Ann Microbiol (2012) 62:939–947 DOI 10.1007/s13213-011-0331-5

ORIGINAL ARTICLE

Assessment of and sensory properties of dahi and yoghurt prepared using bulk freeze-dried cultures in buffalo

Sistla Venkata Naga Vijayendra & Ramesh Chander Gupta

Received: 3 January 2011 /Accepted: 20 July 2011 /Published online: 4 August 2011 # Springer-Verlag and the University of Milan 2011

Abstract are part of the diet in sensory scores of dahi and yoghurt was observed with the many parts of the world and are consumed on a regular supplementation of probiotic cultures. Thus, the present basis. Enhancing the nutritional and therapeutic properties study indicated the probiotic potential of dahi and yoghurt of traditional fermented can improve the health and prepared using selected probiotic strains. physiology of consumers. In the present study, dahi and yoghurt were prepared using buffalo milk. Bulk freeze-dried Keywords Antibacterial activity. Body weight . cultures of dahi and yoghurt, either with or without Bifidobacterium . Acidophilus . Hypocholesterolaemic . supplementation of IandLactoba- Sensory quality. Wister rat cillus acidophilus R, were used to prepare these products. Probiotic properties such as antimicrobial activity in vitro, effect on body weight and serum cholesterol level Introduction in albino rats, and sensory properties of the products were evaluated. Among the products tested, probiotic Fermented foods play an important role in the human yoghurt showed very strong inhibition (>16 mm diameter) diet.Dahi, which has an appearance similar to that of against all the food-borne pathogens tested. Supplementation yoghurt, is a popular fermented dairy product in the Indian of probiotic cultures helped to improve the body weight of subcontinent. It is prepared by fermenting milk from cows, albino rats (Wister) after 30 days of feeding, with a weight buffalos or goats with mesophilic lactic cultures, and its gain of 123.33 and 129.33 g with probiotic and method of preparation and physico-chemical characteristics probiotic dahi, respectively. The serum cholesterol level of arewelldocumented(RatiRaoetal.2006). Dahi is easy to these rats decreased gradually, with a significant reduction digest and has been found to reduce the risk of cardiovas- of 2.63, 4.1 and 4.68 mg/100 ml at the end of 30 days cular problems and cancerous tumors, besides strengthening in rats fed with yoghurt, probiotic dahi and probiotic general immunity (Sinha and Sinha 2000). Yoghurt, a yoghurt, respectively, indicating the hypocholesterolaemic western counterpart of dahi, besides its nutritive value, is effect of the probiotic cultures. No significant difference in believed to be effective in both the prevention and treatment of various illnesses, viz., gastrointestinal disorders, heart diseases and tumor development, in man as well as : S. V. N. Vijayendra R. C. Gupta animals (Deeth and Tamime 1981). Several health Dairy Microbiology Division, National Dairy Research Institute, benefits of dahi and yoghurt have been reported (Arvind Indian Council of Agricultural Research, et al. 2009;Palaetal.2011; Yadav et al. 2008). Excellent Karnal 132001, India reviews have been published revealing the importance of Present Address: nutritional and health benefits (antimicrobial activity, S. V. N. Vijayendra (*) improving lactose metabolism, reduction of serum cholesterol, Food Microbiology Department, increase in body weight, stimulation of immune system, Central Food Technological Research Institute, Mysore 570020, India antimutagenic effects, reestablishing the normal flora in e-mail: [email protected] small intestine, anticarcinogenic properties, etc.) of 940 Ann Microbiol (2012) 62:939–947 probiotic cultures such as Bifidobacterium spp. and lactis ssp. lactis biovar. diacetylacis DRC 10 in 1:1:1 ratio), acidophilus or dairy products containing yoghurt (designated as Y, containing Streptococcus salivarius these cultures and on the technological characteristics of ssp. thermophilus HST and L. delbrueckii ssp. bulgaricus these organisms (Bhadoria and Mahapatra 2011; Gilliland RTS in 1:1 ratio), L. acidophilus R (designated as A), B. 1989;OoiandLiong2010; Ouwehand et al. 2011;Sarkar bifidum I (designated as B) and pathogenic cultures such as 2008;Shah2007;Soccoletal.2010; Stenger et al. 2011). Escherichia coli K-12, Salmonella typhimurium A-KIGM To enhance the therapeutic values and dietary qualities of and Shigella dysenteriae KIGM were collected from the fermented milk products, the use of suitable strains of B. Culture Collection Center of the National Dairy Research bifidum and L. acidophilus, which have greater potential to Institute (NDRI), Karnal, India. synthesize antibiotic-like substances, and essential amino acids, has been advocated (Kim 1988). These micro- Microbiological analysis organisms have GRAS (generally regarded as safe) status, besides having acid- and bile-tolerance and the ability to The viable count of lactococci and yoghurt cultures was adhere to intestinal cells (Dunne et al. 2001). also estimated by the serial dilution method using yeast help to establish normal intestinal flora. Strains of bifidobac- dextrose agar and tryptone glucose vegetable agar, teria and L. acidophilus have been used along with the respectively, and the plates were incubated aerobically mesophilic lactic starters to prepare progurt and acidophilus at 30°C and 37°C for D and Y combinations, products (Kurmann and Rasic 1988). Preparation respectively, for 48 h. To determine the viable count of a fermented milk drink using lactic mesophilic of L. acidophilus and bifidobacteria from DAB and YAB (Lactococcus lactis, Lactococcus cremoris, Lactococcus lactis combinations, appropriate serial dilutions of the sample ssp. diacetylactis)alongwithL. acidophilus and bifidobacteria were spread onto LA agar (Klupsch 1984) and incubated (in a 1:3 ratio) has been reported (Klupsch 1989). anaerobically at 37°C for 48 h. On LA agar plates, Probiotic efficacy may not be the same for all probiotic bifidobacteria appear as elevated, chocolate-brown- strains, and considerable strain-to-strain variation in colored colonies, and L. acidophilus as flat, grayish properties within bacterial species is reported (Crittenden et colonies larger in size than those of bifidobacteria. The al. 2005). The probiotic properties of several bifidobacteria total viable count in all samples was determined by plating and lactobacilli have been evaluated in vitro (Alhaj et al. onto deMann Rogosa Sharpe agar, and incubating at 30°C 2010; Ali et al. 2011; Borpuzari et al. 2007; Delgado et al. and 37°C for dahi and yoghurt samples, respectively. 