1. Introduction

Frozen yoghurt is a dessert that combines the texture of ice cream with the nutritive and healthy properties of yoghurt (Rezaei, et al., 2011). Its process consists in mixing all ingredients to make natural stirred yoghurt with stabilizers/emulsifiers and sugar, then freezing the mix in a conventional ice cream freezer (Tamine and Robinson 2007). Production of the first fermented milks dates back to 7000 BC with origins in the middle and far-east of Asia, making it one of the oldest methods of long term food preservation. A further spreading east of these traditions, by way of Russia and Eastern Europe, by the Tartars, Mongols and Huns occurred during their conquests (Vasiljevic, et al., 2008).

Frozen yoghurt popularity has increased and continues to grow; making it one of the most frequently consumed frozen desserts around the world. As the popularity of yoghurt products continues to grow, manufacturers are continuously investigating value-added ingredients to entice health-conscious consumers (Allgeyer et al., 2010).

Frozen yoghurt’s attractiveness to consumers include providing a low fat replacement for ice cream and the probiotic benefits of the live cultures present in the yoghurt (Streptococcus thermophilus and Lactobacillus bulgaricus). According to Tamine and Robinson (2007), the official standard of identity for frozen yoghurts has not been specified yet in most countries. However, some references specify that the final product should have a minimum of 0.15% titratable acidity (expressed as lactic acid), >3.25% milk-fat, a pH <5, and a minimum yoghurt content >70% (Westerbeek, 1995; Marshall et al., 2003).

According to Tamine (2002), frozen yoghurt can be classified into three categories: soft, hard, or mousse. Soft frozen yoghurt consists of a mix of 80% yoghurt base (cold) with 20% fruit syrup base and stabilizer/emulsifier; hard frozen yoghurt is a mix containing 65% yoghurt base with 35% fruit syrup plus stabilizer/emulsifier; and mousse frozen yoghurt is a mix of the yoghurt with hot mousse base mixture (skim milk, sugar and stabilizer/emulsifier). According to the International Dairy Foods Association (IDFA), a total of 1.481 billion gallons of frozen yoghurt mix were produced in 2010. In addition, frozen yoghurt production increased by 8.1% with 49.7 million gallons produced in 2010 compared with 2009 (NASS, 2012).

Marine lipids have for the last decades received increasing attention because of their beneficial health effects. Since the first evidences regarding the beneficial health effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cardiovascular diseases appeared, many studies have demonstrated that these fatty acids have a positive effect especially on myocardial infarction (Schmidt et al., 1992) and other diseases. Foods containing fish oil are very susceptible to oxidation due to the high content of PUFAs (Nielsen et al. 2007). In recent years, the number of studies describing the health-promoting benefits of omega-3 fatty acids has increased substantially. Some of the reported activities attributed to the omega-3 fatty acids include improving serum cholesterol profiles, stabilizing arrhythmias, reducing inflammation, regulating endothelial cell function, improving insulin sensitivity in patients with Type 2 diabetes, and enhancing the immune response.

Vitamin E is a fat soluble vitamin and recent research suggests that its deficiency is associated with an elevated risk of atherosclerosis and other degenerative diseases. The physiological role of vitamin E centers on its ability to react with free radicals in cell membranes and other lipid environments, thereby preventing PUFAs from being damaged by lipid oxidation (Bramley et al., 2000). Temperature, oxygen and UV light parameters during extraction, encapsulation and storage of vitamin E affect its degradation (Sabliov et al., 2009). Frozen desserts can be used to carry health-promoting constituent’s vitamins and minerals or fiber or nutraceuticals (minor constituents of foods that have been shown to have health-promoting effects).

Polyunsaturated fatty acids (PUFAs) lower the risk of heart disease and vitamin E is an important lipophilic antioxidant that protects cell membranes from oxidation. Our goal in the current research was to isolate and identify of beneficial bacterial strains with properties of probiotic bacteria from Laban Rayeb and develop a successful strategy obtains fermented milks with probiotics that has a satisfactory quality for consumers and also to delivery of ω-3 and to evaluate their effect on sensory characteristics of frozen yoghurt during storage.

