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An assessment of Cuminum cyminum (Boiss) essential oil, NaCl, bile salts and their combinations in probiotic yogurt

Noushin Mohajeri1, Peyman Mahasti Shotorbani2*, Afshin Akhondzadeh Basti3, Zaleh Khoshkhoo4, Ali Khanjari5

1Student of Food Science and Technology, Department of Food Science and Engineering, Tehran North Branch, Islamic Azad University;

2Department of Food Quality Control and Hygiene, Science and Research Branch, Islamic Azad University, Tehran, Iran;

3Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran;

4Department of Food Science and Technology, Tehran North Branch, Islamic Azad University, Tehran, Iran;

5Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

This article is prepared to investigate the impacts of Cuminum cyminum essential oil (CEO), NaCl, bile salts, and their combinations on the viability of Lactobacillus casei in probiotic yogurt.

The water distillation method was used to extract the CEO, and GC/MS was used to determine its constituents. Then, the CEO’s antibacterial activity, together with NaCl and bile salts, was investigated via the microdilution technique by determining the minimum inhibitory concentration (MIC) against L. casei. Further, the stress effects of 50% MIC on CEO, NaCl, and bile salts were examined by comparing the stress treatments with the control in terms of the L. casei population, pH, acidity, and syneresis percentage in probiotic yogurt during storage in the refrigerator for 28 days.

According to the results, the L. casei population and pH decreased in all the treatments during the storage time, such that the intensity of the decrease in the control and CEO treatments was lesser than in other stress treatments (P<0.05). The acidity and percentage of syneresis during the storage time increased for all the treatments, with the increase being less in control and CEO than in the other stress treatments (P<0.05). The control and CEO treatments scored the highest in the sensory evaluation (P<0.05).

Applying stresses below the MIC resulted in the survival of L. casei in the recommended amount (105–106 CFU mL–1) in the probiotic yogurt until the end of 28 days.

Key words: Cuminum cyminum, probiotic, yogurt, Lactobacillus casei, Stress, Essential oil

*Corresponding Author: Peyman Mahasti Shotorbani, Department of Food Quality Control and Hygiene, Science and Research Branch, Islamic Azad University, Tehran, Iran. Email: [email protected]

Received: 27 December 2020; Accepted: 5 February 2021; Published: 11 February 2021

DOI: 10.15586/ijfs.v33iSP1.1990

© 2021 Codon Publications

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Today, particular attention is being given to functional foods, which have nutritional value as well as positive effects on human health. People are excited about eating products containing probiotics. However, products containing probiotics are amongst the ones in which there have been health allegations. These allegations are even advertised in the media during the last few years (Zendeboodi et al., 2020). Various studies on probiotics for humans’ advantages have focused on the innovative formulation, and some even provided valuable information on probiotics linked to health and well-being. (Roobab et al., 2020). Probiotics are microorganisms that improve the gut microbial balance. These often include the Lactobacillus and Bifidobacterium species, as they have a historically prolonged and reliable value. Also, identified as generally recognized as safe (GRAS), all are the predominant inhabitants in the human intestines. (Al-Okbi and Mohamed, 2012; Lucatto et al, 2020). Lactobacillus casei is a gram-positive, mesophilic, microaerophilic, catalase-negative, and spore-free bacterium and a facultative hetero-formative bacterium with high acid production capacity (Fontana et al., 2018). L. casei is a probiotic strain linked with antihypertensive antioxidant, antihypocholesterolemic and anticarcinogenic, characteristics (Balthazar et al., 2018; Garcia et al., 2019).

Fermented milk, such as yogurts, has the potential to act as a medium for producing value-adding materials because of its favorable sensory attributes, nutritional properties, and high-grade harmony, and ample content of essential nutrients; besides, yogurt consumption enhances gut macrobiotic activity, mitigates immune responses, and increases gastrointestinal functionality by adjusting lactose intolerance. Technologists and manufacturers have reviewed distant fortifications by combining probiotics and nutraceutical compounds (Alizadeh Khaledabad et al., 2020; Lucatto et al., 2020).

