Full Text - Effect of refrigerated storage on the probiotic survival and sensory properties of milk/carrot juice mix drink

Effect of refrigerated storage on the probiotic survival and sensory properties of milk/carrot juice mix drink

Mohammad Daneshi¹ · Mohammad Reza Ehsani² · Seyed Hadi Razavi*¹ · Mohsen Labbafi¹

¹University of Tehran, Agricultural Campus, Faculty of Agriculture Engineering and Technology, Department of Food Science, Technology and Engineering, Karaj, Iran
²Islamic Azad University, Science and Research Branch, Department of Food Science & Technology, Tehran, Iran

Financial support: This research was supported financially by Pegah Tehran Dairy Co.

Keywords: carrot juice, functional beverage, non-fermented, probiotic, viability.

Background: There is a genuine interest in the development of probiotic milk and juice based beverages because they are a good-vehicle to deliver probiotic microorganisms to consumers. For this purpose, the viability and metabolism of four probiotic strains (Lactobacillus acidophilus LA5, Bifidobacterium lactis BB12, L. rhamnosus and L. plantarum) were studied in non-fermented milk and carrot juice mix drink. The drinks were evaluated in 5 days interval for viable cell count, pH, acidity, sedimentation and sensory quality during refrigerated storage at 4 ± 2ºC for up to 20 days.

Results: The results showed that all strains had good viability in milk/carrot juice drink (88-98%), but L. acidophilus LA5 seemed more stable than three other strains. The levels of pH and acidity were ranged 5.33-6.6 and 0.13-0.31%, respectively. The drinks inoculated with L. rhamnosus and control (non-probiotic) showed more variation in pH and acidity. The most sedimentation was detected in drinks inoculated with L. rhamnosus, reaching 3.73 mL/10 mL sample. Sensory assessment indicated lowest acceptability in control and milk/carrot juice drink inoculated with L. rhamnosus, respectively.

Conclusion: This study indicated that some probiotic bacteria can be applied by food producers to produce functional drinks with an increased shelf-life.

There is a major trend for consumers to purchase foods which provide excellent nutrition and health benefits, especially those that can prevent disease and/or maintain health, so development of foods that promote health and well-being is one of the key research priorities of food industry (Yoon et al. 2004). In the worldwide functional foods market, dairy products are key products and, among those dairy-based products, functional beverages account for an important fraction of this sector (Rodrigues et al. 2012). This trend has favoured consumption of functional drinks that enriched with physiologically active components such as prebiotics, probiotics, vitamins, minerals, dietary fiber, plant sterol and other functional ingredients (Betoret et al. 2011). Probiotics are defined as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host beyond inherent general nutrition including the improvement of the intestinal microbial balance" (Fuller, 1989; FAO/WHO, 2002). Micro-organisms most commonly used as probiotics belong to the heterogeneous group of lactic acid bacteria (Lactobacillus, Enterococcus) and to the genus Bifidobacterium. These bacteria have been used widely in dairy and non dairy products (Holzapfel and Schillinger, 2002). Milk is a good-vehicle to deliver probiotic microorganisms to consumers.

Traditionally, probiotics have been added to yoghurt and other fermented dairy products (Ranadheera et al. 2010; Rivera-Espinoza and Gallardo-Navarro, 2010). On the other hand, fruits and vegetables have been suggested as ideal media for probiotic growth because they inherently contain essential nutrients, they are good-looking and have good taste (Luckow and Delahunty, 2004; Sheehan et al. 2007). There is a genuine interest in the development of fruit and vegetable juice/milk based functional beverages with probiotics because they have taste proﬁles that are appealing to all age groups and because they are perceived as healthy and refreshing foods (Tuorila and Cardello, 2002; Yoon et al. 2004; Sheehan et al. 2007). Besides being delicious, these beverages are highly nutritious. Fruits and vegetables contain various bioactive compounds with antioxidant activities, such as vitamins A, C and E, which have a high antioxidant capacity (Sánchez-Moreno et al. 2006; Zulueta et al. 2007), and phenolic compounds, which recent studies have shown to be good contributors to the total antioxidant capacity of the foods that contain them (Dillard and German, 2000; Vinson et al. 2001; Zulueta et al. 2007).