2. Materials and Methods

Probiotic strains candidate: Lactobacillus acidophilus r1, Lactobacillus acidophilus r2 and Latctococcus lactis subsp lactis r3 were isolated from traditional Egyptian dairy product Laban Rayeb and used after the selection had been done according to Bergey’s Manual of Determinative Bacteriology, 9th edition (Holt et al., 1994) with confirm the identification by API 50CH system (Biomerieux, Marcy l’Etoile France). The strains were tested for their probiotic characteristic i.e. gastric acid resistance, bile salt tolerance, antibacterial activity, adhesion to human mucus according to Mahrous et al., (2010). The antimicrobial activity was determined by measuring the diameter of the inhibition zone around the wells. The bacterial isolates showing the widest zone of inhibition against Staphylococcus aureus; E.coli ATCC 25922 and Bacillus subtilis NCIB3610 were selected for further studies.

Lactobacillus strains were cultivated in MRS (de Man Rogosa Sharpe) broth (Lab M, IDG, UK) and incubated at 37°C in BBL anaerobic jar (Becton Dickinson Microbiology Systems, Sparks, MD) provided with disposable BBL gas generating pack (CO₂ system envelopes, Oxoid, Ltd., West Heidelberg, Victoria, Canada). Other strains were cultivated in Nutrient broth and incubated at 37°C. Isolates were stored at –20°C in MRS broth supplemented with 25% (v/v) glycerol. For routine analysis, the strains were subculture twice in MRS broth at 37°C for 24 h.

Experimental plain yoghurt was prepared by heating pasteurized whole milk at 72°C for 10 minutes and subsequently cooled to 43°C. It was then divided into three separated containers. Formula 1 inoculated with 2%w/w starter culture yoghurt with no probiotics, Formula 2 inoculated with 2%w/w starter culture yoghurt with 3%w/w of Lactobacillus acidophilus r2, and Formula 3 inoculated with 2%w/w starter culture yoghurt with 3%w/w of Lactobacillus acidophilus r2; with 2% ω-3 with vitamin E (after separated 2% of milk fat). The inoculated mixes were incubated at 43°C until the acidity of 0.80- 0.88 was obtained.

The frozen yoghurt mixes were prepared by combining experimental plain yoghurts (three formulas), sucrose, 11%w/w, guar gum, 0.3%w/w and the mix was standardized to a total solid content of up to 12% by addition of skim milk powder. Weighed guar gum was dissolved into hot water (60°C). Other ingredients (without plain yoghurts) were weighed and mixed in the containers. Hydrated guar gum suspension was poured into the mixes and pasteurized at 85°C for 25 seconds. The mix was divided into three separated containers and mix with the tree formulas of plain yoghurts. The final formula as Formula 1 with no probiotics, Formula 2 containing 3%w/w of Lactobacillus acidophilus r2, and Formula 3 containing 3%w/w of probiotic strain (Lactobacillus acidophilus r2); with 2% ω-3 with vitamin E (50 μg/g). All three yoghurt mixes were each transferred into the ice-cream maker. The finished products were stored at -18°C for further tests.

The frozen yoghurt samples were analyzed for total solids, fat, lactose, protein, titratable acidity (%), pH and acetaldehyde (Robinson et al., 1977).

The α-tocopherol concentration was determined using the method described by Jang and Xu (2009). Oils were extracted from frozen yoghurts using the modified Folch extraction method (AOCS, 1985). All solvents used were HPLC-grade (Fisher Scientific). A sample (0.2 g) of oil was dissolved in 2 mL of hexane in a glass test tube and vortexed. The mixture was transferred to HPLC vials and 25 μL were injected into the HPLC system for analysis (1260 HPLC Liquid Chromatography and Waters), Bondapak C18 3.9×300 mm column. The mobile phase consisted of 0.5% ethyl acetate and 0.5% acetic acid in hexane at a flow rate of 1.5 mL/ min. The fluorescence detector was set at 290 nm excitation and 330 emissions to monitor α-tocopherol. The α-tocopherol concentration was determined and expressed as μg/ g frozen yoghurt.

