World Applied Sciences Journal 19 (10): 1367-1380, 2012ISSN 1818-4952© IDOSI Publications, 2012DOI: 10.5829/idosi.wasj.2012.19.10.2797

Corresponding Author: Mona M.A. Aly, Food Technology Research Institute, Agricultural Research Center, Giza, Egypt.

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Production of Cereal-Based Probiotic Beverages

Amal A. Hassan, Mona M.A. Aly and Soher T. El-Hadidie1 2 2

Deptartment of Food Science, Faculty of Agriculture,1

Ain Shams Univesity, Shoubra El-Kema, EgyptFood Technology Research Institute, Agricultural Research Center, Giza, Egypt2

Abstract: In the present study, rice and millet grains were fermented with ABT-2 starter culture (S. thermophilusL. acidophilus and Bifidobacterium BB-12) to obtain probiotic beverages, fortified with pumpkin and sesameseed milk in order to combine the high nutritive value of seeds and the health benefits of the probiotic culture.The levels of several factors, i.e. starter culture concentration, rice and millet level and the amount of honey,affecting the fermentation process, were established for completing a controlled fermentation for 16 hours.The viable cell counts reached at the end of fermentation time about 4.3 x 10 cfu/ml. Changes were observed9

in acidity and counts of probiotic bacteria as a result of fortification with pumpkin and sesame milk at 10%.Sensory properties were also evaluated. Fermentation with ABT-2 starter culture improved the color, flavor,texture and overall acceptability of the beverages. Slight changes in the count of probiotic, pH and acidityduring storage of the beverages at 4°C were observed. The shelf-life of the rice and millet fermented beverageswas estimated to be 15 days under refrigerated storage.

Key words: Rice Millet ABT-2 starter culture Pumpkin seed Sesame Probiotic Acidity Sensoryproperties Shelf-life

INTRODUCTION Rice (Oryza sativa L.) competes closely with wheat

Most probiotic foods at the markets worldwide are 100 countries including Egypt, when compared with othermilk based and very few attempts are made for cereals; it has the highest food yield. Regarding to wheatdevelopment of probiotic foods using other fermentation and maize, rice supplies more than half of all the caloriessubstrates such as cereals. Their large distribution and for human consumption. Rice is consumed as milled riceimportant nutritive value have focused the attention on after dehulling process and whitening (removal oftheir use as raw materials for the development of new pericarp, bran layer and embryo from brown rice). Afterfermented functional foods [1]. The interest in the whitening parts of rice were removed which has verydevelopment of non-dairy probiotic products is increasing low starch content but has high percentages of oil,due to consumer interest for exotic and different tastes, protein, vitamins and minerals, however, the remained partemerging number of vegan consumers and also because (the endosperm of the milled rice) mainly composed ofsome consumers are lactose intolerant or have milk carbohydrates and proteins [4]. Millet (Pennisetumprotein allergies [2]. Cereal products often ferment typoides) is one of the oldest foods known to human andspontaneously, resulting in improved shelf-life and better possibly the first cereal grain to be used for making bread.nutritional properties compared with the raw material. Today millet ranks as the sixth most important grain in theSingle or mixed cereals are used as a substrate in the world. Millet is equal or superior to grain of wheat, corn,production of fermented foods and the final product can sorghum and rice in protein and oil content, it containsvary according to the microbial population and similar amount of calcium (Ca) and phosphorus (P),fermentation conditions. Fermentation procedures have more iron (Fe) than cereals. In Egypt, millet is mainly usedbeen used to develop new foods with enhanced health in feeding rations as bird seeds and relatively smallproperties [3]. amount of total crop is utilized directly in foods [5].

as the world most important crop. It is grown in more than

Soaking (2hr)

Draining

Cooking(1:3)/30 min

Blending with water

Rice

Rice milk

Millet

Soaking (12 hr)

Draining

Wet milling

Addition of water (1:1)

Filtration

Settling and sedimenting (over night)

Removing the top layer

Adding water to make millet milk

Dehulling

Wet milling (1:1)

Sieving

Pumpkin seed milk

Pumpkin seed

Heat treatment (90°C/5min)

Cooling

Sesame seeds

Soaking (8 hr)

Draining

Wet milling (1:1)

Sieving

Sesame seed milk

Heat treatment (90°C/5min)

Cooling

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Pumpkin (Cucurbita pepo) seeds were found to be rich in Research Center, Ministry of Agriculture Giza, Egypt.oil (about 50%), protein (38%) and alpha-tocopherols Sesame and pumpkin seeds were purchased from local(3g/100g). The seed oil contains an appreciable amount of market. A commercial probiotic starter culture (ABT-2)unsaturated fatty acids (78%) and found to be a rich of was used (Peyma - Chr. Hansen, Badr city, Cairo)linoleic (47%) [6]. Sesame (Sesamum indicum L.) is one of containing S. thermophilus ST-20Y, L. acidophilus LA-5the most important oil seed crops worldwide and has and Bifidoacterium BB-12.been cultivated since ancient Egyptian and used as Preparation of Cereal-based Probiotic beverages: wastraditional health food. Its a good source of protein, Carried out on three stages:minerals and oil. Sesame seeds are used in the making ofTahin (sesame butter) and halawa, for the preparations of Stage 1: Preparation of Rice and Millet Milk as Follows:rolls, crackers, cakes and pastry products. They are usedfor their nutritive, preventive and curative properties.Sesame oil seeds are sources of some phytonutrientssuch as minerals, vitamins, omega-6 fatty acids, flavonoid,phenolic anti-oxidant and dietary fibers with potentanti-cancer as well as health promoting properties [7].