Carrots (Daucus carota L.) as one of the most popularly consumed vegetables are rich in functional food components such as vitamins (A, D, B, E, C, and K) and minerals (calcium, potassium, phosphorus, sodium, and iron). In carrots, β-carotene is present in a high concentration and can be considered as one of the most essential micronutrients because of its antioxidant activity and its property to act as a provitamin A (Knockaert et al. 2012). It has been noted that 100 g of carrot contains between 6 mg and 15 mg of carotenoids, mainly β-carotene (2-10 mg) (Bandyopadhyay et al. 2008; Kun et al. 2008). Thus, an increased intake of carrot may favour the massive synthesis of vitamin A. Moreover, the carotenoids and other antioxidants present in carrot play an important role in the inhibition and/or interruption of oxidation processes, as well as in counter balancing free radical activities (Bandyopadhyay et al. 2008; Kun et al. 2008). Therefore, carrot may protect humans against certain types of cancer and cardiovascular diseases. Additionally, the allergenic effect of carrot is very low or lacking (Bandyopadhyay et al. 2008).

An important portion of carrots wasted every year due to quality defects and lack of food industry consumptions. These carrots can be used in other products like milk or beverage. The use of carrot juice as a natural flavourings agent in the preparation of milk based drink may prove to be beneficial due to highly nutritious nature of carrot which is the richest natural source of β-carotene and contains appreciable amounts of other vitamins, anthocyanin pigments and minerals (Charanjiv et al. 2006). However, carrot juice is very sensitive to microbial spoilage and oxidation and the taste and appearance of it change in short time so it needs special treatment in processing to develop an acceptable and stable beverage.

Studies dealing with the probiotic carrot juice can be found in literature but to our knowledge, this is the first report on the viability of probiotic bacteria in non-fermented milk/carrot juice mix beverage. Also the present work studies the changes of pH, acidity, sedimentation and sensory quality of the product during cold storage. The next aim is determining of suitability of this beverage as a probiotic milk based drink.

Commercial low fat UHT milk (1% fat) was prepared by Pegah Tehran Dairy Co. and used as raw material in this study. Physicochemical characteristics of the used milk are given in Table 1.

Carrots (D. carota L. var Nantes) were purchased from a local store. After being washed thoroughly, they were peeled and chopped (thickness approx. 0.5 cm) and washed again. Then they were blanched in acidified water by citric acid (0.5 g/kg carrots) at 80ºC for 6 min. The juice was prepared using a laboratory juice extractor (PANASONIC^®, Japan). Coarse particles content was separated gravimetrically by centrifugation of carrot juice at 4000 rpm for 20 min at 20ºC by SIGMA^® centrifuge (model 2-16 K, Germany). The supernatant was separated with a pipette and moved to a sterile container. After pasteurization at 90ºC for 1 min, it was filled in opaque glass bottles and kept in refrigerator for next using. The chemical composition of treated carrot juice used in this study is also shown in Table 1.

First, milk was heated up to 60ºC and dry mix of sugar (6.5%) and high methoxy pectin (HMP, 0.2%) (Orana, Denmark) was added to it through a home blender with continuous stirring. Then the centrifugated carrot juice (30%) was poured to it and it was mixed thoroughly to obtain a homogenous texture. Then, the product was removed from blender and heated to 70ºC to increase efficiency of next homogenization process. Homogenization process was conducted at 160/40 bar by a 2-stage homogenizer (APV, Denmark). The milk/carrot juice (MCJ) mixed drink then pasteurized at 80ºC for 5 min and cooled down to 5 ± 1ºC for next stage. Our preliminary investigations showed that the addition of the concentration levels studied for sugar, HMP and carrot juice resulted in desirable sensory attributes.

The following probiotic strains were selected: L. acidophilus LA5 and Bifidobacterium animalis subsp. Lactis BB12 from CHR-HANSEN A/S, Denmark; L. plantarum subsp. plantarum (DSM No. 20179); and L. rhamnosus (ID 100271) from the Microbial Collection of Food and Science Department, Agriculture Campus, Tehran University, Iran.