Today, probiotic yogurt is the most popular and widely consumed probiotic product in the world. The survival of probiotic bacteria in yogurt and similar products is an important challenge during storage in probiotic products. The minimum acceptable concentration of probiotic strains for beneficial and therapeutic effects should be at least 106–107 CFU g -1 or mL in the final product (Azizkhani and Parsaeimehr, 2018). The main problem in production was maintaining the survival rate of probiotic strains during storage of product with due attention to high acidity, oxygen stress, and nutrient deficiencies. The main reasons for reducing the viability of probiotic strains in the stomach were low pH and bile salts in the intestines (El-Shafei et al., 2010). Many studies have been carried out on the viability of probiotics under the stomach’s acidic conditions, bile salts of the small intestine (Sahadeva et al., 2011), and survival rate of probiotics were studied in the cold storage of foods (Mortazavian et al., 2007). Various methods such as microencapsulation, the addition of prebiotics and essential oils (Capela et al., 2006), and different procedures were used to increase the survival rate of probiotic strains during storage of functional products applied stresses were less than the minimum inhibitory concentration (MIC). They produced resistance-inducing genes (Maragkoudakis et al., 2006). Some essential oils improve probiotics’ viability, and others may also decrease the viability of probiotics (Calsamiglia et al., 2007).

In many cases, the viability of probiotic bacteria is not sufficient, and it is necessary to evaluate the viability of probiotic bacteria in yogurt by applying different procedures to increase the resistance of bacterial cells against stresses. Hence, the usage of herbal essential oils and probiotic strains in dairy products is a new strategy to overcome pathogenic bacteria and stimulate probiotics. This study aimed to evaluate the effects of stresses less than the MIC of Cuminum cyminum Boiss. essential oil (CEO), NaCl (NC), and bile salts (BS) or a combination of them on the viability of probiotic L. casei and later we monitored through the storage time, physicochemical and sensory properties of probiotic yogurt.

Materials and Methods

Materials

Cow’s milk (3% fat) was supplied from Pegah, Tehran Plant, and Starter culture of yogurt (Streptococcus thermophilus and Lactobacillus bulgaricus sub spp . delbrukii) was purchased from Hanson Company, Denmark.

Extraction and analysis of the essential oil

The Cuminum cyminum was collected from the Kerman province of Iran, and the Iranian Institute approved its scientific name of Botanical Garden Research. The plant's essential oil was extracted by the water vapor distillation method, and then it was analyzed by colorimeter attached to a mass spectrometer (Model HP-6890, USA). HP-5MS capillary column with 30 m length, 0.25 mm inner diameter, and 0.32 μm inner layer thicknesses was used. The regulated programming for identification and quantification was set up as follows: the temperature was elevated from 60 to 265°C, with a flow rate of 2.5°C per min, and then the column was maintained at 265°C for 30 min. The injection room temperature was 250°C, and the flow rate of helium as a carrier gas was 1 mm/min. Finally, the flame ionization detector (FID) identified the essential oil components with an electrical capacity of 70 eV and an ionization source temperature of 250°C.

Preparation of inoculums

Lactobacillus casei (ATCC39392) was supplied from the Microbial Collection of Pasteur Institute of Iran. It remained in laboratory samples in a glycerol stock at –70°C and transferred in Brain Heart Infusion (BHI) broth (Merck, Germany) at 37°C without shaking. Working cultures were prepared from stock cultures in two successive transfers (1% inoculum) in BHI broth at 37°C for 18 h. L. casei cells were inoculated from working cultures to BHI broth. After 18 h incubation at 37ºC, optical density (OD) (absorbance) of 0.1 at 600 nm, using a Spectronic 20 spectrophotometer (Varian, USA) was applied for determining the population of L. casei. Cell concentration of L. casei was 2.2×1010 CFU mL-1 for inoculation. The enumeration of L. casei was performed according to the serial dilution method and cultivating on (BHI) agar (Merck, Germany) after incubation for 24 h at 37ºC.

Determination of minimum inhibitory concentration (MIC) of Cuminum cyminum Boiss. essential oil, NaCl, and bile salt

A 96-well plate with a volume of 300 μL was used in this experiment. Sequential concentrations of essential oil of C. cyminum Boiss (0, 500, 1000, 2000, 3000, 4000, 5000, 7500, and 10. 000 mg L-1), bile salt (0%, 0.05%, 0.02%, 0.03%, 0.06%, 0.07%, 0.08%, 0.1%, 0.2% and 0.3%) and NaCl (0%, 1%, 2%, 3%, 4% and 5%) were used in De Man, Rogosa and Sharpe agar (MRS) broth (Merck, Germany). Media contained 5% DMSO and were transferred to 96-well plates. Then, 250 μL of different concentrations of essential oil, NaCl, and bile salts along with 20 μL of L. casei suspension (5×106 CFU mL-1) were added to all well. The contents of each well were mixed with a shaker for 2 min. The microplates were closed by Parafilm and then incubated for 24 h at 37°C in an anaerobic jar (Merck, Germany). At the end of incubation time, turbidity or non-turbidity was evaluated in the wells.