Str. thermophilus and Lb. bulgaricus counts and probiotic bacteria counts were enumerated in each fermented milk samples. Each sample was serially diluted to 10^-6 with Ringers’ solution. Appropriate dilutions were prepared using the following media: MRS Agar (Merck, Darmstadt/Germany) for the enumeration of Lb. bulgaricus; was incubated anaerobically at 42±2°C for 3 days (Dave and Shah, 1997); M-17 medium agar for the enumeration of Str. thermophilus was made of the following (g/L of dH₂O): phytone peptone, 5; polypeptone, 5; lactose, 5; beef extract, 5; yeast extract, 2.5; L-ascorbic acid, 0.5; sodium glycerophosphate, 19; MgSO4∗7H₂O 1M solution, 1 ml; and agar, 12. Medium was adjusted to pH 5.4, dispensed into 150-ml screw-cap bottles, sterilized (20 psi, 20 min) and stored in the dark until used; was anaerobically incubated at 37±2°C for 3 days and MRS agar containing D-sorbitol for the enumeration of Lb. acidophilus; was incubated at 37°C for 72 hours anaerobically (Dave and Shah, 1997).

Selective media for total count bacteria Plate count agar (PCA), Coliforms Violet Red Bile Glucose Agar (V. R), Enterobacter spp. Violet Red Bile Glucose Agar (V.R), Entercoccus spp. Streptococcus faecalis Medium (S.F) and yeasts and molds Potato Dextrose Agar (PDA) contained chloramphenichole, the plates incubated at 37°C for 72 hours.

Ten panelists participated in a sensory evaluation of the yoghurt on the basis of appearance, flavour, texture, and overall acceptability. The panelists rated these characteristics according to the hedonic scale where 1 corresponds with “dislike extremely” and 9 corresponds with “like extremely”. Panelists were asked to assess the sensory characteristics of the three different types of frozen yoghurt: frozen yoghurt 1 = plain (non-probiotic) yoghurt, frozen yoghurt 2 = probiotic yoghurt and frozen yoghurt 3=probiotic yoghurt with 2% ω-3 with vitamin E. All participants received a letter of information and consent document, and gave signed consent before participating in the study. Panelists were then given three samples at a time at storage temperature (4°C), a pencil, and a glass of cold water to rinse their mouths between samples.

Data are presented as the mean ± standard deviation, and n represents the number of samples from the frozen yoghurt and the control.

3. Results and Discussion

Table 1. Biochemical identification of isolates bacteria

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Three isolates were selected from Laban Rayeb were identified by morphological & biochemical tests (Gram staining and catalase production, growth at 10, 45°C for 48 h, and CO₂ production from glucose) and API 50CH system (Biomerieux, Marcy l’Etoile France). Gram positive, catalase-negative rods, which grew at 45°C and not grow at 10°C, were considered as thermophilic lactobacilli, while those that didn’t grow at 45°C and grew at 10°C were considered as mesophilic lactobacilli. The identified Lactobacillus strains were Lactobacillus acidophilus r1, Lactobacillus acidophilus r2 and Latctococcus lactis subsp lactis r3 as shown in Table 1.

A potentially successful probiotic strain is expected to have several desirable properties in order to be able to exert its beneficial effects. The selection criteria that have been considered to be relevant for any potential probiotic microorganism are: acid and bile stability; Adhesion to mucosal surfaces; antimicrobial activity and good technological properties (Ouwehand et al., 1999).

The LAB survival in low pH is very important for bearing initial stress in the stomach (Gilliland and Walker, 1989). At the application level, when LAB enter into human body, first constraint is gastric acid with very low pH level around 2- 3 (Minellia et al., 2004). Resistance of isolates for Gastric Juice (In Vitro) was determined according to the method of Pennacchia (2004). All isolates were tested for their survival in low pH level around 2- 3 and Lactobacillus acidophilus r1 was the best isolates for survival in these levels. The acid resistant LAB isolates were moreover tested for bile tolerance according to their survival in MRS broth containing 0.1, 0.2 and 0.3% bile salt and MRS broth without bile salt as a control. Therefore, all isolates were able to grow and survive at bile salt condition after six hours especially Lactobacillus acidophilus r1. The survival at bile salt condition is one of the main criteria for in vitro selection of potentially probiotic bacteria and critical points for the microorganisms. Because some of LAB are able to survive at bile salt condition.

