Introduction

The development of novel functional foods containing probiotic is a highly growing area of the food industry and attracts special interest from the field of nutrition, due to their beneficial properties to human health¹. However, to deliver their health benefits, probiotics must be present in food products above a threshold level (>6 logcfu/g) at the time of consumption, in order to survive the passage through the upper and lower parts of the gastrointestinal (GI) tract². Lactobacillus casei ATCC 393 strain has been reported to have beneficial effects for cholesterol removal³, reduction of the risk of osteoporosis⁴, activity against cancer cell proliferation⁵, beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects⁶, survived the passage through the GI tract in a rat model and potential regulates intestinal microbiota⁷. In addition L. casei ATCC 393 is a strain with satisfying technological characteristics and therefore has numerous applications in the production of food products such as cheese⁸, sausages⁹, fermented milks^4,7,10 and yogurts^11,12.

Prebiotics are short-chain carbohydrates, which are non-digestible by humans¹³ and selectively stimulate the growth and/or activity of one or a limited number of bacteria¹⁴. More specifically they pass by the small intestine to the lower gut where they utilized by probiotic bacteria but not by other intestinal bacteria¹⁵. Most of the studies on prebiotics concern fructooligosaccharides (with plant origin such as chicory roots, garlic, onion etc), galactooligosaccharides (with origin through enzymatic conversion lactose) and inulin¹⁶. However considerable attention has been given in the so called raffinose family oligosaccharides RFOs (α-galactosides), having their origin from seeds of legumes, lentils, peas, beans, chickpeas, mallow composite, and mustard and represented by raffinose, stachyose, verbascose and other oligosaccharides¹⁵. RFOs and especially raffinose and stachyose positively affected probiotic increase (Bifodobacteria and Lactobacillus), Fe bioavailability and brush border membrane functionality in vivo¹⁷. In addition RFOs have been incorporated in fermented milks and provided a beneficial effect in L. acidophilus¹⁸, Bifidobacterium lactis Bb-12¹⁹ and a combined culture of them (1:1)²⁰. Raffinose in a synthetic medium was tested as prebiotic in several lactobacilli (among them L. casei) and in its presence the growth of Lactobacilli was statistically improved¹⁶. In a preclinical study, where the effects of raffinose and Bifidobacterium breve JCM 1192T administration was studied, the results showed that raffinose influences on the cecal proliferation of Bifodobacterium breve JCM 1192T²¹. These results support the concept of combining a probiotic strain with its preferred prebiotic substrate. Therefore the aim of the present study was to evaluate the use of raffinose as prebiotic in the production of probiotic yogurt using traditional yogurt starters and probiotic L. casei ATCC 393 cells.

Materials and Methods

Strains

Lactobacillus casei ATCC 393 (DSMZ, Braunschweig, Germany) was used as probiotic strain. The thermophilic starter, CH-1 (Chr. Hansen, Hørsholm, Denmark) consisted of Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus in a freeze-dried form was also employed in the present study. All cultures were prepared, as described in previous study¹⁰.

Yogurt manufacture

Pasteurized bovine milk (3.7 % fat) was heated at 42 ^oC and raffinose was added in several amounts (up to 2.0 % w/v). Then Lactobacillus casei ATCC 393 cells (1 g/L) were added. After 15 min, all samples were inoculated with the activated CH-1 culture (0.3% v/v). The fermentation was terminated at pH 4.7 and the samples stored at 4^oC for 28 days.

Determination of culture viability

Yogurt samples were added to ¼ Ringer’s solution and the appropriate serial dilutions were prepared. L. casei was enumerated in lithium propionate MRS (LP-MRS) agar (lithium chloride 0.2% w/v and sodium propionate 0.3% w/v) at 37^oC²².

Determination of pH and titratable acidity of yogurts

pH of yogurts was measured using a pH-meter and titratable acidity was determined by titration using 0.1 mol L^-1NaOH and phenolphthalein as an indicator and expressed as g of lactic acid per 100 g yogurt.

Syneresis of yogurts

50mL of unstirred yogurt spread evenly on a Whatman No. 1 filter paper in a funnel. After 5 h at 4^oC, the volume of liquid collected was recorded and multiplied with 2 giving % syneresis.

Sensory evaluation

20 panelists familiar with the consumption of yogurts from the Department of Food Technology, were used to evaluate the produced yogurts for color, sweet odor, smoothness, sweetness, viscosity and overall acceptability, using a 9-point hedonic scale ranging from 1 (“dislike extremely”) to 9 (“like extremely”).

Statistical Analysis

Significance was established at P < 0.05. The results were analyzed for statistical significance by ANOVA, and Tukey honest significant. Coefficients, ANOVA tables, and significance (P < 0.05) were computed using Statistica version 5.0 (StatSoft Inc., Tulsa, OK).