2008; Jiang et al. 2010; Mohammadi et al. 2011;Nagpalet al. 2010), and the safety of probiotic cultures has been Preparation of dahi and yoghurt evaluated in vivo using rats or mice (Chouraqui et al. 2008; Gaudana et al. 2010;RajpalandKansal2009). The For the preparation of dahi, 1 L each of standardized importance of sensory analysis of probiotic foods has been buffalo milk (4% fat) was placed in two individual flasks reviewed recently (Cruz et al. 2010). Very few researchers and heated at 90°C for 15 min. After cooling to room have used buffalo milk for the preparation of dahi and temperature, 1 L milk was inoculated with 0.01% (w/v) yoghurt, either with or without supplementation of probiotic BFD dahi culture combination D and incubated at 30°C, cultures (Ranjan et al. 2006; Rajpal and Kansal 2009). and another 1 L with 0.01% (w/v) BFD dahi culture Therefore, the present work was carried out to assess the combination DAB and incubated at 37°C until probiotic and sensory properties of dahi and yogurt prepared formation (14 h). The set dahi samples were stored at 5°C using buffalo milk and bulk freeze-dried (BFD) cultures, until use. For the preparation of yoghurt, the solids content either with or without B. bifidum and L. acidophilus.Thisis of standardized buffalo milk (4% fat) was increased to 13% the first report on evaluation of probiotic and sensory by adding spray-dried skim milk powder and ground cane properties of dahi and yoghurt prepared with buffalo milk sugar (8% w/v). The milk was heated at 90°C for 15 min using both L. acidophilus and B. bifidum. and, after cooling to 37°C, each lot of 1 L was inoculated either with 0.01% (w/v) of BFD yoghurt culture combina- tion Y or 0.01% (w/v) BFD yoghurt culture combination Materials and methods YAB and incubated at 37°C until the milk had set (12 h). The set yoghurt samples were stored at 5°C until use. Bacterial cultures Evaluation of antimicrobial activity of dahi and yoghurt The bacterial cultures used for the preparation of dahi (designated as D, containing Lactococcus lactis ssp. lactis The antibacterial activity of the dahi and yoghurt prepared C10,Lactococcus lactis ssp. cremoris C1,Lactococcus with or without probiotic cultures was evaluated by agar Ann Microbiol (2012) 62:939–947 941 well assay using E. coli K-12, Salmonella typhimurium A- or without probiotic cultures were evaluated by subjecting KIGM and Shigella dysenteriae KIGM as test organisms. it to sensory evaluation by a panel of experts (n=5) using a Sterile brain heart infusion (BHI) agar (Himedia Labs, 5 point hedonic scale. With a maximum of 20 points, the Mumbai, India) plates were prepared and overlaid with products were categorized as poor (score <10), fair (10–14), 0.5 ml active cell suspension having 50% transmittance at good (14–16), very good (16–18) or excellent (18–20). 430 nm (Spectrocol, CL: 23, Elico, India) of the test cultures individually and spread uniformly. Taking aseptic precautions, Statistical analysis five wells of 6 mm in diameter were made on each plate using a sterile glass borer. Dahi and yoghurt samples were All the results obtained were subjected to statistical analysis centrifuged at 3,000 rpm for 20 min and the clear cell-free using analysis of variance method (Senedcor and Cochran supernatant (CFS) was separated and filtered through a Seitz 1967). The results are presented as means of replicates with filter and kept at 5°C until use. To prepare the control, lactic standard deviation. acid was added to the sterilized buffalo milk (4% fat) to reach 2% acidity, then centrifuged for 20 min at 3,000 rpm; the pH of the supernatant was adjusted to the pH of the CFS of Results and discussion respective dahi and yoghurt samples. CFS (100 μl) of all four products and the control were dispensed into different wells Viability of probiotic cultures and allowed to diffuse for 1 h. All the plates were incubated at 37°C for 24 h, and the zone of inhibition was measured Probiotics are described as ‘live micro-organisms which using Vernier calipers and expressed in millimeters. when administered in adequate numbers confer a health benefit on the host’ (FAO/WHO 2002). To have the Effect of dahi and yoghurt on body weight gain and serum therapeutic benefit, there is a need to maintain a minimum cholesterol in rats number of 106–107 colony forming units (CFU) of viable cells of probiotic cultures in the products until the time of Dahi and yoghurt samples prepared with or without consumption (Klupsch 1989; Boylston et al. 2004). The probiotic cultures (B. bifidum-I and L. acidophilus-R), were United States Food and Drug Administration (US FDA) has evaluated for their influence on weight gain and serum also recommended that the minimum probiotic count in a cholesterol level in male albino rats. To determine the effect probiotic food should be at least 106 CFU/ml (Bhadoria and on body weight gain and serum cholesterol content, 36 male Mahapatra 2011). As indicated in Table 1, the viable count albino (Wistar) rats weighing between 110 and 160 g were of probiotic cultures in probiotic dahi and probiotic yoghurt collected from the stock colony of the small animal house of immediately after their production (zero hour) was well NDRI and housed individually in anodized aluminum cages above the minimum prescribed level. Similarly, Shah et al. with free access to water. These 36 rats were divided into 6 (2010) have indicated a viable count of 7.41 log CFU/ml, groups receiving different diet: control (only rat chow); BM and 6.44 log CFU/ml of B. lactis and L. paracasei in (standardized buffalo milk containing 4% fat); dahi (25 g fermented fruit juices after 6 weeks of storage. plain dahi, without probiotic cultures); probiotic dahi (25 g probiotic dahi,i.e.,dahi prepared with probiotic cultures); Antibacterial activity of dahi and yoghurt yoghurt (25 g plain yoghurt without probiotic cultures) and probiotic yoghurt (25 g probiotic yoghurt, i.e., yoghurt The importance of antibacterial activity of cultured prepared along with probiotic cultures). In addition to the milks, and cultures containing dairy products, in human freshly prepared products, all the rats were provided with rat health has been reviewed (Gilliland 1989). The antibac- chow ad libitum. Control and experimental rats were terial activity of LAB has been ascribed to production of weighed on day zero and at weekly intervals for 30 days. (Gilliland and Speck 1969)and Blood samples were collected on day zero and after every bacteriocins (Klaenhammer 1988;Halamietal.2011). 15 days. The serum was separated from the clotted blood Production of bacteriocins like acidophilin or acidocin samples and the cholesterol content was estimated (Zlatkis et (Abo-Amer 2010;Sahanietal.1977; Maqsood et al. al. 1953). The cholesterol content of various products fed to 2008; Zamfir and Grosu-Tudor 2009; Mkrtchyana et al. albino rats was also analyzed. 2010) and bifidin (Anand et al. 1984;Cheikhyoussefetal. 2010)byL. acidophilus and Bifidobacterium, respectively, Sensory evaluation of dahi and yoghurt has been reported. The antagonistic activity of probiotic lactobacilli and bifidobacteria against various entero and Sensory properties, such as appearance, body and texture, uropathogens like E. coli, Salmonella and Shigella has and flavor of dahi and yoghurt samples prepared either with been studied in vitro (Hütt et al. 2006). 942 Ann Microbiol (2012) 62:939–947