The Lactobacillus bacteria in the frozen cultures were activated by spread plating on Man Rogosa Sharpe (MRS) agar (MERCK, Germany), after incubation anaerobically for 48 hrs at 37ºC (GASPAK, Darmstadt, Germany) but for B. lactis BB 12, spread plating was done on MRS + 0.5% cysteine hydrochloride (CyHcl). Then the colonies were inoculated in MRS-broth or MRS+CYHCL (MERCK, Germany) and incubated at 37ºC for 72 hrs anaerobically and the cell mass were harvested by centrifugation at 5000 x g for 15 min by a refrigerated centrifuge (SIGMA, model 2-16 k, Germany). After centrifugation, the cell pellets were washed in Ringers solution and the necessary inoculums to adding to product was calculated and added to pasteurized milk/carrot juice mixed drink.

After inoculations with different probiotic bacteria (each >10⁶ cfu mL^-1), each probiotic MCJ drink was filled in some sterile glass containers (100 mL), separately and all of them were kept under refrigeration at 4 ± 2ºC for 20 days. Samples were taken at 5 days intervals for microbiological and chemical analysis.

Samples were diluted in sterile Ringer´s solution and viable probiotic strains were determined by pour plate counting in duplicate on MRS agar (MERCK, Germany). Subsequently Lactobacillus and Bifidobacterium were plated into MRS agar and MRS agar + 5 mL/liter medium CyHcl (MERCK, Germany) respectively. Plates were incubated in anaerobic jar + GASPAK System. An anaerobic indicator (ANAEROTEST, MERCK, Germany) was used to control anaerobic conditions. The colonies were counted after 72 hrs of incubation at 37ºC, after which they were expressed as log cfu mL^-1.

Acidity of samples was determined according to the general titration method and based on lactic acid percentage. 10 mL of sample was titrated against 0.1 N NaOH in presence of phenolphthalein. The values of pH were measured by a digital pH meter (Model METTLER TOLEDO, Switzerland). The pH meter was calibrated using standard buffer solutions (Merck) at pH 4.0 and 7.0.

The sediment content was determined according to the standard procedure based on centrifugation. Samples (10 mL) were centrifuged at 1250 rpm for 15 min (SIGMA, model 2-16 k, Germany). The supernatant was decanted and the volume of sediment was determined (ISIRI, 2007).

Sensory quality evaluation of samples was performed by trained panel of assessors on a 9-point Hedonic scale (Amerine et al. 1965). Samples were served in plastic cups at temperature of 20ºC as recommended for sensory evaluation of fermented dairy products (IDF, 1997). Evaluation was based on colour, taste, flavour, consistency and overall acceptability. Each panellist scores were reflected on a hedonic scale of 1 to 9 where 1 = dislike extremely and 9 = like extremely.

Results were expressed as mean ± SD. Values were the average of triplicate experiments. Significant differences between the results were calculated by analysis of variance (ANOVA) with the help of SPSS software version 16 (SPSS Inc., Chicago, IL, USA). Differences at p < 0.05 were considered to be significant.

Viable counts (log cfu mL^-1) of 4 probiotic strains in MCJ drink during storage at 4ºC over 20 days are presented in Table 2. All strains attained viable cell numbers reduction of less than 1 log cfu mL^-1. L. acidophilus LA5 showed a stable level of viable cells (98.8% viability) in milk/carrot juice drink during storage. The other strains including B. lactis BB 12, L. rhamnosus and L. plantarum showed a viability of 91.9, 90.1 and 88.0 percent, respectively (Figure 1). L. acidophilus and L. rhamnosus remained viable above the critical level of 10⁶ cfu mL^-1 in MCJ drink for 20 days. The changes of viable cell counts of all the strains during cold storage are insignificant (p < 0.05) but the viability of L. plantarum decreased slightly and more than other probiotics in the same time. The results also showed viability of B. lactis BB12 in milk and carrot juice mix drink is relatively high during refrigerated keeping for 3 weeks. All the probiotic samples showed a longer shelf life in compare with controls except L. rhamnosus which was less.