Probiotic microorganisms have been studied assuccessful ingredients in the functional food segment.The popularity of probiotic foods is due to the numeroushealth benefits attributes to its intake [8]. A number ofhealth benefits of probiotic products have been proposedincluding: antimicrobial, antimutagenic, anticarcinogenic,antihypertensive properties, reduction in serumcholesterol, alleviation of lactose intolerance, reduction ofallergic symptoms, reduction of diarrhea and stimulationof the immune system [9, 10, 11, 12].

To achieve the benefits from their ingestion, theprobiotic microorganisms must reach the intestine aliveand in sufficient concentration, surviving to harsh Fig. 1: Preparation of rice and millet milkconditions found during the flow through thegastrointestinal tract [13]. A minimum daily intake of Stage 2: Preparation of Pumpkin and Sesame Seed Milk8.9 log of colony forming unit (CFU) or 6-7 log cfu/g or as Follows:1ml of food is generally recommended [14]. The survivalof probiotic microorganisms in food products is stronglyinfluenced by pH and post-acidification, which may occurduring refrigerated storage of fermented products.Other factors such as production of hydrogen peroxide,oxygen level, temperature and food matrix also affect themicrobial viability [15].

The aim of this study was to develop rice and milletprobiotic beverage products using commercial probioticstarter culture (ABT-2). As well as to study the effect ofpumpkin and sesame seeds addition on somephysicochemical, microbiological and sensory propertiesduring refrigerated storage.

MATERIALS AND METHODS

Dried millet (Shandewel 1), brown rice (Giza 181)were obtained from Crops Research Institute, Agricultural Fig. 2: Preparation of pumpkin and sesame seeds milk

Rice or Millet milk

Heat treatment (90 °C/20 min)

Cooling (37 °C)

5% honey

Inculcation with 5% ABT-2 starter culture

Incubation (37°C/16hr)

Plain beverage

Refrigerated storage (4 °C/15 days)

(10% Pumpkin seed ) milk) (10% sesame seed milk)

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Stage 3: Preparation of Cereal – Based Probiotic Microbiological Analysis: Viable counts were determinedBeverage as Follows: in fermented beverages (1 ml), after 3, 5, 7, 9, 12 and 15

Fig. 3: Preparation of probiotic cereal - based beverage refrigerated storage (4°C) during which pH remained

Formula of beverage: Amount of serving above 10 cfu/ml. Refrigerated storage was carried out for(100 ml) 15 days with periodical observation of pH, total acidity

Fermented rice or millet beverage: 85 and viability of starter culture.Sesame or pumpkin milk: 10Honey or sugar: 5 Statistical Analysis: The experimental data were analyzed*Vanilla or fruit flavor ----------- using analysis of variance (ANOVA) followed by

100 Duncan’s multiple range tests (P < 0.05) to determine a

Chemical Analysis: Total solid were determined by analyzed according to user’s guide of statistical analysisdrying the sample at 102±2°C to constant weight. Protein, system [21].ash, fat content was determined according to A.O.A.C[16]. Nitrogen free extract (total carbohydrates) was RESULTS AND DISCUSSIONcalculated by difference. Na, Zn, Fe, K and Ca weredetermined using the flame photometer (Galienkamp, Chemical Composition of Raw Materials: Moisture,EGA 330, England) and Perkin Elmer atomic absorption crude fat, crude protein, ash, crude fiber andspectrophotometer (model 80, England) as described in carbohydrate of brown rice, millet, pumpkin seeds andA.O.A.C [16]. Total phosphorus was determined by sesame seeds flour were determined. The results arespectrophotometer at 650 nm according to the method presented in Table 1 . Moisture, crude fat, crudedescribed in A.O.A.C [16]. Total phenols were determined protein, ash, fiber and carbohydrate of brown rice wereas described by Singleton and Rossi [17]. Gallic acid was 12.23, 1.60, 8.38, 1.38, 1.14 and 87.17%, respectively.chosen as a standard to prepare the standard curve. These results are in agreement with the values reportedPhenols were expressed as mg/100g sample. by Asad [4, 22].