Table 1 Viable count of bulk freeze dried (BFD) cultures, dahi and yoghurt prepared using BFD cultures

Product Culture combinationa Total viable count in BFDC (log CFU/g) Viable count (log CFU/g) in the products TA (%)b

Lactics L. acidophilus R B. bifidum I

Dahi D 11.38 8.24 - - 0.96 Probiotic dahi DAB 11.04 7.21 7.81 7.44 1.22 Yoghurt Y 10.90 8.31 - - 1.38 Probiotic yoghurt YAB 10.94 8.10 7.59 7.09 1.66 a D: Lactococcus lactis ssp. lactis C 10, Lactococcus lactis ssp. cremoris C1,Lactococcus lactis ssp. lactis biovar. diacetylacis DRC 10 (in 1:1:1 ratio); DAB: Culture of D along with L. acidophilus R and B. bifidum I; Y: S. salivarius ssp. thermophilus -HST and L. delbrueckii ssp. bulgaricus RTS (in 1:1 ratio); YAB: Cultures of Y along with L. acidophilus R, B. bifidum I b Titratable acidity percent (expressed as )

The antibacterial activity of dahi and yoghurt, prepared with inhibition zones greater (>16 mm) than that of plain either with or without probiotic cultures, was tested against yoghurt made using culture combination Y (12.5– common food-borne pathogens in vitro and the results are 14.0 mm). Yoghurt samples with or without probiotic presented in Table 2. All the products showed antibacterial cultures exhibited higher inhibitory activity compared to activity, to a variable degree, against all three test that of dahi samples, which can be attributed to the organisms. From the results, it is evident that dahi prepared production of antimicrobial compounds (Dave et al. with culture combination DAB had more antibacterial 1992). Earlier, Singh et al. (1979) proved the antibacterial activity than dahi made with culture combination D. activity of pure and mixed strains of S. thermophilus and L. Inhibition was highest against E. coli. However, Ratnakar bulgaricus against E. coli, P. fragi and Micrococcus flavus. (1989) reported weak inhibitory activity of market dahi However, in vitro studies indicated that L. bulgaricus and S. samples against E. coli, Bacillus subtilis and Staphylococ- thermophilus do not produce any antibiosis against E. coli cus aureus, whereas no inhibition was observed against (Bianchi-Salvadori et al. 1989). Acidophilus yoghurt, Salmonella typhi; dahi samples prepared using Lactococcus containing L. acidophilus and S. thermophilus, suppressed lactis ssp. lactis C10 had a weak antibacterial activity Staphylococcus aureus more effectively than standard against Bacillus subtilis and Salmonella typhi. Prior to this, yoghurt (Attaie et al. 1987). B. bifidum was found to Reddy (1980) reported the antibacterial activity of Strepto- exhibit maximum antagonistic action against several path- coccus lactis ssp. diacetylactis strains DRC1 and DRC2 ogenic microorganisms, such as Staphylococcus aureus, against Staphylococcus aureus S6 and Pseudomonas fragi. Bacillus cereus, E. coli, P. fluorescens, Salmonella typhosa, Similarly, pre-feeding with probiotic dahi containing L. Shigella dysenteriae in decreasing order (Anand et al. acidophilus and L. casei ameliorated Salmonella enteritidis 1984). The enhanced antibacterial activity of dahi and infection by stimulating specific and non-specific immune yoghurt containing B. bifidum and L. acidophilus may be response, and lowered colonization of the gastrointestinal due to the production of bacteriocins. Isolation of L. tract as well as leading to translocation of Salmonella acidophilus strains with antimicrobial activity against enteritidis and a modulated immune response in experi- enteropathogenic E. coli, from market dahi samples was mental mice (Jain et al. 2008, 2009). reported (Borpuzari et al. 2007). Mobarez et al. (2008) have Yoghurt prepared with culture combination YAB indicated that the antimicrobial activity of L. acidophilus exhibited very strong inhibition against all the test cultures, isolates against Gram positive bacteria like Bacillus cereus