Based on existing standards and from a health view-point, it is very important that probiotic strains retain their viability and functional activity throughout the shelf life of product. Some probiotic strains do not grow well in milk. In such cases the presence of plant-based ingredients may improve the growth of probiotic cultures in milk such as tomato juice, peanut milk, soy milk, carrot and cabbage juice (Nadal et al. 2010). Today, most available dairy products with probiotic bacteria are fermented milks such as yoghurt. However, little is known of the stability of probiotics in non-fermented products. One of the main advantages of using non-fermented milk products as carriers of probiotics is the absence of fermentation end-products. Organic acids and flavour compounds have a negative impact on the survival of probiotic LAB. Sweet acidophilus milk is an example of a sweet milk product with added probiotic L. acidophilus (Mattila-Sandholm and Saarela, 2003). In this research, the viable cell counts of the four probiotic bacteria in the non-fermented MCJ drink ranged from 6.75 to 5.78 log cfu mL^-1 after 20 days of cold storage at 4ºC. The L. plantarum, L. rhamnosus and B. lactis showed slightly reduction in cell count whereas L. acidophilus counts remained stable at 6.6 log cfu mL^-1.

It was shown that L. acidophilus LA5 is a slow-growing organism and survives well in milk and dairy products (Ostlie et al. 2003). The L. acidophilus LA5 strain ferments lactose to DL-lactic acid and it is very stable and has a high resistance towards acids in fermented dairy products. The optimum pH for its growth is between 5.5-6 (Chr. Hansen A/S, 2008). In this research, during the storage time, pH values of three probiotic MCJ drinks were almost constant (except of L. rhamnosus), whereas the initial pH value was about 6.5, it did not changed significantly after 20 days of cold storage at 4ºC. The main factors for loss of viability of probiotic organisms have been attributed to the decrease in the pH of the medium and accumulation of organic acid as a result of growth and fermentation (Yoon et al. 2004). So, high viability of the probiotics in this study might be due to constant pH throughout the 3 week - storage period and/or nutrient content of carrot juice-supplemented milk. Whilst there are a few researches about non-fermented probiotic foods, the effect of refrigerated storage on the viability of probiotics has been studied by many researchers. Mortazavian et al. (2007) investigated the effect of cold storage temperature on the viability of probiotics in yogurt and reported that the highest viability of L. acidophilus after 20 days, was observed in less cold storage temperature (2ºC), whereas for B. lactis the highest viability was obtained when yogurt was stored at 8ºC. Saccaro et al. (2009) noted a reduction of more than 2 log 10 cycles /mL in the counts of L. acidophilus at the end of the storage period in yogurt. L. acidophilus has no adverse effects on the taste, appearance, or palatability of the product. Furthermore, it is able to survive in the product until consumption (Hoppe and Larsen, 2008).