Physical Analysis are presented in same (Table 1). Millet flour containedMeasurement of pH, Acidity and Viscosity During 8.40, 12.0, 7.20, 1.95, 3.0 and 71.46% moisture, protein,Fermentation: The pH of the fermented samples was fat, ash, fiber and carbohydrate respectively. The revealedmonitored during fermentation by measures of pH-meter. results are in agreement with Ali et al. [23], Belton andThe viscosity was measured by a dynamic viscometer Taylor [24]. Data presented in Table 1 indicated also(Brookfield Model RVDI, USA). Titratable acidity was that dehulled pumpkin seeds contained 8.05 moisture,determined as lactic acid percentage by titrating with 33.44 crude protein, 46.50 crude fat, 3.02 ash, 3.12 crude0.1 NaOH, using phenolphthalein as an indicator [18]. fiber and 13.42% total carbohydrates, respectively.

days of storage. Differential media used for enumerationof S. thermophilus, L. acidophilus and BifidobacteriumBB-12 where those previously described by Martin-Dianaet al. [19].

Sensory Properties: Ten tasters trained are usually usedin the Food Technology Research Institute assessed theproduced eight beverage samples. The eight beveragesamples were scored for color, taste, texture, flavor andoverall acceptability. Score were based on a hedonicscale of 1 to 9 where: 1 = dislike very much (very bad) and9 = like very much (excellent) [20].

Estimation of Shelf-Life: The self life of the fermented riceand millet beverages was defined as the period of

above 4.0 and the number of viable cell counts was6

significant difference among samples. The data were

Results of the proximate composition of millet flour

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Table 1: Chemical composition (% dry basis) of raw materials

Parameter Brown rice Millet Pumpkin seed Sesame seed L.S.D

Moisture 12.2 3 8.40 8.05 6.55 0.9525a b c d

Protein 8.38 12.00 33.44 29.76 0.8639d c a b

Fat 1.60 7.20 46.50 51.17 0.6917d c b a

Ash 1.38 1.95 3.02 4.19 0.0490d c b a

Crude fiber 1.14 3.00 3.12 4.07 0.3626c b b a

Total carbohydrate 87.17 71.46 13.42 10.41 0.8273a b c d

Data are averages from three independent trialsData in the same row with different small letters are significantly different (p<0.05)

Table 2: Physical properties of different beverage samples

Sample pH Titratable acidity (%) Dry matter % Viscosity CP

(1) 7.83 0.08 9.92 601a e g b

(2) 4.55 0.54 10.41 772f a f a

(3) 5.29 0.31 12.24 240c c d g

(4) 5.30 0.32 14.17 119c c b e

(5) 7.11 0.16 9.18 500b d h c

(6) 4.34 0.59 11.06 600g a e b

(7) 5.26 0.45 13.44 390d b c f

(8) 5.20 0.41 14.20 440d b a d

L.S.D 0.03 0.03 0.02 3.78

Data are averages from three independent trialsData in the same column with different small letters are significantly different (p<0.05)1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesameseed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed milk 8= fermented milletbeverage + 10% sesame seed milk

These results are in agreement with those obtained by Regarding to viscosity, addition of probiotic bacteriaAlfawaz [25]. Data in Table 1 also indicated that the caused increasing in viscosity of the producedproximate composition of sesame seeds which contained beverages; meanwhile, adding pumpkin and sesame milk6.55, 29.76, 51.17, 4.19, 4.07 and 10.41% moisture, protein, caused decrement in viscosity. Sesame seed milk fortifiedfat, ash, crude fiber and carbohydrates, respectively. From beverages had the lowest viscosity. The viscositythe above mentioned data indicated that cereal grains behavior was related to the pH, yielding maximum valueshave high carbohydrate, meanwhile pumpkin and sesame at the lowest pH. So, the increase in viscosity takes placeseeds have high fat, protein and ash content. at acidic pH values [26]. In the same Table, dry matter

Physical Properties: The physical properties of the added and the fortification with sesame and pumpkin seedbeverage samples studied in this work are shown in milk. Dry matter ranged between 9.92 and 14.17 % forTable 2. These quality parameters (pH, titratable acidity, sample 1 and 4, respectively.dry matter and viscosity) are related to stability ofbioactive components in plant derived products. Chemical Composition of Different Beverages: ChemicalAdding probiotic starter culture caused decreasing in pH composition of different produced beverages is presentedvalue of the beverage relative to control, at the same time in Table 3. Percentage of protein content was 1.27 andtitratable acidity increased. Fortification with pumpkin 1.35% for control rice and millet beverages (sample 1 andand sesame seed milk increased the beverage pH value. 5), respectively. Protein content was observed to increaseThis implies that pumpkin and sesame seed milks are less about two times from that for control as a result ofacidic. Thus an addition of pumpkin and sesame seed milk fortification with pumpkin and sesame seed milk. Proteinwill increase pH value of the beverages. This shows that content were 3.11 and 3.63% for fermented rice beveragethese probiotic beverage blends will acceptable or + 10% pumpkin seed milk and fermented rice beverage +suitable for people with ulcers and other related problems 10% sesame seeds milk, respectively. Fat content wasthan probiotic beverage alone since it is less acidic. significantly (p<0.05) increased as pumpkin and sesame

increased in the produced beverages as probiotic starter

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Table 3: Chemical composition (% dry basis) of different beveragesSample Energy Kcal. Protein Ash Fat Total carbohydrate Total phenols mg/100g(1) 36.49 1.270 0.089 0.187 9.16 23.61f c f a b

