Production of synbiotic Doogh enriched with Plantago psyllium mucilage

Abstract

Physical instability and loss of viability of probiotic bacteria are the most important problems in production of synbiotic Doogh. Some plant hydrocolloids have been recognised as effective components to prevent these problems. In this study the effect of Plantago psyllium mucilage (PPM) (0, 0.15%, 0.30%, 0.75% (w/w) on the physicochemical, microbial, and sensory properties of Doogh samples was evaluated by measuring phase separation, viscosity, flow behaviour, probiotic viability and sensory parameters. The results revealed that the stability of samples containing PPM were higher than samples without this hydrocolloid. By increasing the amounts of PPM, the viscosity of treated samples were increased compared to control sample. Herschel-Bulkley rheological model was an appropriate model for describing the flow behavior of Doogh formulated with PPM and the power-law rheological model was suitable model for describing the flow behavior of control samples. PPM had non-digestible food ingredients and improved the viability of Lactobacillus casei; therefore, this herbal mucilage may have prebiotic potential. Finally, the samples treated with 0.30% PPM on the 15^th day were chosen as the best formulation for the production.

Introduction

Now a days, consumers of dairy products like to use healthful dairy products such as probiotic and synbiotic Doogh containing active physiological compounds (Sedaghati et al. 2016). Probiotics are live microorganisms which when used in an adequate amount could confer a health benefit. Probiotics must survive during food stages and to improve the probiotic viability, indigestible oligosaccharides, like psyllium being used, as prebiotic ingredients. Prebiotic oligosaccharides are fermented in the intestinal tract by microbiota and reveal consequently allocate numerous natural benefits. Probiotics revealed health benefits such as anti-cancer, anti-microbial, anti-allergic and immune–stimulating properties. By using suitable substrates of probiotics, their survival can be increased in fermented milk products. The term synbiotic is used when a product contains both probiotics and prebiotics (Batista et al. 2017; Karim et al. 2017).

Lactobacillus spp. and Bifidobacterium spp. are the most prominent bacterial species used in probiotic products (Ahmadi et al. 2019), and Lactobacillus casei PTCC 1608 (L. casei) has been recognized in previous studies as one of the standard probiotic strains (Zomorodi et al. 2011). Nevertheless, the main problem with synbiotic Doogh is that probiotic viability dwindles in the final product during storage and then in the gastrointestinal tract (Ziaolhagh and Jalali 2017). Generally, 10⁶ cfu/mL or cfu/g of viable probiotic cells is accepted as the minimum level that confer a beneficial health effect to the host (Iravani et al. 2015). Previous studies showed that some hydrocolloids can act as prebiotic components to improve the viability of the probiotics (Karlton-Senaye et al. 2015; Ziaolhagh and Jalali 2017).

Another issue with Doogh is its acidic nature leading to casein aggregation and phase separation (Khanniri et al. 2019; Li et al. 2018). Different hydrocolloids, such as tragacanth gum, guar gum and high methoxyl pectin (HMP), have been applied to stabilize Doogh by creating a strong three-dimensional network for trapping water and caseins (Li et al. 2018; Pirsa et al. 2018).

Plantago psyllium is an annual plant belonging to the Plantaginaceae family; it is mostly found in Pakistan, India, Iran, and some parts of Europe. The seed husk contains a high concentration of fibrous hydrocolloids with ability to absorb water resulting in the formation of a colorless gel. The polysaccharide present in the Plantago psyllium mucilage (PPM) consists of L-arabinose, D-xylose and D-galacturonic acid. L-arabinose is a well-known promoter for probiotic bacteria growth, and the presence of this component in PPM can improve probiotic bacteria survivability (Guo et al. 2008). Also, the PPM powder may improve the stability of Doogh, as it boosts the formation of a powerful gel (Gharibzahedi et al. 2013). PPM is an anionic polysaccharide carrying a negative charge due to ionized carboxyl groups. In an acidic medium, most of the –COO⁻ groups are converted into –COOH groups; consequently, hydrogen bonds form between hydrophilic groups and increased crosslinking (Hashemi et al. 2015). In addition, Ca⁺ ions in fermented milk can form covalent cross-links between free carboxyl and amino groups along neighboring polymer chains, thus reducing the negative charge. Therefore, the interaction between Ca⁺ ions and negatively charged components could lead to a stronger network in Doogh (Guo et al. 2008; Hashemi et al. 2015).