The optimum pH for growth of Bifidobacterium is 6-7. Therefore, the neutral pH of the non-fermented MCJ drink can prevent the decline of bifidobacteria populations. In this study, the highest viability after L. acidophilus LA5 was observed in B. lactis BB12 (Figure 1). Hughes and Hoover (1995) suggested the possibility to enhance the survivability of bifidobacteria in skim milk at refrigerated temperature with keep the pH unchanged. On the other hand, B. lactis BB12 do not grow well in milk (Ostlie et al. 2003) and loss of bifidobacteria viability due to low proteolytic activities, low betagalactosidase activity and low availability of nutrients, has also been reported by Martínez-Villaluenga et al. (2006), Kun et al. (2008), and Charalampopoulos and Pandiella (2010) in dairy products. Since bifidobacteria strictly anaerobic, so the dissolved oxygen levels in the product have also been cited to be important factors affecting the survival of probiotic bacteria in dairy products (Shimamura et al. 1992). Kun et al. (2008) reported that bifidobacteria were found to be capable of growing well on pure carrot juice without nutrient supplementation but its growth in milk is generally slow. Bifidobacteria produce acetic and lactic acids under the fermentation process where two moles of glucose result of three moles of acetate and two moles of lactate. They can ferment galactose, lactose and fructose because of possessing Fructose 6 phosphate phosphoketolase (F6PPK) (Shah, 1997). The growth and activity of bacteria in non- fermented probiotic foods have been studied by some researcher. Zareba et al. (2011) demonstrated that the differences in the volatile profiles of probiotic supplemented non-fermented milk and milk fermented by B. animalis subsp. lactis BB-12 indicated the formation of volatile compounds not only during the fermentation process, but also during storage at low temperature. In this research, the MCJ drink was a good medium to keep viability of L. rhamnosus more than 90% during cold storage, attaining viable cell numbers of 6.75-6.08 log cfu mL^-1 after 20 days. After storage for 15 days at 4ºC a faster reduction in viable cells was observed for L. rhamnosus. However, after 20 days, the viable cell count of L. Rhamnosus was still above 6 log cfu mL^-1. Unlike the L. acidophilus, L. plantarum and B. lactis that showed a stable pH in the storage period, L. rhamnosus showed a further decrease in pH from 6.47 to 5.33. L. rhamnosus is a facultative heterofermentative bacterium that ferments hexoses such as lactose and fructose tolactic acid, and also pentoses to a mixture of lactic and acetic acids (Oliveira et al. 2012). It has been shown that L. rhamnosus survive well in dairy products (Ostlie et al. 2003; Chr. Hansen A/S, 2008; Rivera-Espinoza and Gallardo-Navarro, 2010). Alegre et al. (2011) reported L. rhamnosus remained viable in orange juice and apple wedges over 12 and 4 weeks of storage at 4ºC, respectively. The survival rate of this organism in probiotic yoghurts was found to be more stable than L. acidophilus (Saccaro et al. 2009). L. plantarum is an important species in the fermentation of various plant and some animal products and it is known to produce antimicrobial substances, e.g. plantaricin, that are active against certain bacteria and pathogens (Cebeci and Gürakan, 2003). L. plantarum has the coding capacity for the uptake and utilization of many different sugars, uptake of peptides, and formation of most amino acids. The high galactosidase and glucosidase activities detected, and relatively low activities toward other carbon sources, suggest that L. plantarum strains prefer glucose and lactose for their carbon and energy requirement (Georgieva et al. 2009). L. plantarum indicated the ability to adapt to many different conditions (De Vries et al. 2006) but in the present study, the lowest cell survival was observed for L. plantarum with a reduction of 0.8 log cfu mL^-1 after 20 days in cold storage.

The international standard FIL/IDF describe that the probiotic products should be contained minimum of 10⁶ viable probiotic bacteria per gram of product at the time of consumption for health and functional claiming (Samona and Robinson, 1991; Roy, 2005), so the studied MCJ drinks in this research could be probiotic after 20 days keeping in cold storage if the provided initial cell counts in the product should be increased to 10⁷-10⁸ cfu mL^-1. The present results showed that some strain of probiotic bacteria can prolong the shelf-life of MCJ drinks in comparison with non-probiotic drinks. In this study, fermentation was limited by keeping the samples in refrigerator but it was reported that bacteria cells have some fermentative activity even during storage at 6ºC (Nighswonger et al. 1996). Lactic acid bacteria not only improve health when consumed, but they can also play a protective role against pathogens in the product itself during storage by competing with pathogens for nutrients, producing organic acids and bacteriocins (biopresevation). Biopreservation is the extension of storage life and enhancing of safety of foods using the natural or controlled microflora and/or their antimicrobial products (Rodgers, 2001; Alegre et al. 2011).