(2) 44.40 2.752 0.257 0.440 6.96 36.13d d e ef a

(3) 70.50 3.119 0.416 2.700 6.91 35.19c b b ef a

(4) 72.07 3.635 0.312 3.070 6.77 25.50a c a f c

(5) 38.39 1.350 0.095 0.476 8.09 30.98f e e b b

(6) 46.38 2.073 0.234 0.918 7.83 37.83e d d bc a

(7) 64.04 3.453 0.475 2.365 7.25d 35.72b a c e a

(8) 72.81 3.510 0.389 2.750 7.55 31.40b b b cd b

LSD 0.05 -- 0.163 0.040 0.058 0.38 2.68Data are averages from three independent trialsData in the same column with different small letters are significantly different (p<0.05)1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesameseed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed milk 8= fermented milletbeverage + 10% sesame seed milk

seed milk fortification. Fat content of the control rice and depending on the kind of milk used in its preparationmillet beverages were 0.18 and 0.47%, respectively. (defatted, low fat or whole milk). According to the result,Fermented rice beverage + 10% sesame and pumpkin seed the fermentation of rice or millet resulted in a non-dairymilk were 2.70 and 3.07%, respectively. Similar findings probiotic beverage with lower caloric value compared towere observed for fermented millet beverages. The commercial available fermented dairy products. Asincrement in fat content of produced beverages attributed noticed in the same Table, total phenols content of theto high fat content of oily pumpkin and sesame seeds. produced probiotics beverages were higher than that of

Ash content of produced fermented probiotic control (without fermentation). Total phenols increasedbeverages increased about two times relative to controls. to reach 36.13 and 37.83 mg/100g for plain probiotic riceFermented rice and millet beverages + 10% sesame milk and millet beverages, respectively. These resultshad ash content amounted in 0.31 and 0.38%, supported by Tseng et al. [29] who reported that totalrespectively. Ash content increased significantly may be phenolic content of fermented rice beverage was higherattributed to fermentation process and fortification with than that using rice solely. They also mentioned that totalpumpkin and sesame seed milk. Hughes and Hoover [27] phenolics were the major naturally occurring antioxidantreported that bifidobacteria produce nutrients and component.improve lactose tolerance, calcium absorbability anddigestibility. Total carbohydrate content of fermented rice Minerals Content: Table 4 shows minerals i.e. sodiumand millet beverages decreased relative to control (Na), potassium (K), phosphorus (P), calcium (Ca), ironbeverages. Carbohydrate content ranged from 6.77 to (Fe) and zinc (Zn) content of different probiotic9.16%, respectively. These differences could be generally beverages. Results indicated that minerals contentattributed to fermentation process, since lactic acid increased as a result of pumpkin and sesame seed milkbacteria form exoplyaccharides which have been of added. Samples 4 and 8 contained the highest value of Nainterest for their technological potential as thickening (190.6 mg/L, 114.0 mg/L) followed by sample (6) 76.1 mg/L.agent or as a source of ropines and for their potential as The lowest value was 30.5 mg/L for sample (5) followed bydietary fiber [28]. On the other hand, the energy values of 32.0 mg/L for sample (1). Potassium content ranged 114.7produced beverages were found between 36.49 and to 284.1 mg/L. The highest value for sample (8). Notable72.81 kcal/100ml. caloric values of different beverages increase in Ca content of produced beverages as a resultwere increased by pumpkin and sesame seed fortification. of sesame milk added, this means sesame seeds are richThe caloric value was increased to reach 70.50 and source of calcium. The highest value 457 mg/L for sample72.07%, for fermented rice beverages + 10% pumpkin milk (4) followed by 270 for sample (8), increasing percentageand sesame milk, respectively. Meanwhile, caloric values for that samples were 124.08 and 174%, respectively.were increased to reach 64.04 and 72.81% for fermented Regarding to iron content data showed that iron contentmillet beverages + 10% pumpkin milk and sesame seed of sample (8) and (4) were 18.9 and 18.2 mg/L, followed bymilk, respectively. The caloric value of commercial (3) and (7) which were 17.5 and 16.1 mg/L, respectively.available probiotic products, which are dairy based Meanwhile, the obtained values of iron content in (2) andproducts, falls in the range from 60 to 120 kcal/g, (6) were 15.4 and 14.3 mg/L, respectively.