Table 2 Antibacterial activity of dahi and yoghurt prepared using Product Inhibition zone (mm) BFD cultures. Results are the average of five observations ± Escherichia coli K12 Salmonella typhimurium Shigella dysenteriae standard deviation (SD). Means A-KIGM KIGM without a common letter within ab bc bc type of products or among Control (lactic acid) 10.50±0.3 10.00±0.1 10.00±0.2 different pathogens are signifi- Dahi 12.00±0.5 ac 10.34±0.4 bd 11.08±0.3 ce cantly different (P<0.05) Probiotic dahi 16.00±0.2 ad 14.12±0.3 be 14.20±0.3 be Yoghurt 13.50±0.1 ae 12.50±0.2 bf 14.00±0.5 cg Probiotic yoghurt 18.50±0.4 af 16.10±0.3 bg 17.60±0.6 ch Ann Microbiol (2012) 62:939–947 943 and Staphylococcous aureus and Gram negative bacteria like P. aeruginosa and Klebsiella pneumoniae may be due to the production of bacteriocin-like substance. Recently, Nagpal et al. (2010) reported antibacterial activity of several lactobacilli against Bacillus cereus, E. coli, Staphy- lococcus aureus and Salmonella spp. When compared to the control (2% lactic acid), the greater inhibitory activity of dahi and yoghurt observed in the present study indicates the presence of inhibitory substances other than lactic acid. Conversely, in a study by Makras and de Vuyst (2006), bifidobacteria were found to exert strong inhibitory activity towards Gram-negative indicator bacteria, namely Salmo- nella enterica serovar Typhimurium SL1344 and E. coli C1845k and the inhibitory mechanism was shown to be dependent on the lowering of the pH of the medium by the production of organic acids, in particular acetic acid and lactic acid. Although this strain produced a bacteriocin, the contribution of such an inhibitory substance to the antibacterial activity towards Gram-negative pathogenic Fig. 1 Effect of dahi and yoghurt on body weight gain of male albino bacteria was found to be negligible. However, Cheikhyous- rats. C Control group fed with rat chow, BM group fed with raw sef et al. (2008) noticed enhancement of antagonistic action buffalo milk, Dahi group fed with dahi, PD group fed with probiotic towards Salmonella ssp. in the combination of mixed dahi, Yoghurt group fed with yoghurt, PY group fed with probiotic yogurt culture with bifidobacteria than with yoghurt yoghurt cultures alone, and indicated that organic acids are not the sole inhibitory factors present in several bifidobacteria. with dahi or yoghurt. Yoghurt prepared with culture Production of a bacteriocin-like inhibitory substance combination Y had more growth-promoting activity, with (BLIS) with inhibitory activity against a wide range of a mean body weight gain of 119.83 g in rats, than did dahi Gram positive and Gram negative bacteria by B. infantis prepared with culture combination D, with a mean body BCRC 14602 was reported (Cheikhyoussef et al. 2009). weight gain of 107.16 g. However, the effect was reversed BLIS showed high temperature stability up to 121°C for in rats fed with products containing probiotic culture 15 min with no loss of activity and had pH stability in the combinations of DAB and YAB, with body weight gains range of 4–10, making it useful for applications in food of 129.33 and 123.33 g, respectively. processing technologies and food safety. Recently, Chen et al. It is interesting to note that the addition of B. bifidum (2010) reported that human lactoferrin expressed by L. casei and L. acidophilus helped to improve body weight gain in can elevate antibacterial activity in the gastrointestinal both groups (dahi and yoghurt) and this effect was tract of mice. significantly greater with dahi than with yoghurt samples. The data obtained in this experiment revealed that the Effect of dahi and yoghurt on body weight and serum differences observed in body weight gain among rats fed cholesterol in Wister rats with various diets were significant. From the data, it can be inferred that fermented milk products clearly have a Experiments were performed to evaluate the effect of role in improving body weight; similar observation in rats feeding of various cultured dairy products such as dahi, were recorded earlier (McDonough et al. 1985;Hitchinset probiotic dahi, yoghurt and probiotic yoghurt on body al. 1983). However, the rats given milk grew slightly weight gain and serum cholesterol level in male albino faster than those given dahi or acidophilus milk, and there (Wister) rats and the results are presented in Fig. 1 and was no difference in growth rates of rats given acidophilus Table 3, respectively. milk or dahi (Chawla 1982). The differing results were During the 1st week, the rats fed with rat chow (control attributed to the specific strains of L. acidophilus used, the group) grew slightly faster than those fed with cultured host animal employed, the number of bacteria fed, etc. products. However, after 4 weeks the control group had McDonough et al. (1985) opined that the stimulation of lowest weight gain (84 g) as compared to the weight gain of growth by feeding yoghurt might be due to the increasing rats fed with other diets, which was in the range of 90– absorption and utilization of one or more minerals. 130 g. The rats fed with standardized buffalo milk had According to Mantere-Alhonen (1995), the efficiency of lower weight gain (99.66 g) when compared to rats fed probiotics like and propionic acid 944 Ann Microbiol (2012) 62:939–947

