactobacilli MRS Agar" that was developed by the researchers (deMan Rogosa and Sharpe, 1960) [11].
The monitoring of the growth consists of diluting of 1 ml of the set yogurts and its serial diluting. The seeded plates were incubated in a thermostat at 37°C for 72 hours. The nutrition medium MPC was prepared by dissolving of 38.85 g medium in 1000 ml distillated water, and then it's sterilization was performed in an autoclave (Instrumentaría Sutjeska) for 15 minutes at 121°C. The pH of the nutritional medium after the sterilization should be 6.9 ± 0.1. After the incubation was finished, the number of the grown colonies was determined and they were counted with the Colony reader (pbi internacional F4). Thereafter, 1 ml of the diluted solution was planted on nutritional medium (MRS agar with maltose- Liofilchems.r.l. - Bacteriology product) in Petri 90
plates. Relates to the total number of bacteria starting from the moment of inoculation with the starter culture (day zero), then day one, day seven, day twenty one and day twenty eight we observed constant increase of the number of cells.
2.2. Enumeration of the total number of Lb. acidophilus in experimental samples
Previously a serial diluting was conducted, and then the diluted solution was planted in a Petri plate with nutritional medium MRS agar with maltose. This medium represents a selective medium for bacteria L.acidophilus, which suppresses the growth of S.thermophillus and B.bifidum for easily counting the number of colonies of the probiotic culture L .acidophilus. The Petri plates were placed in a thermostat (Instrumentaría Sutjeska, Serbia) for 72 hours at 37 °C. The nutritional medium was prepared on the same way as it was previously for the assessment of total number of bacteria, plus 20% of maltose solution which was sterilized with filtration through a 0.22 ^m filter. After the incubation was finished on the Petri plate one can see clear white circle formations of Lb.acidophilus. All microbiological analyses were carried out in triplicate.
Results and discussion
Days in storage
Variant Repetitions 0 1 7 14 21 28
x106 x10'° x10'° x106 x106 x10s
cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml
I 13 58 44 55 41 32
"K" II 19 82 60 58 23 18
III 11 70 54 42 35 20
X 14.33 70.00 52.67 51.67 33.00 23.33
Table 1. Dynamic of the total number of L.acidophilus in control variant "K"
Cow's milk proved a good substrate for the survival of L. acidophilus. A range of oligosaccharides was estimated by using various methods to determine their prebiotic effect (Kalpan and Hutkins 2000) [12]. Assessment of the growth of the bacteria Lb. acidophilus was conducted on two set yogurts with prebiotics "V1" and "V2", as well as to the control variant "K" (Table 1, 2 and 3).
Starting from the moment of the inoculation with the starter culture (F-DVS ABT 10- Probio-Tec.) marked as day zero, the growth of the bacteria Lb.acidophilus was constantly increasing. Lb.acidophilus was present even on day twenty eight of storage.
Variant Repetitions Days in storage
0 1 7 14 21 28
x106 cfu/ml x10'° cfu/ml x10'° cfu/ml x106 cfu/ml x106 cfu/ml x10s cfu/ml
"V1" I 43 72 58 66 40 34
II 39 108 87 63 52 31
III 67 102 85 70 65 49
X 49.67 94.00 76.67 66.33 52.33 38.00
Table 2. Dynamic of the total number of L.acidophilus in variant "V1"
During the first week in storage the average number of living cells of L. acidophilus was higher in the natural- set yogurt with prebiotics 100.00 - 80.33 x 10'°cfu/ml compared with the control variant (p<0.05). Thus, the influence of the prebiotic on the growth of the bacteria has been proved. In Table 4 average values of total number of Lb.acidophilus in all three variants has been shown. After the inoculation the number of living cells of L. acidophilus increased, and during the time of storage it reduced. Comparing the average values of variants V1 and V2, set yogurt with Fibregum P inclusion (variant V2), proved better growth and viability of the probiotic bacteria, especially in the first week of storage 106.00 - 85.00 x 10'°cfu/ml (p<0.05). Such obtained results were also found in better agreement with the other workers in dairy science [3,12,13,14,15].