Therefore, this study aimed to improve the probiotic viability and stability of Doogh using PPM powder to improve its acceptability of the consumers.

Materials and methods

Materials

Plantago psyllium was purchased from a local market in Tehran (Iran). Fresh milk (2.5% fat) was also purchased from a local milk producer in Tehran. Cultures of L. casei PTCC 1608 were brought from the Iranian Research Organization for Science and Technology Company (IROST) in Tehran. Lyophilized L. casei (more than 10⁷ cfu/mL) was incubated in MRS for 24 h at 37 °C using a CO₂ incubator (Memmert, Munich, Germany). MRS Agar and Broth (Man, Rogosa and Sharpe Broth) were provided by Merck (Darmstadt, Germany). Starter culture containing of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus was procured from Danisco (Copenhagen, Denmark). Sodium hydroxide was supplied by Sigma-Aldrich Chemie GmbH (Munich, Germany). Other chemicals and reagents used in this study were of the analytical grade and obtained from Merck (Darmstadt, Germany).

PPM powder extraction

Following Pawar and Varkhade (2014), an aqueous method was used to extract the mucilage of Plantago psyllium. The impurities were removed from the psyllium seeds; afterwards, 300 g of the seeds were milled using a laboratory mill (A11 IKA, Staufen, Germany) and passed through a sieve (600 μm) to separate the husk. The husk was then mixed with water in a ratio of 1:50 and left at room temperature for 24 h to absorb the water. The next stage was centrifuging at 30,240 g for 10 min. The resulting gel was mixed with 1:3 deionized water (DI), and the hydrocolloid sediment was isolated through centrifugation at 3360 g for 5 min. Finally, the hydrocolloid was washed with DI three times and then freeze-dried (FD10, Tehran, Iran).

Preparation of the Doogh samples

Fresh milk (2.5% fat) was pasteurized at 95 °C for 10 min and then cooled to 40 °C. Afterwards, the starter culture was added to the milk at the rate of 0.02% (w/v) and incubated at 43 °C to reach the acidity of 0.58% (pH around 4.02) and was then cooled. Then, PPM powder (0, 0.15%, 0.30%, 0.75% w/v) and 0.7% salt were dissolved in DI water (90 °C for 10 min). After that, the yoghurt (around 40% of the Doogh formulation) was diluted with the dilution mixture of the stabilizer and salt. The prepared Doogh was homogenized (T18 IKA, Staufen, Germany) at 16,262 g for 30 s at 60 °C. 250 ml polyethylene terephthalate bottles were then filled and the samples were pasteurized at 80 °C for 15 min. Then, the Doogh samples were inoculated using L. casei (8 × 10⁷ cfu/mL) after cooling to the fermentation temperature (37 ± 1 °C) for 8 h. The Doogh samples were stored at 4 °C for 30 days and all tests were performed in triplicate.

Physico-chemical analysis

The pH of the samples was measured using a digital pH meter (AZ 86,502, Taipei, Taiwan). Titrable acidity was measured by titrating the samples with 0.1 N NaOH (AOAC, 1990). The viscosity was assessed using a rheometer (MCR 301, Anton paar, Austria) with CC27 spindle. The samples were poured into the measuring vessel and sheared from 1.0 to 500 (1/s) at 20 °C. The obtained date was fitted to the Power Law model (Eq. 1) and the Herschel-Bulkley model (Eq. 2) to analyze the rheological properties of Doogh samples.

The Power Law model:

$$\text{t}=\text{k}{\left(\gamma \right)}^n$$ 1

The Herschel–Bulkley model:

$$\text{t}={\text{t}}_0+\text{k}{\left(\gamma \right)}^n$$ 2

where t is the shear stress (Pa), t₀ is the yield stress, γ is the shear rate (s⁻¹), k is the consistency index (Pas) and n is the flow behavior index (Vukic et al. 2018).

To measure the phase separation, the Doogh samples were equally poured into the test tubes and stored to 5ºC for 30 days. The volume of the separated serum (transparent phase) to total volume ratio of the samples was calculated in percentage (Khanniri et al. 2019).