Table 3 Effect of feeding dahi and yoghurt prepared using Major diet fed and cultures used Serum cholesterol level (mg/dl) BFD cultures on the serum cholesterol level of male albino Period (days) rats of feeding dahi and yoghurt prepared using BFD cultures. 01530 Results are the average of six observations ±SD. Means Group A without a common letter Rat chow 59.55±1.1 aa 61.85±1.3 be 62.06±1.1 ch within a group are significantly Buffalo milk 58.92±0.9 ab 65.18±1.2 bf 72.54±1.5 ci different (P<0.01) Dahi 60.28±1.2 ac 63.18±1.0 bg 65.41±1.2 cj Group B Probiotic dahi 60.56±1.1 aa 58.46±0.9 bc 56.55±1.0 ch Yoghurt 58.38±0.8 ab 56.98±1.0 bf 55.75±0.9 ci Probiotic yoghurt 59.21±0.7 ac 57.75±0.6 bg 54.53±0.8 cj bacteria might be due to the occurrence of useful 100 ml serum at the end of the experiment, whereas an metabolites (lactic and propionic acids), improved feed increase of only 2.4 mg cholesterol per 100 ml was efficiency, increased resistance to disease, the elimina- observed in the control group fed exclusively with rat tion of unfavorable drug effects, and/or the inhibition of chow. The rise in serum cholesterol is due mainly to the the growth of harmful microorganisms in the intestinal milk fat present in the diets. The rats fed with yoghurt tract. A significant increase in weight gain (31.7%) in (prepared with culture combination Y), dahi and probiotic Swiss mice fed with L. acidophilus MA27/6R compared yoghurt containing bifidobacteria and L. acidophilus to the control group was noted (Bernardeau et al. 2002). registered a steady and gradual decrease in serum Similarly, appreciable body weight gain in albino mice cholesterol until the end of the experimental period. fed with L. acidophilus E1 was reported (Borpuzari et al. The decrease was in the order of 2.63, 4.1 and 4.68 mg/ 2007).Feedingofalbinoratswithprobiotics,particu- 100 ml in rats fed with yoghurt (with culture combination Y), larly B. breve and L. paracasei, along with yoghurt probiotic dahi (with culture combination DAB) and probiotic significantly improved body weight gain over the control yoghurt (with culture combination YAB), respectively. It is (Ali et al. 2011). In contrast, a weight-gain suppressive interesting to note that feeding dahi-containing therapeutic effect in diet-induced overweight rats by L. gasseri cultures decreased serum cholesterol level significantly more BNR17 isolated from human was reported than feeding dahi prepared with culture combination D. A (Kang et al. 2009). These latter authors noted a signifi- similar pattern was observed with yoghurt prepared with cantly lower percent increase in body weight and fat pad culture combination Y or YAB. mass in the BNR17 fed group. Kansal and Chawla (1984) reported a reduction in A striking correlation exists between dietary saturated cholesterol level from 58 mg to 39 mg/100 ml plasma in fat or cholesterol intake and serum cholesterol level. rats fed with dahi for a period of 90 days. The increased High serum cholesterol is a major factor of coronary serum cholesterol level in rats fed with supplemented butter heart disease. Nair and Mann (1977) described that the oil was lowered by the addition of yoghurt (Kaul 1984). effective agent present in yoghurt may be inhibiting the Homma (1988) reported lower serum cholesterol levels in hydroxymethyl glutaryl CoA reductase, the regulatory rats fed orally with bifidobacteria. In contrast, Kim (1988) enzyme in cholesterol synthesis. In vivo and in vitro ascribed the hypocholesterolemic effect to lactobacilli cholesterol removal by L. acidophilus and bifidobacteria rather than to bifidobacteria. A hypocholesterolaemic wasreported(MinTze2006). Very recently, Ooi and effect, along with an inverse relationship between faecal Liong (2010) reviewed the cholesterol-lowering effects of excretions of bile acids and the level of total cholesterol probiotics and prebiotics under in vivo and in vitro in blood plasma, in rats fed on a cholesterol-enriched conditions. However, little information is available on diet with buffalo milk yoghurt and soy-yoghurt containing B. the effective dosage of probiotics and prebiotics needed to longum Bb-46 has been reported (Abd El-Gawad et al. exert hypocholesterolemic effects. 2005). Total plasma cholesterol was suppressed to the extent In the present study, experimental animals became of 22 to 28% in hyperlipidaemic rats fed lyophilized hypercholesterolemic and their serum cholesterol level rose probiotic dahi containing L. acidophilus for 30 days (Vibha from 58 to 72 mg/dl on feeding standardized buffalo milk Sinha and Hariom 2007). An increase in cholesterol content for 30 days. The serum cholesterol level in rats fed with in diet-induced hypercholesterolemic rats fed on probiotic dahi went up to 65 mg from the initial level of 60 mg per dahi containing L. acidophilus and B. bifidum of only 39% Ann Microbiol (2012) 62:939–947 945 was noted, compared to 62% and 68% with dahi and be due to its high acidity, which might have masked the buffalo milk, respectively (Rajpal and Kansal 2009). In a original flavour of the dahi,andtovariationinthe recent in vitro study, a reduction of up to 48% in individual tastes of the judges. cholesterol level by B. animalis subsp. lactis Bb12 was The differences in sensory scores of yoghurt prepared reported (Alhaj et al. 2010). Consumption of probiotic with culture combination Y and YAB were found to be yoghurt containing L. acidophilus and B. lactis significantly insignificant at the 1% level, which indicated that decreased serum total cholesterol in comparison with addition of L. acidophilus and B. bifidum did not change ordinary yoghurt in mild-to-moderately hypercholesterolemic the sensory properties of the yoghurt. Similarly, supplemen- human subjects (Ataie-Jafari et al. 2009). Albino rats fed with tation of ice cream with bifidobacteria was found to have little yoghurt incorporated with probiotics significantly lowered effect on its flavor, texture or other sensory characteristics cholesterol concentration before pregnancy and the reduced (Mohammadi et al. 2011). These observations are in cholesterol concentration continued to be significant after accordance with the findings of Klupsch (1989). However, parturition (Ali et al. 2011). probiotic buffalo curd (dahi) supplemented with B. longum NCTC11818 had significantly higher (P<0.05) Sensory evaluation of dahi and yoghurt sensory scores for properties such as taste and mouth-feel, resulting in a significantly higher (P<0.001) overall Sensory analysis represents a critical step at various acceptability (Jayamanne and Adams 2004). This may be stages during food product development (Cruz et al. due to the variation in number and type of strains or 2010). Very few reports are available on the sensory storage conditions. quality of dahi and yoghurt supplemented with L. acido- philus and or bifidobacteria. On the basis of organoleptic evaluation, probiotic dahi prepared with a freeze-dried Conclusions culture combination of DAB was rated as very good, with an overall score of 17.36 (Table 4), whereas dahi samples The dahi and yoghurt supplemented with therapeutic prepared with BFD culture combination of D were cultures exhibited higher inhibitory activity against com- considered excellent with a score of 18.18. Yoghurt mon food-borne pathogens when compared to the activity samples prepared with culture combinations of Y and of the products prepared using conventional cultures. The YAB were rated as excellent. Analysis of variance rat feeding experiments revealed that products containing pertaining to the sensory evaluation of dahi samples therapeutic cultures performed well in improving body prepared with BFD dahi culture combination of D weight and were able to reduce serum cholesterol levels revealed that the sensory score was significantly higher significantly. No significant differences in the sensory compared to the score of dahi samples prepared with quality of the products prepared with or without probiotic culture combination of DAB at the 1% level. The reason cultures were observed. This indicates the scope of using for low score (8.02) for flavor of dahi prepared with these cultures in the preparation of commonly consumed culture combination of DAB compared to the score of fermented foods like dahi and yoghurt to increase the dahi prepared with a culture combination of D (9.62) may probiotic quality of these products.