Results and Discussion

L. casei ATCC 393 has been proposed for the production of several dairy products due to its excellent technological properties and the health benefits related to its consumption. However, there are some additional important requirements that have to be fulfilled by each probiotic in order to be suitable for food applications. The probiotic culture should be prepared in a dry state in order to comply with the commercial needs of industry and also should be capable to survive the harsh conditions of the gastrointestinal tract of humans²³. These two requirements have been extensively studied and fulfilled by L. casei ATCC 393. More specifically L. casei ATCC 393 cells have been prepared in dried form either using freeze drying²⁴ or spray drying¹⁰ with good survival rates. In addition their capability to survive the harsh conditions of the gastrointestinal tract has been proved by several studies either in free or microencapsulated form^10,25. Therefore, in the present study, the use of prebiotics in combination with L. casei for yogurt production was studied in order to further increase the previous mentioned characteristics of L. casei cells. To the best of our knowledge, this is the first report concerning the production of yogurt using prebiotics such as reffinose with L. casei providing a complete research assay for ease industrial exploitation.

Effect of prebiotics on pH and titratable acidity of yogurts

The addition of raffinose did not affect the fermentation time for the production of probiotic yogurts (data not shown). The physicochemical characteristics of yogurts with or without raffinose during 28 days of storage are presented in Table 1. Titratable acidity was continuously increased during storage up to 1.09-1.18 g/100 g of yogurt and was significantly (P < 0.05) affected by the storage time. In contrast, no significant differences were observed between products produced with and without raffinose. In the same way, the pH values of yogurts decreased rapidly during the first week of storage but after that remained constant to values 4.05-4.10.

Effect of prebiotics on syneresis of yogurts

Whey separation (wheying-off or syneresis) is defined as the expulsion of whey from the yogurt’s protein matrix which then becomes visible as surface whey. This phenomenon adversely affects consumer perception of yogurt, as they consider that there is a microbiological problem²⁶. Yogurt manufacturers, in order to reduce or eliminate this problem, usually use stabilizers such as starch, gelatin and vegetable gum or increase the level of milk solids and especially of the protein percentage²⁷. Spontaneous syneresis, which is contraction of gel without the application of any external force, is the usual cause of whey separation and is related to an unstable network, which can be due to an increase in the rearrangements of the gel matrix or it can be induced by damage to the weak gel network (e.g., by vibration or cutting)²⁸.

Table 1: Effect of storage and raffinose content on physicochemical characteristics of probiotic yogurts with L. casei

In the present study the use of raffinose as prebiotic did not affect the syneresis of the yogurts resulting to similar values of syneresis compare to yogurts without prebiotics (Table 1). More specifically the syneresis ranged from 33% to 40% during the 28 days of storage at 4^oC. Only the use of high raffinose content 2% w/v led to significantly (P<0.05) higher values of syneresis ranging from 37% to 43%. These results are very important for the dairy industries since the addition of raffinose did not affect the textural characteristics of yogurts as proved by the syneresis values.

Effect of prebiotics on survival of L. casei ATCC 393 cells

The microbiological analysis of the produced yogurts (Figure 1) showed the viability of probiotic culture during storage. After yogurt production the viable cells of L. casei were 5.3 x 10⁸ cfu g^-1 of yogurt. After 28 days of storage at 4⁰C, in all cases the viable numbers of L. casei remained in values higher than 10⁶ cfu g^-1 that is necessary to confer the probiotic character of the products. In addition in all cases a significant (P<0.05) decreased in viable L. casei cells was observed to values ranging between 3.0 x 10⁸ and 4.0 x 10⁸ cfu g^-1 of yogurt. However in the yogurt with 1% w/v raffinose a significant (P<0.05) increase in the viable cells of L. casei was observed to 9.7 x 10⁸ cfu g^-1 of yogurt.

Figure 1: Effect of storage (28 days at 4oC) and raffinose content on viability of L. casei in probiotic yogurts   Click here to View figure

Sensory evaluation of yogurts

The results of sensory evaluation of the new products are presented in Figure 2. The results showed that, in most cases, no significant differences were observed between yogurts produced with the addition of raffinose. However, the samples with raffinose presented significantly (P < 0.05) higher values in all cases compared to the control sample (without raffinose). Sample with 1% w/v raffinose received significantly (P<0.05) higher scores in overall acceptability compared to all other samples. These findings indicated the high industrial potential of the proposed technology since sensory evaluation showed the acceptance of the new yogurts from the panelists and no defect was mentioned by them.

Figure 2: Sensory evaluation of probiotic yogurts produced using different raffinose concentrations   Click here to View figure

Conclusions

In conclusion, the use of raffinose as prebiotic in yogurt production is very promising since no defects in the production of yogurts observed. Similarly, the proposed technology resulted in significantly higher cell counts during production and refrigerated storage of yogurts over products without raffinose. Noticeably, the probiotic levels ranged above the minimum requirement for conferring a probiotic effect, especially when 1% w/v raffinose was used, and the new products were of improved sensory characteristics, while the overall high quality was ascertained by the organoleptic tests. Since probiotic culture is important to survive the passage through the upper and lower parts of the gastrointestinal (GI) tract², future studies will give more insight into the role of prebiotic to protect L. casei cells also under gastrointestinal conditions. Finally since there is evidence that prebiotics based on raffinose, RFOs, stimulate the growth of Lactobacilli and as a consequent suppress the growth of pathogens such as Escherichia coli and Salmonella enteric serovar Typhimurium²⁹, future studies may also focus on the evaluation of symbiotic effect of raffinose prebiotics and probiotic L. casei ATCC 393 cells on various pathogens in dairy products.

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