Microbial analysis

For the microbiological analyses, 10 ml of the mixed samples were homogenized into a sterile beaker with 90 mL of sterilized saline solution (0.95% w/v) to obtain the initial dilution (1/10). By applying this dilution, a number of decimal dilutions were prepared using the same diluent. For the enumeration of the L. casei, the dilutions were plated in depth in MRS agar supplemented with vancomycin (10 mg/L) using the Pour Plate method for counting the number of colony-forming L. casei present in the liquid samples. The plates were placed in a CO₂ incubator for 72 h at 37 °C. The results were expressed as log cfu/g (Colombo et al. 2014).

Sensory analysis

The sensorial properties (color, taste, flavor, texture, and general acceptance) of samples were tested by ten trained panelists (5 women and 5 men, age 20–30) using a 5-point hedonic scale ranging from 1 (dislike extremely) to 5 (like extremely). These parameters were analyzed using chi-square test on the 30th day of the storage. The Doogh samples were prepared in numbered containers released to panelists at 4 ± 1ºC. Panelists used water after each test to wash their mouth (Arsenos et al. 2002).

Statistical analysis

The experiments were performed in triplicates, and the significant differences between means were analyzed using the one-way ANOVA and LSD post-hoc test (SPSS, version 22, 2016). The nonparametric data were analyzed using the Kruskal–Wallis test.

Results and discussion

Changes in pH and acidity

Figure 1a presents the pH values of the different Doogh samples during cold storage. The pH values of the Doogh samples were in the range of 4.09–4.18, and the addition of the PPM powder significantly decreased the pH values of the samples (p < 0.05). On the first day, the highest pH was detected for the control sample. On the 30th day, the samples containing 0.75% of the PPM powder displayed the lowest pH values. The pH values of all the samples decreased during the storage and the samples showed a significant difference during the cold storage (p < 0.05).

Fig. 1.

Effect of Plantago Psyllium mucilage (PPM) powder (Control, 0.15%, 0.30% and 0.75%) on pH (a), acidity (b) of Doogh samples during storage at 4 °C for 30 days

The acidity of the Doogh samples was in the range of 0.30%-0.52% and increased significantly (p < 0.05) during 30 days of storage (Fig. 2b). The results revealed that the acidity values of the treated samples increased significantly compared to the control samples (p < 0.05). On the 1^st day, the least acidity was detected for the control sample, while the sample containing 0.75% of the PPM powder showed the highest acidity on the 30^th day.

Fig. 2.

Effect of Plantago Psyllium mucilage (PPM) powder (Control, 0.15%, 0.30% and 0.75%) on Viscosity a Flow curves b of Doogh samples during storage at 4 °C for 30 days

The pH value and acidity of Doogh are critical factors for quality determination. Codex alimentarius (2018) has determined a maximum pH of 4.5 and a minimum acidity of 0.30% for Doogh. In the present study, the pH value and acidity of Doogh corresponded to the Codex-recommended limits. Moreover, the pH values and acidity of Iranian Doogh samples were in the range of 3.03–4.27 and 0.4%-1.67% in the study by Sari et al. (2018). Ziaolhagh and Jalali (2017) reported a range of 4–4.2 for the pH and 0.35%-0.42% for the acidity of bio-Doogh containing wild thyme essence and xanthan gum. In our research, the pH values of all the samples decreased while their acidity increased during the storage period. These changes in pH values and acidity might be related to the lactose fermentation as well as lactic acid and other organic acids produced by probiotic Lactic acid bacteria (LAB) like L. casei and non-probiotic LAB. Similar findings showing a reduction in the pH values of synbiotic Doogh during cold storage were recorded by Batista et al. (2017). In the presence of PPM powder, pH values decreased while acidity increased significantly; therefore, PPM powder might increase bacterial growth and acid production (Sari et al. 2018). Ziaolhagh and Jalali (2017) reported that the acidity of bio-Doogh containing wild thyme essence and xanthan gum increased during fermentation. The increasing trend of acidity in the presence of PPM powder in the treated samples corresponded to the probiotic viability results. Samples with higher probiotic viability revealed the highest acidity and the lowest pH values. It seems that PPM powder provides a valuable source of nutrients for L. casei, since it contains significant concentrations of arabinoxylan (arabinose 22.6% and xylose 74.6%), which is a well-known growth promoter for probiotic bacteria. Probiotic microorganisms probably consumed arabinoxylan and carbohydrates to produce small amounts of organic acids (Nami et al. 2017).