Table 4 Sensory evaluation of dahi and yoghurt prepared using Product Attribute Sensory score Overall score BFD cultures. Results are the average of five observations ± Allotted Obtained SD. Means without a common letter within same type of Dahi Appearance 5 0.73 18.18±0.7 a product (dahi or yoghurt) are Body and texture 5 4.83 significantly different (P<0.01) Flavor 10 9.62 Probiotic dahi Appearance 5 4.61 17.36±0.5 b Body and texture 5 4.73 Flavor 10 8.02 Yoghurt Appearance 5 4.82 19.29±1.0 c Body and texture 5 4.64 Flavor 10 9.82 Probiotic yoghurt Appearance 5 4.84 19.30±0.8 c Body and texture 5 4.66 Flavor 10 9.80 946 Ann Microbiol (2012) 62:939–947

Acknowledgment S.V.N.V. acknowledges the fellowship received Chen HL, Lai YW, Chen CS, Chu TW, Lin W, Yen CC, Lin MF, Tu from NDRI, Karnal to carry out this study. The help received from Dr. MY, Chen CM (2010) Probiotic expressing K. Jayaraj Rao, Sr. Scientist, Dairy Technology division, NDRI, for human lactoferrin elevates antibacterial activity in the gastroin- statistical analyses of the data is acknowledged. testinal tract. Biometals 23:543–554 Chouraqui JP, Grathwohl D, Labaune JM, Hascoet JM, Montgolfier I, Leclaire M, Giarre M, Steenhout P (2008) Assessment of the References safety, tolerance, and protective effect against diarrhea of infant formulas containing mixtures of probiotics or probiotics and prebiotics in a randomized controlled trial. Am J Clinical Nutr Abd El-Gawad IA, El-Sayed EM, Hafez SA, El-Zeini HM, Saleh FA 87:1365–1373 (2005) The hypocholesterolaemic effect of milk yoghurt and Crittenden R, Bird AR, Gopal P, Henriksson A, Lee YK, Playne MJ soy-yoghurt containing bifidobacteria in rats fed on a (2005) Probiotic research in Australia, New Zealand and the cholesterol-enriched diet. Int Dairy J 15:37–44 Asia-Pacific region. Curr Pharm Des 11:37–53 Abo-Amer AE (2011) Optimization of bacteriocin production by Cruz AG, Cadena RS, Walter EHM, Mortazavian AM, Granato D, Lactobacillus acidophilus AA11, a strain isolated from Egyptian Faria JAF, Bolini HMA (2010) Sensory analysis: relevance for cheese. Ann Microbiol. doi:10.1007/s13213-010-0157-6 , probiotic, and synbiotic product development. Comp Alhaj OA, Kanekanian AD, Peters AC, Tatham AS (2010) Rev Food Sci Food Saf 9:358–373 Hypocholesterolaemic effect of Dave RI, Dave JM, Sannabhadti SS (1992) Antibacterial activity of subsp. lactis (Bb12) and trypsin casein hydrolysate. Food buffalo milk dahi prepared using S. thermophilus. Indian J Dairy Chem 123:430–435 Sci 45:607–610 Ali ARA, Metwally AMM, Mahmoud AH, Attia HF (2011) Effect of Deeth HC, Tamime AY (1981) Yoghurt: nutritive and therapeutic feeding probiotics on rats’ immunity and health conditions during aspects. J Food Prot 44:78–86 pregnancy. Food Nutr Sci 2:96–104 Delgado S, O’Sullivan E, Fitzgerald G, Mayo B (2008) In vitro Anand SK, Srinivasan RA, Rao LK (1984) Antibacterial activity evaluation of the probiotic properties of human intestinal associated with Bifidobacterium bifidum I. Cult Dairy Prod J 19 Bifidobacterium species and selection of new probiotic candidates. J (9):6–8 Appl Microbiol 104:1119–1127 Arvind, Sinha PR, Singh NK, Kumar R (2009) Effect of Acidophilus- Dunne C, O’Mahony L, Murphy L, Thornton G, Morrissey D, casei dahi (Probiotic curd) on lipids in 1,2-dimethylhydrazine O’Halloran S, Feeney M, Flynn S (2001) In vitro selection induced intestinal cancer in rats. Int J Probiot Prebiot 4:195–200 criteria for probiotic bacteria of human origin: correlation with in Ataie-Jafari A, Larijani B, Alavi Majd H, Tahbaz F (2009) vivo findings. Am J Clin Nutr 73:386S–392S Cholesterol-lowering effect of probiotic yogurt in comparison FAO/WHO (2002) Guidelines for the evaluation of probiotics in food. with ordinary yogurt in mildly to moderately hypercholesterol- Food and Agriculture Organization of the United Nations and