Variant Repetitions Days in storage
0 1 7 14 21 28
x10° cfu/ml x10'" cfii/ml x10'" cfu/ml x10° cfu/ml x10° cfu/ml x10= cfu/ml
"V2" I 49 82 69 73 55 42
II 59 126 96 79 66 49
III 71 110 90 81 72 55
X 59.67 106.00 85.00 77.67 64.33 48.67
Table 3. Dynamic of the total number of L.acidophilus in variant "V2"
Variants Days in storage
0 1 7 14 21 28
x106 cfu/ml x10'° cfu/ml x10'° cfu/ml x106 cfu/ml x106 cfu/ml x10s cfu/ml
"K" 14.33 70.00 52.67 51.67 33.00 23.33
X nn.im
"V1" 49.67a 94.00a 76.67a 66.33a 52.33a 38.00a
"V2" 59.67b 106.00b 85.00b 77.67b 64.33b 48.67b
X 54.67 100.00 80.83 72.00 58.33 43.33
Table 4. Average value of the total number of L.acidophilus in variants "V1" and "V2"
abValues followed by a letter are significantly different (P < 0.05) from the control.
Conclusion
The obtained results suggest that prebiotic Fibregum has a stimulating effect on Lactobacillus acidophilus growth and development. It was proved that its inclusion makes it possible to obtain a product with a high number of viable cells of the probiotic microflora in the final product. From our research, we can conclude that the storage faze with all series is finished by the fourth hour, and then we see an increase in the biomass. Then, from the two species of prebiotics, Fibregum P proved better stimulating effect on probiotic growth and function.
Acknowledgment
The authors are highly thankful to the dairy industry authority for allowing to use the valuable facilities from the dairy lab as well as to the management team of the Clinical Hospital, Dr. Trifun Panovski, Bitola, for providing the opportunity to do a part of the experimental work.
References
[1] Kojic M. i sor. (2000); Savremeni trendovi u Mlekarstvu (ZbornikRadova). Beograd 115120
[2]Wallowski I., Rechkemmer, G. et al., (1999); Protective role of probiotics and prebiotics in colon cancer. American Journal of Clinical Nutrition, 73, 451-455
[3] Shah, N. P. "Probiotics and prebiotics. AgroFood Industry Hi-Tech 15.1 (2004): 13-16.
[4] Campbell, J.M.; Fahey, G.C.; Wolf, B.W. Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. J. Nutr. 1997, 127, 130-136.
[5] Moon, N. J. (1983), Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures. Journal of Applied Bacteriology, 43: 215-230.
[6] Rubin H. E. (1987) Toxicological model for a two-acid system. Appiled and Environmental Microbiology, 36 (4), 623-624.
[7]Liong,M. T., and N. P. Shah. "Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. Journal of dairy science 88.1 (2005): 55-66.
[8] Trowell, H. and Burkitt, D. "Physiological role of dietary fiber: a ten-year review. Bol. Asoc. Med. P. R. 1986; 78: 541-544
[10]. RESHETNIK E. I. & UTOCHKINA E. A. (2013). HEALTHY FOOD PRODUCTS WITH PROBIOTIC AND PREBIOTIC PROPERTIES. Foods and Raw materials
[11] De Man, J.C., Rogosa, M., Sharpe, M. Elisabeth. 1960. A Medium for the Cultivation of Lactobacilli. J. Appl. Bact.; 23:130-135.
[12] Kaplan H, Hutkins R. 2000. Fermentation of fructooligosaccharides by lactic acid bacteria and bifidobacteria. Appl Environ Microbiol 66 (6): 2682-4.
[13] Gibson GR, Ottaway PB, Rastall RA. 2000. Prebiotics. Oxford, U.K.: Chandos Publishing Ltd. p 1-20.
[14] Roberfroid MB. (2001). Prebiotics and probiotics: Are they functional foods? Am J Clin Nutr 71(suppl.):1682S-7S.
[15] Trombeva Dona et al., (2010), Impact of prebiotic Eqacia HV on the stability of Bifidobacterium Bifidus in fermented cow's milk, Medicus, Vol. XIV (2).