Phase separation

Table 1 presents the serum separation of different Doogh samples during cold storage. The serum separation of the Doogh samples increased significantly during storage (p < 0.05). On the 1^st day, the lowest phase separation was observed for the samples containing 0.75% of the PPM powder. On the 30th day, the control samples and those treated with 0.15% PPM powder exhibited the highest phase separation. The reducing effect of the PPM powder on the phase separation depended on its concentration, as higher concentrations of PPM powder had a stronger reducing effect.

Table 1.

Phase separation percentage of Doogh samples containing different concentrations of Plantago Psyllium mucilage (PPM) (Control, 0.15%, 0.30% and 0.75%) powder during 30 days of storage^a,b

Sample	1	15	30
Time (day)
Control	23.30 ± 0.10 aB	36.56 ± 0.60 aA	39.90 ± 0.40 aA
0.15%	16.53 ± 0.20 abB	29.96 ± 0.35 aA	33.20 ± 0.50 aA
0.30%	9.96 ± 0.15 bB	19.97 ± 0.15 bA	21.50 ± 0.40 bA
0.75%	0.001 ± 0.01 cAB	0.001 ± 0.01 cAB	3.26 ± 0.10 cA

^aMeans within each column followed by different letters (a–c) show significant different (P < 0.05) between treatments at the same time

^bMeans within each row followed by different letters (A–B) show significant different (P < 0.05) at a treatment during storage period

The results indicated that serum separation in Doogh samples treated with high amounts of PPM powder was reduced significantly. Generally, the steric repulsive interactions of casein micelles in milk (pH 6.7) cause micelles to stay in the suspended state. Because of milk acidification to a pH value around 4, as is the case in the preparation of Doogh, the native stabilization mechanism of casein micelles fails. For stabilizing Doogh, the addition of stabilizer is required to prevent the precipitation of milk protein and the subsequent macroscopic whey separation (Hashemi et al. 2015). It seems that the formation of a powerful gel, the increase in the hydrogen bonding interactions between hydrophilic groups and the creation of covalent cross-links between the Ca⁺ ions and negative charge components in PPM powder can reduce the serum separation in treated samples. The Doogh samples with higher concentrations of PPM powder had a lower phase separation. Hashemi et al. (2015) also reported the capacity of hydrocolloids to prevent the phase separation of Doogh during storage and showed that the psyllium seed hydrocolloid, high methoxyl pectin, and gellan, alone or in a binary mixture, can reduce Doogh phase separation. Khanniri et al. (2019) also reported that the Doogh samples containing a mixture of locust bean gum and CMC were more stable compared to the hydrocolloid-free counterpart.

Viscosity and rheological properties

Figure 2a presents that there was no significant difference in the apparent viscosity of the Doogh samples during cold storage (P > 0.05). Nonetheless, the treated samples displayed a significant increase in their apparent viscosity during cold storage compared to the control counterpart (p < 0.05). The sample with the 0.75% PPM powder on the 30th day had the highest apparent viscosity, while the control showed the lowest viscosity. A higher viscosity was observed for the samples containing the higher amounts of PPM which can be due to the presence of arabinoxylan with 1–4 linkages in the xylan backbone of the PPM. PPM powder has a high molecular weight which makes it a suitable viscosity enhancer (Guo et al. 2008; Hashemi et al. 2015). In accordance, a higher apparent viscosity was reported for Doogh samples which were enriched with inulins and modified starch (Karim et al. 2017).

Figure 2b presents the flow curves of the relationship between the shear rate values and apparent viscosity. The viscosity of the Doogh samples decreased as the shear rate increased; therefore, they can be considered as non-Newtonian fluids. At low shear rate values, the differences between the apparent viscosity of the treatments and controls depended on the PPM concentrations. The samples with higher concentrations of PPM presented a large difference compared to the controls. Meanwhile, at high shear rate values, the differences between the apparent viscosity of the treatments and controls declined.