emic subjects. Ann Nutr Metab 54:22–27 World Health Organization Working Group report. FAO, Rome Attaie R, Whalen PJ, Shahani KM, Amer MA (1987) Inhibition of Gaudana SB, Dhanani AS, Bagchi T (2010) Probiotic attributes of growth of Staphylococcus aureus during production of acid- Lactobacillus strains isolated from food and of human origin. Br ophilus yoghurt. J Food Prot 50:224–228 J Nutr 103:1620–1628 Bernardeau M, Vernoux JP, Gueguen M (2002) Safety and efficacy of Gilliland SE (1989) Acidophilus milk products: a review of potential probiotic lactobacilli in promoting growth in post-weaning Swiss benefits to consumers. J Dairy Sci 72:2483–2494 mice. Int J Food Microbiol 77:9–27 Gilliland SE, Speck ML (1969) Biological response of lactic streptococci Bhadoria PBS, Mahapatra SC (2011) Prospects, technological aspects and lactobacilli to catalase. Appl Microbiol 17:797–800 and limitations of probiotics—a worldwide review. Eur J Food Halami PM, Badarinath V, Manjulatha Devi S, Vijayendra SVN (2011) Res Rev 1:23–42 Partial characterization of heat-stable, antilisterial and cell lytic Bianchi-Salvadori B, Camaschella P, Cislaghi S (1989) Effect of bacteriocin of Pediococcus pentosaceus CFR SIII isolated from a yoghurt lactic acid bacteria and bifidobacteria on translocation of vegetable source. Ann Microbiol 61:323–330 Escherichia coli into the lymph system. Microecol Ther 18:137– Hitchins AD, McDonough FE, Wong NP, Hargrove RE (1983) 142 Biological and biochemical variables affecting the relative values Borpuzari RN, Borpuzari T, Deuri B (2007) Probiotic characteristics for growth and feed efficiency of rats fed yoghurt or milk. J Food of Lactobacillus acidophilus strains isolated from fermented Sci 48:1836–1840 milk. J Food Sci Technol 44:158–160 Homma N (1988) Bifidobacteria as a resistance factor in human Boylston TD, Vinderola CG, Ghoddusi HB, Reinheimer JA (2004) beings. Bifidobact Microflora 7:35–43 Incorporation of bifidobacteria into cheeses: challenges and Hütt P, Shchepetova J, Lõivukene K, Kullisaar T, Mikelsaar M (2006) rewards. Int Dairy J 14:375–387 Antagonistic activity of probiotic lactobacilli and bifidobacteria Chawla K (1982) Comparative studies on the hypocholestermic effect against entero- and uropathogens. J Appl Microbiol 100:1324–1332 of milk, dahi, acidophilus milk and acidophilus milk powder. Jain S, Yadav H, Sinha PR, Naito Y, Marotta F (2008) Dahi containing MSc dissertation, Kurukshetra University, Kurukshetra probiotic Lactobacillus acidophilus and Lactobacillus casei has a Cheikhyoussef A, Pogori N, Tian FW, Chen W, Zhang H (2008) protective effect against Salmonella enteritidis infection in mice. Interaction of Bifidobacterium and yoghurt mixed culture with Int J Immunopathol Pharmacol 21:1021–1029 Salmonella associated cultures growth. Biotechnology 7:563–568 Jain S, Yadav H, Sinha PR (2009) Probiotic dahi containing Lactobacil- Cheikhyoussef A, Pogori N, Chen H, Tian F, Chen W, Tang J, Zhang lus casei protects against Salmonella enteritidis infection and H (2009) Antimicrobial activity and partial characterization of modulates immune response in mice. J Med Food 12:576–583 bacteriocin-like inhibitory substances (BLIS) produced by Bifido- Jayamanne VS, Adams MR (2004) Survival of probiotic bifidobac- bacterium infantis BCRC 14602. Food Control 20:553–559 teria in buffalo curd and their effect on sensory properties. Int J Cheikhyoussef A, Cheikhyoussef N, Chen H, Zhao J, Tang J, Zhang Food Sci Technol 39:719–725 H, Chen W (2010) Bifidin I—a new bacteriocin produced by Jiang J, Hang X, Zhang M, Liu X, Li D, Yang H (2010) Diversity of Bifidobacterium infantis BCRC 14602: purification and partial bile salt hydrolase activities in different lactobacilli toward amino acid sequence. Food Control 21:746–753 human bile salts. Ann Microbiol 60:81–88 Ann Microbiol (2012) 62:939–947 947