The ability of the probiotics present in dairy products that inhibit the growth of pathogens confirms the health benefits. Our study suggests that probiotics are helpful in the protection and improvement of some pathogenic bacteria. Table 2 showed the antibacterial effect of the three isolates against some pathogenic microorganisms as Staphylococcus aureus; E. coli ATCC 25922 and Bacillus subtilis NCIB3610. The results showed that all isolates had ability to inhibit the pathogenic bacteria and Lactobacillus acidophilus r2 was the highest ability in inhibition the growth of Staphylococcus aureus; E. coli ATCC 25922 and Bacillus subtilis NCIB3610 so it was used in the production of our product. Consuming these dairy products can help human healthcare and can also protect against occurrences of diarrhea and food contaminations (Walencka, 2008).

Table 2. Antimicrobial effect of isolates bacteria

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Table 3. Adhesion of Lactobacillus acidophilus r1, Lactobacillus acidophilus r2 and Latctococcus lactis subsp lactis r3 to intestinal mucous of isolates bacteria

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Many different intestinal mucosal models have been used to assess the adhesive ability of probiotics. In this study we had focused on the adhesion to intestinal mucous which is overlying the enterocytes. For the adherence assay, the crystal violet method was selected (Vesterlund et al., 2005). Table 3 indicates that all isolates strains had the ability to adhere on intestinal mucous to various extents. The strongest in vitro adhesion was observed for strains of Lactobacillus acidophilus r2 compared to the reference probiotic strain Bifidobacterium bifidum (B). From the obtained results it is clear that there are many differences in the adhesive characteristics among strains and species in the genus Lactobacillus these results agreed with several literatures (Jonsson et al., 2004; MacKenzie et al., 2009).

From the obtained results frozen yoghurt were made by using Lactobacillus acidophilus r2 which has shown significant promise in all probiotics characteristics. The formulas of prepared frozen yoghurt were: Formula 1 inoculated with 2%w/w starter culture yoghurt with no probiotics, Formula 2 inoculated with 2%w/w starter culture yoghurt with 3%w/w of Lactobacillus acidophilus r2, and Formula 3 inoculated with 2%w/w starter culture yoghurt with 3%w/w of Lactobacillus acidophilus r2; with 2% ω-3 with vitamin E (after separated 2% of milk fat). The following analyses were tested in these formulas.

Table 4 showed that the best features of this product were the high levels of protein (8.2±0.02; 8.5±0.15 & 8.3±0.03%) in the three formulas 1; 2&3, respectively. Titratable acidity in zero time of formula 1, 2& 3 were observed to be 0.81, 0.85& 0.89%, respectively. According to ISI (1974), the maximum lactic acid acidity should be 0.8 percent. After 4 weeks there were increasing in titratable acidity as 0.89; 0.96& 0.99 in the three formulas 1; 2&3, respectively. The difference in acidity was due to addition of probiotic Lb. acidophilus r1. Titratable acidity found increased and pH decreased with time after 4 weeks mainly due to sugar fermentation and conversion of lactose to lactic acid. Similar findings were reported Tamine and Robinson, 1985; Shin et al. 1991 and Salwa et al. 2001 for yoghurt.

Table 4. Chemical analysis of frozen Yogurts

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Lactose concentrations were detected in all formulas to determine if additional organisms (Lactobacillus acidophilus r1) increased lactose hydrolysis or not. Lactose concentration was decreased after 4 weeks especially in formula 2 &3. The differences were observed in lactose concentration due to the difference in levels of acidity. Thompson and Mistry (1994) observed no significant changes in lactose concentrations in frozen yoghurt mix when stored frozen for 1 and 3 months. Gilliland and Kim found that lactose decreased from 6.26% in uninoculated yoghurt mix to 4.23% in inoculated. Our results generally agree reports Hekmat and McMahon (1992).