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Table 4: Mineral profile of different produced beverage samples (mg/L)Sample Na K Fe Zn Ca P Na/K Ca/P(1) 32.0 114.7 14.7 2.7 130 180 0.28 0.73(2) 63.3 117.5 15.4 2.7 204 200 0.53 1.02(3) 68.0 172.3 17.5 6.1 208 241 0.39 0.99(4) 190.6 258.8 18.2 2.9 457 300 0.73 1.52(5) 30.5 205.4 12.4 2.0 50 100 0.20 0.60(6) 76.1 210.0 14.3 2.05 100 160 0.36 0.62(7) 75.9 275.8 16.1 4.4 110 200 0.30 0.55(8) 114.0 284.1 18.9 3.2 270 230 0.31 1.191= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesameseed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed 8= fermented millet beverage+ 10% sesame seed milk.

Table 5: Sensory evaluation of different beveragesSample Color Taste Flavor Texture Acceptance(1) 7.90 ± 0.66 7.55 ± 0.98 7.75 ± 0.82 7.87 ± 0.80 8.07 ± 0.61b bc bc bc b

(2) 8.61 ± 0.31 8.65 ± 0.24 8.60 ± 0.32 8.70 ± 0.47 8.64 ± 0.51a a a a a

(3) 8.20 ± 0.34 8.10 ± 0.32 8.02 ± 0.68 7.26 ± 0.47 8.25 ± 0.41ab ab b c b

(4) 7.15 ± 0.34 6.83 ± 0.59 7.25 ± 0.49 8.35 ± 0.41 7.42 ± 0.29c de c ab c

(5) 6.20 ± 0.26 6.35 ± 0.71 7.25 ± 0.49 7.45 ± 0.49 6.20 ± 0.34d e c cd e

(6) 8.00 ± 0.75 7.45 ± 1.04 8.260 ± 0.68 8.54 ± 0.45 7.86 ± 0.47b ab b a d

(7) 7.50 ± 0.61 7.75 ± 0.83 7.75 ± 0.83 7.05 ± 1.27 6.9 ± 0.16c bc bc d f

(8) 6.20 ± 0.88 6.50 ± 0.48 6.20 ± 0.43 7.70 ± 0.52 6.15 ± 0.34d e d bd e

LSD 0.05 0.50 0.52 0.56 0.60 0.38Data in the same column with different small letters are significantly different (p<0.05).1= unfermented rice beverage 2= fermented plain rice beverage 3= fermented rice beverage + 10% pumpkin seed milk 4= fermented rice beverage + 10% sesameseed milk 5= unfermented millet beverage 6= fermented plain millet beverage 7= fermented millet beverage + 10% pumpkin seed milk 8= fermented milletbeverage + 10% sesame seed milk

Zinc contents in samples (3) and (7) increased with the sensory properties of beverages when sesame seedpumpkin seed milk added amounted in 6.1 and 4.4 mg/L. milk fortification decreased. Fortification with pumpkinThis means pumpkin seeds are good source of zinc. seed milk had more acceptable than sesame seed milk; thisModerate amount of zinc was observed for samples may be because the beany taste of the later. The probioticfortified with sesame milk. Antony and Chandra [30] juices were found to contain aromas flavor (sour)reported that mineral contents of finger millet were (Ca) characteristic of functional ingredients [32].313.1, (P) 467.2, (Fe) 6.53, (Cu) 1.01, (Zn) 2.02 and (Mn) In general, the presence of pumpkin and sesame seed9.86 mg/100g on dry weight. Arora et al. [31] reported that milk increased the nutritional quality of beverages.content of pearl millet seeds were 123.67, 15.90, 2.13, 0.84, Sesame seed milk had a negative influence on the0.50 and 0.35 mg/100g, on dry matter basis for fermented product taste and flavor but at the same time itphosphorous, calcium, iron, zinc, copper and manganese, had positive effect on the beverage texture. These defectsrespectively. can overcome by adding different flavor components

Sensory Evaluation: This study evaluated the peaches. Molin [33] reported that to make the fermentedacceptability for the color, taste flavor and texture of cereal gruel more acceptable to consumers in theprobiotic rice and millet beverages. Results of the sensory industrialized world, it mixed into a fruit drink.evaluation of fermented beverages on a hedonic scalefrom 1 (very bad) to 9 (excellent) are shown in Table 5. Fermentation of Rice and Millet by the CommercialData revealed that there are generally significantly Starter Culture ABT-2:different (p < 0.05), which indicates that the different Changes in pH and Titratable Acidity: The chemicalbeverages have distinctively different tastes. Addition of quality attributes (pH and titratable acidity) of theprobiotic bacteria (commercial ABT-2 starter) at 5 % level, fermented cereal- based beverage during productionhave more acceptable than that of control beverage, while process are shown in Figs. 4 and 5. Significant differences

vanilla, chocolate or fruit juice or jam i.e. straw berries and

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Fig. 4: Changes in pH values of probiotic non - diary of cereal media. These results are in harmony withbeverages samples during fermentation process those obtaine by Afaneh et al. [34] and Mårtensson et al.