Kang JH, Yun SI, Park HO (2009) Effects of Lactobacillus gasseri Pala V, Sieri S, Berrino F, Vineis P, Sacerdote C, Palli D, Masala G, BNR17 on body weight and adipose tissue mass in diet-induced Panico S, Mattiello A, Tumino R, Giurdanella MC, Agnoli C, overweight rats. J Microbiol 48:712–714 Grioni S, Krogh V (2011) Yogurt consumption and risk of Kansal VK, Chawla K (1984) Comparative studies on the depression colorectal cancer in the Italian EPIC cohort. Int J Cancer. in hepatic cholesterol genesis in rats fed the full cream cow’s doi:10.1002/ijc.26193 milk, dahi and acidophilus milk. Indian J Nutr Diet 21:54–57 Rajpal S, Kansal VK (2009) Probiotic dahi containing Lactobacillus Kaul J (1984) A study on the hypocholesteremic effect of yoghurt in acidophilus and Bifidobacerium bifidum attenuates diet induced rats. PhD Thesis, Kurukshetra University, Kurukshetra, India hypercholesterolemia in rats. Milchwissenschaft 64:21–25 Kim HS (1988) Characterization of lactobacilli and bifidobacteria as Ranjan P, Arora S, Sharma GS, Sindhu JS, Singh G (2006) Sensory applied to dietary adjunts. Cult Dairy Prod J 23(3):6–9 and textural profile of curd (dahi) from enriched buffalo Klaenhammer TR (1988) Bacteriocins of lactic acid bacteria. milk. J Food Sci Technol 43:38–40 Biochimie 70:337–349 Rati Rao E, Vijayendra SVN, Varadaraj MC (2006) Fermentation Klupsch HJ (1984) Säure Milcherzeugnisse-Milchmischgetränke und biotechnology of traditional foods of the Indian subcontinent. In: Desserts. Mann, Gelsenkirchen-Buer Shetty K, Paliyath G, Pometto A, Levin RE (eds) Food Klupsch HJ (1989) The technology for producing fermented milk biotechnology, 2nd edn. CRC Taylor and Francis, Boca Raton, products using L. acidophilus and bifidus bacteria. Eur Dairy pp 1759–1794 Mag 1:14–21 Ratnakar AK (1989) Antibacterial activity of fermented milk products Kurmann JA, Rasic JLJ (1988) Technology of fermented special and culture capsules. MSc Dissertation, Kurukshetra University, products. Int Dairy Fed Bull 227:101–109 Kurukshetra, India Makras L, De Vuyst L (2006) The in vitro inhibition of Gram-negative Reddy NS (1980) Studies on the antimicrobial substance produced by pathogenic bacteria by bifidobacteria is caused by the production S. lactis subsp. diacetylactis. PhD Thesis, Kurukshetra University, of organic acids. Int Dairy J 16:1049–1057 Kurukshetra, India Mantere-Alhonen S (1995) Propionibacteria used as probiotics—a Sarkar S (2008) Effect of probiotics on biotechnological characteristics of review. Lait 75:447–452 yoghurt: a review. Br Food J 110:717–740 Maqsood S, Hasan F, Masud T, Imran M (2008) Preliminary Senedcor WG, Cochran GW (1967) Statistical methods, 6th edn. characterization of bacteriocin produced by Lactobacillus acid- Oxford-IBH, New Delhi ophilus TS1 isolated from traditional dahi. Ann Microbiol 58:617– Shah NP (2007) Functional cultures and health benefits. Int Dairy J 622 17:1262–1277 McDonough FE, Wong NP, Wells P, Hitchings AD, Bodwell CE (1985) Shah NP, Ding WK, Fallourd MJ, Leyer G (2010) Improving the Stimulation of rat growth by yoghurt—effect of vitamins and stability of probiotic bacteria in model fruit juices using vitamins minerals. Nutr Rep Int 31:1237–1245 and antioxidants. J Food Sci 75:M278–M282 Min Tze L (2006) In vivo and in vitro cholesterol removal by Shahani KM, Vakil JR, Kilara A (1977) Natural antibiotic activity of lactobacilli and Bifidobacteria. PhD thesis, Victoria University, Lactobacillus acidophilus and bulgaricus. II. Isolation of acid- Australia ophilin from Lactobacillus acidophilus. Cult Dairy Prod J 12:8– Mkrtchyan H, Gibbons S, Heidelberger S, Zloh M, Limaki HK (2010) 11 Purification, characterisation and identification of acidocin Singh J, Khanna A, Chander H (1979) Antibacterial activity of LCHV, an antimicrobial peptide produced by Lactobacillus yoghurt starter in cow and buffalo milk. J Food Prot 42:664–665 acidophilus n.v. Er 317/402 strain Narine. J Antimicrob Agents Sinha PR, Sinha RN (2000) Importance of good quality dahi in food. 35:255–260 Indian Dairyman 52:45–47 Mobarez AM, Hosseini Doust R, Sattari M, Mantheghi N (2008) Soccol CR, Vandenberghe LP, Spier MR, Medeiros ABP, Yamaguishi Antimicrobial effects of bacteriocin like substance produced by CT, Lindner JD, Pandey A, Soccol VT (2010) The potential of L. acidophilus from traditional yoghurt on P. aeruginosa and S. probiotics: a review. Food Technol Biotechnol 48:413–434 aureus. J Biol Sci 8:221–224 Stenger MR, Reber KM, Giannone PJ, Nankervis CA (2011) Mohammadi R, Mortazavian AM, Khosrokhavar R, Gomes da Cruz A Probiotics and prebiotics for the prevention of necrotizing (2011) Probiotic ice cream: viability of probiotic bacteria and enterocolitis. Curr Infect Dis Rep 13:13–20 sensory properties. Ann Microbiol. doi:10.1007/s13213-010-0188-z Vibha Sinha PR, Hariom Y (2007) Antiatherogenic effect of probiotic Nagpal R, Kumar A, Arora S (2010) In vitro probiotic potential of dahi in rats fed cholesterol enriched diet. J Food Sci Technol lactobacilli isolated from indigenous fermented milk products. Int 44:127–129 J Probiotics Prebiotics 5:103–110 Yadav H, Jain S, Sinha PR (2008) Oral administration of dahi Nair CR, Mann GV (1977) A factor in milk which influences containing probiotic Lactobacillus acidophilus and Lactobacillus cholesteremia in rats. Artherosclerosis 26:363–368 casei delayed the progression of streptozotocin-induced diabetes Ooi LG, Liong MT (2010) Cholesterol-lowering effects of probiotics in rats. J Dairy Res 75:189–195 and prebiotics: a review of in vivo and in vitro findings. Int J Mol Zamfir M, Grosu-Tudor S (2009) Impact of stress conditions on the Sci 11:2499–2522 growth of Lactobacillus acidophilus IBB 801 and production of Ouwehand AC, Svendsen LS, Leyer G (2011) Probiotics: from strain to acidophilin 801. J Gen Appl Microbiol 55:277–282 product. In: Kneifel W, Salminen S (eds) Probiotics and health Zlatkis A, Zok B, Boyle GJ (1953) Estimation of serum cholesterol. J claims. Wiley-Blackwell, Oxford. doi:10.1002/9781444329384.ch3 Lab Clin Med 41:486