Acetaldehyde concentrations were increased in all formulas (1; 2&3) from zero to 4 weeks from 6.47±0.02; 6.89±0.01 &6.99±1.01 (μg/mL), respectively at zero time to 7.33±0.03; 8.85±0.02 & 8.95±0.05 (μg/mL), respectively after 4 weeks. The higher levels of acetaldehyde in formula 2 and 3 were thought to be due to the fast metabolic activity of starter bacteria with probiotic bacteria compared with formula 1. Recently, Martin et al. (2011) reported on the influence of the oxidoreduction potential value on the production of aroma compounds, as it can modify the metabolic pathways of the yogurt bacteria. Oxidative conditions contribute to the stability of the acetaldehyde during the yogurt storage, whereas reducing conditions provide the opposite effect.

Means and standard deviations of α-tocopherol content during 4 weeks of storage of frozen yoghurt are illustrated in Table 5. Initial α-tocopherol contents in the three formulas 1, 2 &3 were 18.21 ± 1.15, 20.15 ± 0.05 and 42.13 ± 1.13 μg/ g frozen yoghurt, respectively. After 4 weeks of storage, α-tocopherol contents in the three formulas1, 2 &3 were reduced to 15.40±1.15; 16.25±0.05 and 39.13±1.13 μg/ g frozen yoghurt, respectively; however α-tocopherol content in formula 3 was still greater than the other formulas. A number of factors such as oxygen, light, heat, alkali, trace minerals, and hydroperoxides can cause decomposition of vitamin E vitamers (Bramley et al., 2000). The decrease in α-tocopherol content during refrigerated storage may be primarily caused by oxygen dissolved in the yoghurt matrix, and reaction with hydroperoxides produced by initial lipid oxidation reactions. α-Tocopherol is expected to have chain-breaking antioxidant activity in yogurts as well as in human body tissues. Burton et al. (1985) showed that α-tocopherol donates its phenolic hydrogen atom to peroxyl radicals arising and in the process becomes an α- tocopheroxyl radical.

Table 5. Alpha tocopherol content of frozen Yogurts

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Table 6. Total plate count for lactic acid bacteria of frozen yoghurt samples

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The dairy products, especially frozen yoghurt are good vehicle to transfer probiotics to the human intestinal tract. Consumption of probiotic bacteria via dairy food product is an ideal way to re-establish the intestinal micro-flora balance. Survival of cultures in frozen yoghurt has great importance for the healthy properties of the product (Tamine and Robinson, 1999). Table 6 showed lactic acid bacteria counts in the three trials of the frozen yoghurt. The initial count of probiotic in Formula 2 was 1.6x10⁸ cfu/ ml and that of Formula 3 was 1.8x10⁸ cfu/ml. The counts of probiotic in the two formulae decreased gradually during storage. After four weeks, the counts of probiotic in Formula 2 and Formula 3 were 2.6x10⁷ and 3.2x10⁷cfu/ml respectively. However the bacterial contents of the two formulae were still higher than 1.0x10⁶ cfu/ml, the number regulated by Thai FDA (2008) for the probiotic products. Lopez et al. (1998) observed only a slight decline in lactic acid bacteria in three batches (pH = 4.32, 5.09, and 5.53) of commercial frozen yogurt stored at –23°C for 1 yr. Modler and Villa- Garcia (1993) reported a 2 log¹⁰ cfu/ml loss in B. longum concentrations from acidification of frozen yogurt caused by fermentation and refreezing. Holcomb et al. (1991) observed no decrease in B. longum in frozen yogurt that was frozen at –5°C for 6 h.

