Functional dairy beverages have represented a continuously growing sector within dairy foods in the past few years (Tirloni et al., Reference Tirloni, Vasconi, Cattaneo, Moretti, Bellagamba, Bernardi and Stella2020). There have been improvements in the processing of ingredients, launch of a considerable variety of beverages (whey-based, probiotic, herbal-milk, fat-rich etc) and development of beverages specific for age groups (for children, adults, old age persons). Today, a variety of dairy-based beverages are available globally, however, the market place for many of these beverages is still limited to local areas. It has been reported that probiotic dairy beverages have gained a prominent place in the functional dairy beverages market (Fazilah et al., Reference Fazilah, Ariff, Khayat, Rios-Solis and Halim2018). Dairy beverages are prepared from milk or its derivatives, with or without the addition of other ingredients and must contain at least 51% of the dairy base formulation. In addition to this, the beverage can be subjected to a fermentation process using yoghurt culture/probiotic culture. This not only results in the production of lactic acid upon utilization of lactose but also makes the product more nutritious and digestible. Putative health benefits of probiotics have been proposed (see review by Ibrahim et al., Reference Ibrahim, Gyawali, Awaisheh, Ayivi, Silva, Subedi, Aljaloud, Siddiqui and Krastanov2021) and in addition the dairy product market has also witnessed development of products with a combination of probiotics and prebiotics, also known as ‘synbiotic’ dairy products (Chand et al., Reference Chand, Kumar, Kumar, Deshwal, Rao, Tomar and Sharma2021; Zahed et al., Reference Zahed, Khosravi-Darani, Mortazavian Farsani and Mohammadi2021).
Fermented milk beverages are universally accepted and enjoyed, and can be given added functionality by the incorporation of probiotics, prebiotics and other health promoting ingredients from herbal and plant sources (Sharma et al., Reference Sharma, Rao and Singh2021a). It is suggested that beverages of this sort could help in combating various intestinal disorders as well as hypertension problems, and consumption may be correlated to improved digestion, lactose utilization and prevention of colon cancer (Sarwar et al., Reference Sarwar, Aziz, Al-Dalali, Zhao, Zhang, Din, Chen, Cao and Yang2019; Ibrahim et al., Reference Ibrahim, Gyawali, Awaisheh, Ayivi, Silva, Subedi, Aljaloud, Siddiqui and Krastanov2021). However, the main problem associated with probiotic/prebiotic incorporated product is the viability of micro-organisms in the product by the end of the storage period. Also, the product fermented by Lactobacillus strains is considered to be sour in taste as the product contains 0.6–0.8% lactic acid owing to lactose metabolism (Jirasatid and Nopharatana, Reference Jirasatid and Nopharatana2020; Sharma et al., Reference Sharma, Ozogul, Bartkiene and Rocha2021b). Thus, there exists the need for sweetener addition so as to make it sensorially acceptable to the consumers and to make sure that consumers get viable bacteria from the product even at the end of shelf-life of the product. The present work was designed to optimize the level of sugar and water in low-calorie synbiotic milk beverage (LCSMB) incorporated with indigenous probiotic strain and a combination of inulin and oligofructose. Further, the technological composition and probiotic survivability were evaluated in LCSMB stored in different packaging materials at refrigeration temperature.
Materials and methods
Experimental design and preparation of milk beverage
In this work, synbiotic yoghurt was prepared as per the method described by Chand et al. (Reference Chand, Kumar, Kumar, Deshwal, Rao, Tomar and Sharma2021) using L. rhamnosus 610 and S. thermophilus 74. Water and sugar levels were optimized on the basis of physico-chemical, sensory and probiotic count for the preparation of low calorie synbiotic milk beverage. About 5–7 l of beverage was prepared and samples were aseptically filled (at ambient temperature) and stored in four different packaging materials; polypropylene (PP), high impact polystyrene (HIPS), high density polyethylene (HDPE) and glass under refrigeration conditions (4 ± 1 °C). Characteristics of the materials are reportd in online Supplementary Table S1. The samples were further analysed for different parameters during storage. The methodology for the preparation of LCSB is described in online Supplementary Figure S1.
Analytical methods
The pH of samples was determined using a pH meter (PHAN Lab India Model, Labtek Engg. Pvt. Ltd., India). Total solids (%), crude protein (%), titratable acidity (% lactic acid), fat (%), lactose content (%) and ash (%) content of the product were estimated as described in AOAC (2000). Colour values (L*, a*, b*) of samples were measured using Hunter Lab Colourimeter (Mini Scan XE Plus, Hunter Associates Laboratory, Reston, Virginia, USA). The apparent viscosity of the LCSB samples was determined using a dynamic rheometer (MCR 52, Anton paar, Ostifildern, Germany) employing a parallel plate configuration (CP-75) of 75 mm diameter at 20 °C.
Microbiological analysis
The lactic acid count of LCSMB samples was determined using De Man, Rogosa and Sharpe agar by pour plate method (Guo et al., Reference Guo, Wang, Yan, Chen, Liu and Zhang2009). Total coliform count and yeast and mould count in samples were determined using violet red bile agar and potato dextrose agar, respectively (IS:5401, 2002).