The control and treated Doogh samples were of a non-Newtonian fluid type, showing a shear-thinning behavior which can be due to the high amount of dry matter and the increased internal interaction between the particles in Doogh (Koksoy and Kilic 2004). In the treated samples containing PPM powder with a lower flow behavior index (n), there was a sudden decrease of viscosity at low shear rates because of the sudden reduction in particle size (McClements 2004). As a result of the electrostatic interaction between casein micelles and PPM, the treated samples with higher PPM were more resistant to the low shear rate compared to the control sample (Laurent and Boulenguer 2003). These observations were similar to the results of previous studies using hydrocolloids in acidic milk beverages (Vukic et al. 2018; Khanniri et al. 2019).

The Power Law and Herschel-Bulkley parameters are reported in Table 2. The rheological behavior of the control sample had a high correlation coefficient with the Power Law model, while the treated samples had a high correlation coefficient with the Herschel-Bulkley model; consequently, this model can be regarded as the most appropriate model for describing the rheological behavior of the treated samples. The flow behavior index (n) was less than 1.0 indicating that Doogh is a pseudo-plastic fluid. The results also revealed that the samples with a higher concentration of Plantago psyllium rendered a lower flow index and a higher consistency index.

Table 2.

Parameters of the Herschel Bulkley model and Power law of Doogh samples with different concentrations of Plantago psyllium mucilage (PPM) (Control, 0.15%, 0.30% and0.75%)

t = k(γ)n Power law model				t = t0 + k(γ)n Herschel–Bulkley model
	k(Pa.sn)	n	R2	t0	n	k(Pa.sn)	R2
Control	0.423	0.603	0.996	0.190	0.690	0.060	0.993
0.15%	0.259	0.530	0.988	0.323	0.630	0.130	0.990
0.3%	0.545	0.480	0.988	0.557	0.580	0.280	0.996
0.75%	2.470	0.403	0.975	2.330	0.530	1.160	0.998

The flow behavior modeling of the Doogh samples clearly showed that the suitable model changed from the Power Law model for the control samples to the Herschel-Bulkley model for the treated samples as stabilizer concentration increased. The electrostatic interactions between the positive charge of casein groups and anionic polysaccharide in Plantago Psyllium can cause higher strengths against shear stress; hence, an initial stress would be needed to cope with the new binding (Laurent and Boulenguer 2003). In accordance with our results Karim et al. (2017) reported that Doogh is a non-Newtonian fluid which its rheological behavior can be described by the Herschel-Bulkley model.

Changes in the viability of L. casei

Figure 3 shows the variations in the L. casei cell count in the Doogh samples during cold storage. The number of L. casei (log cfu/mL) in the control sample was in the range of 3.32 to 7.8 log cfu/mL. The number of L. casei showed a significant decrease during the cold storage (P < 0.05). The viability of L. casei in the treatments was significantly more than that in the control sample on the 15^th and 30^th days (P < 0.05). The highest number of L. casei cells was observed on the first day (7.8 log cfu/mL) and the lowest on the 30^th day (3.32 log cfu/mL) in the control samples. The treated samples with 0.75% PPM also showed a standard level of L. casei on test days (6 log cfu/mL).

Fig. 3.

Viability of Lactobacillus casei in Doogh samples containing Plantago psyllium mucilage (PPM) powder (Control, 0.15%, 0.30% and 0.75%) during storage at 4°C for 30 days

Improving the viability of probiotic bacteria in dairy probiotic products during storage has been one of the most important challenges in recent years. Our findings suggested that the number of probiotic bacteria went down in the control and treated samples during storage. Iravani et al. (2015) reported that bacteriostatic and/or bactericidal factors, such as low pH, organic acids, high redox potential, hydrogen peroxide, molecular oxygen, bacterial competition, and changing temperatures during storage can decrease the viability of probiotic micro-organisms. The results showed that the viability of L. casei was significantly improved during the storage as the PPM concentration was increased. The higher concentrations of PPM resulted in a higher number of L. casei. The presence of arabinoxylan as non-starch polysaccharides in PPM enhances the growth of the probiotic bacteria during storage (Hashemi et al. 2015; Karlton-Senaye et al. 2015). The effect of PPM on the improvement of viability can be due to the formation of a cross-link matrix between the mucilage and whey protein in the Doogh samples. This matrix can protect probiotic cells from the effects of improper water activity by forming a mucilage gel. This matrix can also protect microparticles in Doogh against acid digestion during storage (Karlton-Senaye et al. 2015). These observations are in agreement with the findings of Karim et al. (2017), who reported that prebiotics, such as inulin, are capable of increasing the viability of probiotic bacteria in Doogh. Karlton-Senaye et al. (2015) also observed that the addition of different gums improved the survivability of Lactobacillus strains in milk drinks.