The changes of pH and acidity of the different probiotic MCJ drinks studied in this work were shown in Figure 2. The pH of carrot juice before adding to milk was about 5.7. After adding of 30% the carrot juice to low fat milk, there was significant decrease in pH of milk from 6.7 to 6.48. During cold storage of control samples (without any probiotics) at 4ºC, the pH did not show significant change in first week but it is decreased to 5.85 in 3^rdweek. On the other hand, there is an insignificant increase (p ≤ 0.05) in the pH of probiotic carrot flavoured milk except L. rhamnosus inoculated sample in days of 5 and 10 in comparison with first day. L. acidophilus LA5 showed a 0.13 decrease in pH of MCJ drink during storage time. Despite the slight increase in the acidity during storage, the pH of the drink remained unchanged over the 3 week storage, indicating that the buffering capacity of the beverage was high. A similar result was reported by Nualkaekul and Charalampopoulos (2011) for Lactobacillus plantarum in fruit juices stored under refrigeration. Over the total storage period, the most changes in pH were seen in L. rhamnosus included drinks, which it decreased from 6.47 in day 1 to 5.33 in last day. The acidity of milk/carrot juice drinks was about 0.13 based on % lactic acid which it is less than milk in production day. L. rhamnosus showed highest rate of change in acidity and after 10^th day, the differences between this sample and other probiotic MCJ drinks became significant (p ≤ 0.05). However, with increasing the acidity of L. rhamnosus included drink and control sample, they spoiled after 10 and 15 days, respectively.

The sedimentation values ranged from 0.35 in B. lactis BB12 to 3.73 mL/10 mL in L. rhamnosus, after keeping in cold storage for 20 days (Figure 3). The sedimentation values for L. plantarum and L. acidophilus were 0.4 and 0.5 mL/10 mL, respectively. The results indicated a higher amount of sediments in MCJ inoculated with L. rhamnosus compared to other MCJ drinks. The sedimentation value is related directly to acidity and with increasing of acidity, the amount of sedimentation increase, too (Figure 3). Charanjiv et al. (2006) reported that the sediments of carrot flavoured milk ranged from 0.1 to 0.2 mL/10 mL in different samples during 4 days keeping at 4ºC.

The results of sensory evaluation are given in Table 3. The scores allocated for colour, flavour, taste and consistency showed that during the first 5 days of the storage period all samples had the highest sensory acceptability. The sensory properties declined in the samples of control and L. rhamnosus added MCJ after 5^th day as acidic or spoiled flavour and taste increased. However, after 15 days of storage, three other samples showed good sensory evaluation with gaining more than 70% of total score. In recent samples, the lowest taste acceptability observed after 20 days storage in L. acidophilus followed by B. lactis and L. plantarum. The colour scores were highest as compared to flavour, consistency and taste scores. Figure 4 shows the overall acceptability of milk/carrot juice samples during storage. The overall acceptability scores of control (blank) and L. rhamnosus were found to be less than 5 after 5 and 10 days storage, respectively, presumably due to the chemical changes and production of organic acids through the activity of microorganisms resulting to increase the acidity. MCJ drink inoculated with L. acidophilus, B. lactis and L. plantarum showed higher sensory acceptability over 20 days storage (scores ≥ 6). The findings for blank MCJ drinks are in conformity with the reports from Charanjiv et al. (2006) who showed that carrot flavoured milk remained in good condition for 4 days under refrigeration.

The present study was undertaken to identify suitable probiotic bacteria which can survive in milk/carrot juice drink during storage at 4ºC. All four strains showed an acceptable viability with less than one log cfu mL^-1 reduction at refrigerator temperature for 20 days. The MCJ drinks inoculated with L. acidophilus LA5, L. plantarum and B. lactis BB12 showed a longer shelf life (3 weeks) in comparison with L. rhamnosus and non-probiotic MCJ drinks with 1 and 2 weeks, respectively. If the count of each bacterial strain has to be as recommended (6 log₁₀ cfu mL^-1), the microbial load in the inoculums of the probiotic bacteria has to be increased to 7-8 log₁₀ cfu mL^-1 to achieve such a target at the end of the storage period of the product. So, after considering all the results it can be suggested that L. acidophilus LA5, L. plantarum and B. lactis BB12 are suitable probiotics for exploitation in fresh milk/carrot juice drink.

The authors would like to thank the Department of Food Science, Technology and Engineering, Agricultural Campus, University of Tehran for the support provided for this work.

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