Sensory evaluation
Sensory evaluation method used the 9-point hedonic scale where 9 = extremely liked and 1 = extremely disliked. Sensory panel members (age group between 25 years and 60 years) willing to consume the beverage and having adequate knowledge were selected from ICAR-National Dairy Research Institute. Evaluation followed the procedures described in Sharma et al. (Reference Sharma, Singh, Deshwal, Rao and Kumar2021c).
Statistical analysis
The experiment was performed thrice with the readings taken in duplicate. Data was analysed using two-way analysis of variance (ANOVA) followed by Tukey's comparison test to establish the significance of differences among the mean values at 5% level of significance using SPSS version 20.0 software of M/s IBM Corporation.
Results and discussion
Optimization of water and sugar level for preparation of low-calorie synbiotic milk beverage
Physico-chemical characteristics of LCSMB prepared with different levels of water and sugar are presented in online Supplementary Table S2. The addition of water at 40% showed significantly (P < 0.05) higher pH value than 30% and 35%. The addition of water might have changed the pH of the beverage due to a decrease in the concentration of hydrogen ions. Similar results were reported by Jirasatid and Nopharatana (Reference Jirasatid and Nopharatana2020) in acidophilus milk added supplemented with coconut sugar syrup. However, irrespective of the sugar level, a beverage with 40% water had significantly (P < 0.05) lower acidity followed by 35% water suggesting that the addition of water also had a major role in maintaining the pH and acidity of the beverage samples. The apparent viscosity of the samples decreased with the addition of sugar syrup due to the decreased total soluble solids. 30 and 6% level of water and sugar, respectively in beverage had significantly (P < 0.05) higher viscosity than 7 and 8% level. This might be due to the viscous nature of syrup and the hygroscopic nature of sugar. Viscosity changes in the milk beverages are also due to whey protein-casein micelles interaction, thus resulting in changes in pH in different formulations. Decreased whey protein and casein micelle interactions owing to the water dilution effect bring pH to increased level while viscosity is increased. Also, the alterations in the interactions between macromolecules present in the food matrix such as fat, proteins with the changes in food formulation are held responsible for viscosity changes (Haji Ghafarloo et al., Reference Haji Ghafarloo, Jouki and Tabari2020).
Colour values are also reported in online Supplementary Table S2. The L* value did not vary significantly (P > 0.05) with change in sugar level, however, beverage containing 30% water (73.93 ± 0.55) had significantly (P < 0.05) higher L* values than 35% (70.12 ± 0.42) and 40% water (60.45 ± 0.36). A reduction in the lightness of the samples with an increase in the level of water could be attributed to a decrease in protein content (Kaur et al., Reference Kaur, Chawla, Mishra and Sivakumar2019). Increasing the water level decreased the a* value but increased the b* value. The change in colour scores with the addition of different levels of water and sugar was releated to their corresponding decreased appearance scores (sensory evaluation) in the present investigation. A similar correlation has been established by El-Sayed and El-Sayed (Reference El-Sayed and El-Sayed2020) while studying the quality characteristics of thandai during storage.
The probiotic count did not vary significantly (P > 0.05) in the samples with different sugar levels (online Supplementary Table S2). However, beverage samples with 30% water had significantly (P < 0.05) higher probiotic count than 35 and 40% water. Similar results were reported by Wang et al. (Reference Wang, Liang, Wang and Guo2018) in synbiotic lassi during storage (Sharma et al., Reference Sharma, Ozogul, Bartkiene and Rocha2021b, Reference Sharma, Singh, Deshwal, Rao and Kumar2021c).
Sensory scores revealed that irrespective of the water level, higher appearance scores were observed with samples containing 8% sugar level (online Supplementary Table S3). Higher scores for an appearance with the addition of sugar syrup in lassi have also been reported by Cavalcante et al. (Reference Cavalcante, Campos and Timoni2016). Significantly (P < 0.05) higher flavour score was also observed as the water and sugar levels increased in the beverage samples. The sourness of the samples decreased as sugar level increased from 6 to 8% at constant water level indicating that sugar could mask the effect of sourness significantly. Beverage with a 40% water level was highly acceptable at a particular sugar level. However, among different sugar levels, a beverage with 8% sugar and 40% water had significantly higher overall acceptability score. Based on these results, low-calorie synbiotic milk beverage was prepared with the addition of 40% water and 8% sugar in low-calorie synbiotic yoghurt.
Storage stability of low-calorie synbiotic milk beverage in different packaging materials
The low-calorie synbiotic milk beverage prepared as just described had 14.66% total solids, 2.24% protein and 3.76% lactose (online Supplementary Table S4). The pH of the beverage was around 4.81 with 8.91 log cfu/ml of the probiotic count. Further physico-chemical, microbiological and sensory characteristics were evaluated during storage at 4 °C. In this respect it is important to note that spoilage of the product occurred after day 9 in PP and HDPE and after day 12 in HIPS. Only the product stored in glass remained acceptable through to day 15.