Fig. 5: Changes in titratable acidity (% lactic acid) of values of the two types of fermented beverage underprobiotic non-diary beverages samples during study were increased during the fermentation processfermentation process (16 h) (Fig. 5). Total acidity content ( as percent lactic

were noticed between the two fermented substrates to 0.54% (16h) and from 0.35% (0h) to 0.59% (16h) at theemployed for their performances during the fermentation end of fermentation process for fermented plain rice andprocess. The pH decreased markedly at 4 to 8h until about millet beverage samples, respectively. However, fermented12 h and further decreased afterward. The maximum plain millet beverage samples had significantly higheracidification (pH) ranged from 4.34 to 4.55 according to (p>0.05) acidity than that of fermented plain rice beveragethe fermented substrate employed. The time to reach the samples. These results may be related to fermenteddesired pH (4.5) of the fermented rice beverage samples substrate employed. Generally, the production of aciditywas 1.6 times higher than with the fermented millet was low at the early culture period but increasedbeverage samples. The results demonstrated a slow considerably from 4 h fermentation until 16h.These resultsacidification profile. The time required to reach pH 4.5 for are in agreement with those reported by Tangular andfermented plain rice beverage samples was 16 h while it Erten [39], Ramos et al.[40] and Colakoglu and Gursoywas reduced to 10h for fermented plain millet beverage [41]. Wongkhalaung et al. [42] reported that the titratablesamples. This implies that rice substrate is less acidic acidity of fermented hydrolyzed rice with L. casei andthan millet substrate, thus the use of millet as a substrate L. acidophilus was produced up to 0.6% lactic acid, whilefor fermentation will encourages greater capability of Wongkhalaung and Boonyaratanakornkit [43] achievedproducing acid and decrease the pH value of fermented 0.86 % acidity as lactic and pH 3.48 for 24 h whenmillet beverage. For all fermented non-dairy beverage fermenting rice milk with mixed cultures of L. acidophilus

samples, pH values significantly reduced (p>0.05)during the fermentation process (16h) from 5.32±0.02 to4.55 ± 0.01 and from 5.30±0.02 to 4.34±0.01 for fermentedplain rice and millet beverage samples, respectively,which could be due to the presence of the three bacterialstrains of ABT-2 starter culture, S. thermophilus ST-20Y,L. acidophilus LA-5 and Bifidobacterium BB-12. Whenall data were analyzed together using ANOVA (Fig. 4),it could be observed that a significant effect of fermentedsubstrate employed on final pH (p>0.05) with lower valuesobserved in the fermented millet beverage samples andhigher values in the fermented rice beverage samples afterfermentation time (16h). The different fermentation ratescould be attributed to strain specificities and differences

[35, 36] who reported that after much longer fermentation(16h) of an oat base with commercial mixed dairy cultures,pH reached 3.9-4.5, while Marklinder and Lonner [37]achieved pH 4.3-4.5 for 6 h when fermenting oat mealsoups with L. plantarum strains. Angelov et al. [1]applied oat as substrate for lactic acid fermentation withdairy starter cultures and found that 8h was anappropriate fermentation time to reach the desired pHlevels of 4.0-4.5. Coda et al. [38] achieved pH 3.72 to 4.03for 8h when fermenting cereals (rice, barely, emmer, oator soy flour) and concentrated red grape must withL. plantarum strains for making vegetable yogurt-likebeverages. The results showed that the titratable acidity

acid) significantly increased (p>0.05) from 0.32% (0h)

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and L. casei subsp. rhamnosus. The increase in aciditywith decrease in pH as fermentation proceed mayeliminate or discourage the growth of most spoilage andpathogenic microorganisms that cannot withstandsuch condition hence making the probiotic non-dairy,cereal-based beverages safer for consumption and alsohelp to increase the shelf- life.

Assessment of the Two Types of Probiotic BeveragesDuring Refrigerated Storage: In this study rice and milletwas used as the main substrates for the commercial starterculture ABT-2 and pumpkin and sesame seed milks wereadded after fermentation to investigate their effects on thesurvival and acidifying activities of probiotic culturesduring refrigerated storage at 4±1°C for 15 days. Fig. 6: Changes in pH values of probiotic rice beverage

Changes in pH and Titratable Acidity: The effects ofpumpkin and sesame seed milk addition on pH andtitratable acidity values of fermented rice and milletbeverages during refrigerated storage for up to 15 daysare shown Figs. 6-9. As expected, the storage timesignificantly affected the acidity level in the three typesof rice beverages; titratable acidity increased, whilst thepH was decreased. Differences between pH values offermented plain rice beverage samples at the first ninedays of storage were insignificant (p>0.05), thereafter,the pH values significantly decreased at the twelfthand fifteenth day of storage reaching 4.42 and 4.32,respectively (Fig. 6). In parallel with pH values, thetitratable acidity values of fermented plain rice beveragesamples at the first two days of storage were insignificant Fig. 7: Changes in pH values of probiotic millet beverage(p>0.05). After that, the acidity values of the beverage samples during refrigerated storage (4°C)samples significantly increased (p>0.05) from 0.54% to0.57% after ninth day of storage until the fifteenth dayof storage (Fig. 8).