Sensory properties

The sensory quality assessment of popular products such as Doogh is one of the most important tests for determining its consumer preferences. Table 3 presents the sensory scores of samples on the 30^th day of the storage. The results revealed that there were no significant differences (P > 0.05) in terms of the taste, odor and color of the Doogh samples. Although the addition of PPM powder to the Doogh samples slightly changed their flavor and color, the panelists could not recognize any significant differences between the control and treated samples. The highest and lowest taste scores belonged to the samples containing 0.30% PPM and the control samples, respectively.

Table 3.

Sensory evaluation of Doogh samples with different concentrations of Plantago psyllium mucilage (PPM) (Control, 0.15%, 0.30% and 0.75%) on 30^th day of storage

Sample	Control	0.15%	0.3%	0.75%
Taste	3.1 ± 0.39 a	3.3 ± 0.54 a	3.5 ± 0.32 a	3.3 ± 0.49 a
Odor	3.4 ± 0.61 a	3.4 ± 0.47 a	3.6 ± 0.40 a	3.2 ± 0.62 a
Color	3.4 ± 0.51 a	3.2 ± 0.56 a	3.3 ± 0.46 a	3.1 ± 0.52 a
Texture	2.6 ± 0.46 b	2.8 ± 0.44 b	3.5 ± 0.44 a	1.5 ± 0.62 c
General acceptance	2.4 ± 0.58 c	3.1 ± 0.57 b	4.1 ± 0.62 a	2.7 ± 0.65 bc

Different letter within columns shows a significant difference (p < 0.05)

Sensory evaluation showed significant differences between the samples containing PPM and the control sample in terms of texture and general acceptance (P < 0.05). The best texture and highest general acceptance were observed in the samples treated with 0.30% PPM and the worst texture and the lowest general acceptance were found with the samples treated with 0.75% PPM and control, respectively. The relatively stable appearance in samples containing PPM powder can be attributed to the lower phase separation during storage. However, the highest concentrations of PPM powder (0.75%) were found to adversely affect the texture and consumer acceptance of the samples. Hashemi et al. (2015) also concluded that the use of psyllium seed hydrocolloid had a desirable effect on the sensory characteristics of Doogh. Ziaolhagh and Jalali (2017) also showed that the addition of hydrocolloids such as xanthan gum could improve the texture of Doogh.

Although the number of L. casei in the treated samples containing 0.30% PPM on the 30^th day were below the standard levels of probiotic bacteria (10⁶ cfu/mL), those of L. casei in the 0.30% treatment on the 15^th day were at the recommended standard level of probiotic bacteria. Moreover, the treatment containing 0.30% PPM had a significant reduction effect on phase separation and improved stability on day 15. Therefore, synbiotic Doogh samples containing 0.30% PPM on the 15^th day can be recommended as the optimal sample only.

Conclusion

In the present study, adding PPM to Doogh decreased pH values and increased acidity during the storage period. Using this hydrocolloid also led to a significant reduction in the phase separation and an increase in the viscosity. The sample containing 0.75% PPM could maintain the minimum acceptable level of L. casei on the 30^th day of storage; however, its overall acceptability score was not good. Although the Doogh samples containing 0.30% PPM had the highest overall acceptability score in comparison with the other samples, they had acceptable levels of L. casei on the 15^th day. Overall, further studies are required to formulate a synbiotic Doogh with a high overall acceptability and standard probiotic viability for 30-day storage.

Funding

The Islamic Azad University (Iran, Tehran) was supported the research.

Data Availability

Data are available upon request from the authors.

Declarations

Conflicts of interest

There in no conflicts of interest.

Ethics approval

We did not use biological material in this research and the study was performed in accordance with the ethical standards.

Consent to participate

Individuals participated in this study with consent and voluntarily.

Consent for publication

The consent of others to publish and use their content is considered in this article.

Code availability

Software application are available upon request from the authors.