The overall rate of reduction for Lactobacillus bulgaricus and Streptococcus thermophilus in the frozen yoghurt was found during the storage time but this decrease was reduced in formula 2 & 3. This result was in agreement with Gilliland and Kim (1984) & Chandan and Shahani, 1992 that due to the frozen yoghurt environment is not optimum for survival of bacteria. The freezing process of the mix may cause a loss of ¹⁄₂ to 1 log cycle in viable counts. The obtained data suggested that incorporation of probiotic strain and ω-3 caused protection of LAB against freezing process. The decline in bacterial counts, as a result of freezing, is likely due to the freeze injury of cells, leading eventually to the death of cells.

The rate of decrease of count bacteria on Plate count agar (PCA) in the frozen yoghurt was also observed Table 7 especially in the occurrence of probiotic strain (formula 2,3) compared with the control (formula 1); the same observation was found in the three trials for occurrence of Coliforms on Violet Red Bile Glucose Agar (V. R); Enterobacter spp. on Violet Red Bile Glucose Agar (V.R); Entercoccus spp. Streptococcus faecalis Medium (S.F) and yeasts and molds Potato Dextrose Agar (PDA) contained chloramphenichole. According to PFA (2004) the acceptable value of coliform count in yoghurt should not be more than 10/ml. These organisms are killed during pasteurization and if they are present in the product they are the result of post pasteurization contamination. The low coliform organism indicates that there was no post processing contamination and proper care was taken during processing and this probiotic product could be making without having contamination and can be stored for extra period of time.

Table 7. Other microbial counts of frozen yoghurt samples

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Table 8. Sensory evaluation of probiotic frozen yoghurts

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Activity of yoghurt culture and probiotic bacteria causes specific changes in the chemistry of the product that affect sensory characteristics of the product. Carbonyl compounds, such as lactic and acetic acids, acetaldehyde, acetone, and diacetyl result from fermentation of lactose and proteins and contributes to sensory attributes in frozen yoghurt (Chandan et al., 2006). Consumers have shown a great interest in frozen yoghurt because of its potential as a low fat replacement for ice cream and the perceived probiotic effects of the lactic acid bacteria used in its manufacture (Hughes and Hoover, 1991). Table 8 shows mean sensory scores of overall sensory evaluation of the 3 formulae of frozen yoghurts was carried out among 10 panelists, using 9-point Hedonic Scale Method. According to acceptability of each group, there was low difference in the mean scores of aroma, texture, sourness, sweetness, melting, and the overall acceptability among the 3 formulae. The overall aroma of all three mixes was acceptable. However, the aroma of mix 1 was rated 7.85±0.2 and for formula 2 & 3 were 7.95±1.2 &8.02±2.02, respectively. On the other hand, there was difference in the texture of the three formulas (7.46±1.02; 7.75±1.2 and 7.95±0.2, respectively). Overall scores for body and texture suggested that addition of omega 3 were better in textural quality. Overall scores for mixes 2 and 3 ranged from 7.98±1.05 to 8.01±1.2, respectively, which were equivalent to “very good” on the sensory scale. No serious body and texture defects were detected by the sensory panel in all formulas but formula 3 had higher scores. The colour of the frozen yoghurt was as natural milk colour and fresh appearance it is the same observation was detected by (Ranganadham and Gupta 1987).

In conclusion, addition of probiotic bacteria with omega-3 and vitamin E did not overlap with the overall acceptance of the frozen yoghurt.

4. Conclusions

There is growing consumer interest in food products that can provide health benefits. The frozen dairy desserts might be a good source for probiotic cultures, due to their composition and low storage temperatures. Frozen storage of the product has little or no effect on culture survival, and bacterial cultures remained at levels sufficient to offer the suggested therapeutic effects. Supplementation with probiotic bacteria has little effect on flavor or compositional characteristics of frozen yogurt. However, fermentation to a lower pH (5.6) does significantly increase the acid flavor of the product.

Our study showed the potential in the development of probiotic frozen yoghurt products which provide the advantage of viable probiotic in the products with omega-3 and vitamin E. The probiotic L. acidophilus r2 in addition to the traditional yoghurt cultures grew well in the frozen yoghurt and were stable to the freezing process, and survival remained high during 4weeks of storage at -18°C. The finished products contained high levels of protein and had good flavor and body characteristics. These additions can be adapted to larger production scales.

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