The addition of pumpkin and sesame seed milk tothe plain rice beverage affected the initial pH value from(4.55 to 5.29 and 5.30) and the titratable acidity from(0.54% to 0.31% and 0.32%), respectively. The mean pHvalues of the fermented rice beverage samples containing10% pumpkin seed milk slightly decreased at the third dayof storage but this was statistically insignificant (p>0.05).On day six, pH values of fermented pumpkin-containingrice beverage samples showing a 0.06 pH unit dropscompared to the beginning of storage period. The meanpH values of the fermented pumpkin - containing ricebeverage samples significantly declined (p>0.05) at theend of storage reaching 5.15 compared to the mean pH Fig. 8: Changes in titratable acidity (% lactic acid) ofvalues at the first day of storage (Fig. 6). In parallel with probiotic rice fermented rice beverage duringpH values, differences between titratable acidity values of storage

samples during refrigerated storage (4°C)

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Fig. 9: Change in titratable acidity ( % lactic acid) of refrigerated storage. Akalin et al. [49] found that probioticprobiotic millet beverage samples during microorganisms reduced pH values of different types ofrefrigerated storage (4°C) yoghurts from 4.51 to 4.40, after 28 days of refrigerated

the rice beverage samples containing 10% pumpkin seed From data reported above it was observed that pHmilk at the first six days of storage were significant values of all beverage samples decreased while titratable(p>0.05) reaching 0.32%. At the end of storage the acidity acidity values were increased during refrigerated storagevalues of fermented pumpkin – containing rice beverage and these differences were found to be significantsamples significantly increased (p>0.05) reaching 0.35% (p>0.05) statistically. This is can be explained by furthercompared to the first day of storage (Fig. 8). metabolic activity of starter culture during refrigerated

There were no significant differences (p>0.05) in storage [50].pH values of fermented sesame containing rice beveragesamples throughout storage period, while differences Survival of Lactic Acid Bacteria in the Probiotic Ricebetween titratable acidity values of fermented sesame- Beverage During Refrigerated Storage: The probioticcontaining rice beverage samples at the first six days of rice beverage was produced in the follow conditions:storage were significant (p<0.05), thereafter the titratable initial pH of 5.32, fermentation temperature of 37°C,acidity values of samples remained unchanged until the with inoculums level of 5% of commercial starter culturelast day of storage reaching 0.34% (Fig. 8). In general, (ABT-2) and 16h of fermentation. After fermentationfermented plain rice beverage samples had the lowest pH process, pumpkin and sesame seed milk in a ratio of 10%and the highest titratable acidity values at the first and (v/v) were added separately to the fermented plain ricelast day of storage, respectively, compared to other types beverage and all the three types of probiotic rice beverageof rice beverages (Figs. 6&8). The same trend was also (fermented plain rice beverage, fermented plain riceobserved in fermented millet beverage samples. Figs. 7&9 beverage containing 10% pumpkin seed milk andshow variation in pH and titratable acidity values of fermented plain rice containing 10% sesame seed milk)fermented millet beverage during refrigerated storage for were stored for up to 15 days at 4±1°C. The results of theup to 15 days. Data indicated that storage time bacterial counts after the production and duringsignificantly affected the acidity level in the three types refrigerated storage of the fermented rice beverageof millet beverage; titratable acidity increased, whilst samples were illustrated in Figs. 10-12. On thethe pH was decreased. Probiotic fermented beverage production day (zero time), the addition of pumpkinprepared from millet only without additives had the and sesame seed milk to fermented plain ricelowest pH values ranged between 4.34 and 4.4 and the beverage significantly increased (p<0.05) the count ofhighest titratable acidity values ranged between 0.59% L. acidophilus from the initial level 9.66 log cfu/ml to 9.73and 0.63%, respectively, throughout storage period as and 9.80 log cfu/ml, respectively, while the count ofcompared with the two other types of millet beverages Bifidobacterium BB-12 was significantly decreased(Figs. 7&9). Shah [10,44] observed similar decreases in pH (p<0.05) from 9.87 log cfu/ml to 9.66 and 9.74 log cfu/ml,values during storage of commercial yogurts containing respectively. However, there were no significant (p>0.05)

L. acidophilus and B. bifidium without any effect on cellcounts, while the increase in acidification in the probioticyogurt was mainly due to the growth of L. debrueckiispp. bulgaricus, S. thermophilus during refrigeratedstorage [45].

The pH level above 4 is generally required for afermented oat beverage throughout storage [46].Results in Figs. 6&7 showed that the pH remained above4.0 for fifteen days of refrigerated storage for fermentedthree types of rice or millet beverages. These results arein line with those reported by Colakoglu and Gursoy [41]and Paseephol and Sherkat [47]. Guo et al. [48] reportedthat the pH of the fermented buffalo milk containingL. casei decreased from 5.02 to 4.00 after 30 days of

storage at 4°C.