The pH of the samples decreased significantly during storage in each of the four packaging materials (Table 1). The post acidification might have been the main reason for the decline in pH of samples (Mani-López et al., Reference Mani-López, Palou and López-Malo2014) and this increased acidity from the beginning to the end of the storage period might be due to the increased metabolic activity of probiotic organisms with time. Throughout the storage period, the rate of decreased pH and increased acidity was least in case of samples stored in glass. Glass packaging material is reported to offer superior barrier performance to gases and vapours and provides high stability over time (Glušac et al., Reference Glušac, Stijepić, Durdević-Milošević, Milanovic, Kanuric and Vukic2015).This might have protected samples against the decreased pH and thus, an increase in acidity.
Effect of packaging materials on physico-chemical characteristics and microbial count of low calorie synbiotic milk beverage during refrigerated storage
All the values are mean ± sd (n = 9); *NA, Not assessed.
abcd Mean values in a row with at least one similar superscript do not differ significantly (P > 0.05).
ABCD Mean values in a column with at least one similar superscript do not differ significantly (P > 0.05).
The changes in colour value (L*, a* and b*) during storage (Table 1) suggested that the darkening of products could be due to non-enzymatic browning reactions (Paul-Sadhu, Reference Paul-Sadhu2016). Also, chemical reactions in the presence of oxygen might have caused decrease in lightness of the beverage. Higher bacterial growth in milk might result in rapid use of oxygen by bacteria, which in turn would have caused changes in the colour of milk. Also, colour of milk could change after prolonged storage which can be correlated to the change in the a* values with increasing bacterial numbers in the present investigation. The yellow colour of the milk and milk products was related directly to the growth of certain micro-organisms (Mani-López et al., Reference Mani-López, Palou and López-Malo2014).
In general, the microbial count increased with advancing storage period (Table 1). Packaging material had no effect (P > 0.05) on promoting or inhibiting the growth of probiotic bacteria. However, the probiotic count was observed to decrease significantly (P < 0.05) in all the samples from day 3 onwards. This might be because the acidic environment can harm probiotic organisms (Somashekaraiah et al., Reference Somashekaraiah, Shruthi, Deepthi B and Sreenivasa2019). The penetration of oxygen through packaging material might also be responsible for the decrease in the probiotic count. Among all packaging materials, only samples stored in glass were observed with probiotic growth of 7.2 ± 0.28 up to day 15 of storage, since spoilage occurred in the otehr samples after day 9 or 12 (Table 1). According to FSSAI, the viable number of organisms in food with added probiotic ingredients shall be more than or equal to 7 log cgu/mL.
Total viable count (log cfu/ml) did not show any significant (P > 0.05) difference in the samples throughout the storage period, irrespective of packaging materials. Coliform was not observed in any of the samples at any time. This indicates that hygienic conditions were practised during the preparation of the beverage. Yeasts and moulds were not observed up to day 6 of storage, but were present thereafter. Samples stored in glass packaging material were observed with a lower count of yeast and mould because of less oxygen penetration.
Sensory evaluation
Sensory evaluation during storage is reported in Figure 1 and online Supplementary Table S5. The appearance of the product deterioriated gradually in all materials and especially when stored in PP and HDPE. This decrease in appearance scores might be related to the increase in total viable count. Flavour of the product also showed a somewhat similar trend (Fig. 1). Samples stored in glass retained the flavour better than HIPS and HDPE (P < 0.05) which might be due to less oxygen permeability of glass, resulting in the delayed occurrence of deteriorating reactions which lead to off-flavours (Ishibashi and Shimamura, Reference Ishibashi and Shimamura1993). The decrease in body and texture scores of beverage samples with storage might be correlated to increased acid production after day 6 of storage, thus resulting in whey separation of the product. Generally, sourness increased significantly (P < 0.05) in all the samples throughout the storage period. The sourness is related to lactic acid accumulation. Thus, relating to our results, exposure of lactic acid bacteria to oxygen during storage might have caused sourness in samples. There was no significant (P > 0.05) difference in the overall acceptability scores of the samples stored in different packaging materials till day 6 of storage, and only small differences thereafter. The evidence for improved performance of glass relies on the fact that spoilage occurred before day 15 in all other materials, and so requires substantiating.
Changes in sensory evaluation of low calorie synbiotic milk beverage stored in different packaging materials at refrigeration temperature.
In conclusion, for the preparation of a low-calorie synbiotic beverage, 40% water and 8% sugar can be added to synbiotic yoghurt to yield a product with acceptable sensory quality and good probiotic viability. The study compared four packaging materials and among these, glass followed by HIPS cups were found as better packaging material with shelf life of 15 and 12 d at 5 ± 1 °C, respectively.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029922000164
Acknowledgements
The first author gratefully acknowledges the ICAR-JRF Fellowship awarded to Prittam Chand to carry out this research work.