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Fig. 10: Viability of S. thermophilus in probiotic rice beverages with ABT-2 starter culture during refrigerated storageat 4°C

Fig. 11: Viability of L. acidophilus in probiotic rice beverages with ABT-2 starter culture during refrigerated storage at4°C

Fig. 12: Viability of Bifidobacterium BB-12 in probiotic rice beverages with ABT-2 starter culture during refrigeratedstorage at 4°C

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differences between the fermented plain rice beverage and The reduction in the count of L. acidophilus duringthe rice beverage containing pumpkin or sesame seed milk the cold storage may be due to the production ofin terms of the viable cell counts of S. thermophilus. antimicrobials such as bacteriocins, H O or organic acids.

Generally, the viability of S.thermophilus decreased Interestingly, Al-Otaibi [52] mentioned that thegradually about 1 log cycle and significantly (p<0.05) populations of bifidobacteria decreased significantlyin the three types of fermented rice beverages, during cold from the production day to the end of refrigerated storagestorage (Fig. 10). During storage, the viable cell counts of period for three weeks at 5±1°C in the tested eightthe bacteria ranged from (9.68, 9.78 and 9.82 log cfu/ml) commercial probiotic fermented milk products. The authorat the beginning of storage to (8.01, 8.04 and 8.02 log also reported that only two of the tested productscfu/ml) at the last day of storage for the fermented plain maintained 10 cfu/ml viable count of bifidobacteria tillsrice beverage, rice beverage containing pumpkin seed the end of cold storage period. According to Kurman andmilk and rice beverage containing sesame seed milk, Rasic [58], the viable level of bifidobacteria must be aboverespectively. The same trend was also observed in the 10 cfu/ml to provide therapeutic benefits. All the resultantviable cell counts of L. acidophilus and Bifdobacterium three types of rice beverages fulfilled this requirementBB-12 (Figs. 11 & 12). However, at the last day of storage for viability of bifidobacteria till the end of cold storagethere were no significant (p>0.05) differences between period. The viability losses or the reduction ofthe three types of fermented rice beverages in terms of bifidobacterial count may be due to the decrease of pHthe viable cell counts of S. thermophilus and values, post process acid production [59], sensitivity toBifidobacterium BB-12, while significant (p<0.05) oxygen [60] and metabolites such as hydrogen peroxidedifferences were found between fermented plain rice and ethanol and to bacteriocins produced by lactic acidbeverage and fermented rice beverage containing bacteria [61]. In the present study, good cell viability waspumpkin or sesame seed milk in terms of the viable cell kept throughout storage. These results are in agreementcounts of L. acidophilus. Similar pattern was also with those obtained by Medina and Jordano [61] andobserved for probiotic millet beverage samples (data not Lankaputhra et al. [62] who found poor viability ofshown). Bakirci and Kavaz [51] mentioned that the total bifidobacteria in yoghurt during storage. The coliformviable counts of S. thermophilus, L. acidophilus and and pathogenic bacteria and molds and yeasts countsBifidobacterium spp. decreased slightly during the were not detected in all the three tested rice beverages onrefrigerated storage, but remained at sufficient levels the production day and during the refrigerated storage at(>6 log cfu/g) for up to 14 days. In addition, Al-Otaibi 4±1°C for 15 days.[52] found that the viable counts of L. acidophilus ofthe commercial probiotic fermented milk products CONCLUSIONreduced about 1-2 log cycles, during refrigeratedstorage period. Speck [53] reported that 10 to 10 viable Shelf-life study revealed that during 15 days storage8 9

cells of L. acidophilus should be ingested daily to at 4°C, pH and acidity of rice or millet-based beverageensure that consumers receive health benefits. All the remained above 4 and lower than 1%, respectively,three types of rice beverages under study fulfilled this while numbers of S. thermophilus, L. acidophilus andrequirement for viability of L. acidophilus at the end Bifidobacterium BB-12 remained above 8 log cfu ml .of the storage. Moreover, several studies have shown This study shows a new possibility to make an acceptablethat yogurt bacteria (S. thermophilus and L. debrueckii fermented product based mainly on rice or millet which arespp. bulgaricus) survive well in yogurt throughout suitable substrates that’s can support high cell viabilitythe shelf life [49, 54]. Olson and Aryana [55] found that during cold storage for 15 days for different probioticthe L. acidophilus counts in plain yogurt tended to strains. In addition these produced beverages combines,decrease from 6.84 to 4.43 log cycles during 8 weeks of therefore interesting nutritional qualities and probioticstorage time. The present results were better than those characteristics. This kind of study can facilitate theobtained by Mousavi et al. [56] for probiotic pomegranate development of new, fermented, non-dairy, nutritionallyjuice. The authors reported the reduction of 3 log cycles well-balanced food products with unique physicalfor L. plantarum and L. debrueckii after 14 days of storage properties.at 4°C. High survival rates of L. casei in non-dairyfermented products stored refrigerated was reported by REFERENCESPereira et al. [57], who studied L. casei fermentation andsurvival in cashew apple juice. 1. Angelov, A., V. Gotcheva, R. Kuncheva and

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