Adaptation and challenging conditions

Cultures were at the logarithmic phase. Bacterial cells were separated by centrifugation (Hettich, Germany) and resuspended in fresh BHI broth (non-adapted control culture). The inoculation content of L. casei was 1 × 1010 CFU mL-1. Adaptation time was conducted in the same medium at 37°C (I) for 120 min with 1 mL included in 100 mL of 5% DMSO (CEO), (II) 10 g per 100 mL in Mueller Hinton broth medium (Merck, Germany) (NC), (III) 0.05 g per 100 mL in Mueller Hinton broth medium (BS), (IV) 1 mL in 100 mL containing 5% DMSO plus 10 g per 100 mL in Mueller Hinton broth medium (CEONC), (V) 1 mL in 100 mL containing 5% DMSO plus 0.05 g per 100 mL in Mueller Hinton broth medium (CEOBS), (VI) 10 g per 100 mL plus 0.05 g per 100 mL in Mueller Hinton broth medium (NCBS) and (VII) 1 mL in 100 mL containing 5% DMSO, 10 g per 100 mL plus 0.05 g per 100 mL in Mueller Hinton broth medium (CEONCBS). After centrifugation, adapted and non-adapted cells were inoculated to yogurt (108 per mL). Enumeration of L. casei was conducted by serial dilution method (Most Probable Number) at 0, 7, 14, 21, and 28 days of storage to evaluate survival rate. All the plates were incubated at 37°C for 48 h. Furthermore, all experiments have been done twice.

Physicochemical properties of yogurt samples

The experiments for physicochemical properties were pH, acidity%, and syneresis%. pH and titratable acidity concluded based on the method described by Yangilar and Yildiz (2018). Moreover, the percentage of syneresis was averaged according to the method defined by Wacher-Rodarte et al. (1993). Five milliliters of the yogurt sample was centrifuged at 2.208 g for 20 min at 4°C, and the volume of isolated whey was calculated after 1 min. Lastly, we displayed the Syneresis rate (%) as the separated whey volume per 100 g of yogurt (Wacher-Rodarte et al., 1993).

Sensory evaluation

Sensorial tests were executed based on a 5-point Hedonic scale. The lowest score was intensely disliked, and the highest score was 5 as remarkably like samples (Shahdadi et al., 2015). Sensory properties were measured as follows: flavor, texture, and overall acceptability. Sensory evaluations were carried out during 28 days of storage. Ten trained panelists performed judgments.

Analytical study

All experiments were performed in completely randomized design as triplicates and the result was reported as mean ± SD. The comparisons of data mean were performed by Tukey test. Two-way ANOVA was used for determination of significance or non-significance of data (P<0.05).

Results

The chemical combination of Cuminum cyminum Boiss. essential oil

Accordingly, 15 compounds were identified that constitute 100% of the essential oils in total. The most abundant essential oil component was Propanal, 2-methyl-3-phenyl. The concentration of them was 24.2%. Next, Gamma-Terpinene, Phenylethanediol, and 2-Beta-Pinene had the highest level of essential oil components, with 18.94%, 18.88%, and 12.59%, respectively (Table 1).

Table 1. GC/MS results of Cuminum cyminum (Boiss) essential oil

No. Name Area (%)
1 α-Pinene 1.12
2 Sabinene 0.74
3 2-Beta-Pinene 12.59
4 β-Myrcene 0.93
5 l-Phellandrene 1.10
6 Cymene 6.91
7 Limonene 1.73
8 Gamma-terpinene 18.94
9 (E)-4-(Cyclohex-1’-enyl)but-2-en-1-ol 1.88
10 Propanal, 2-methyl-3-phenyl- 24.22
11 2-Caren-10-al 8.80
12 Phenylethanediol 18.89
13 Gamma-cadinene 0.57
14 Trans-beta-farnesene 0.72
15 Carotol 0.86

MIC results

The MIC values of the CEO, NC, and BS against L. casei were 1%, 4%, and 0.3% (v/v), respectively.

Survival of Lactobacillus casei

The results showed that the interaction effects of treatment and storage time on the L. casei population (Figure 1) were significant (P<0.05). The viability of L. casei decreased in all treatments except for control and sample under CEO stress at 7 days of storage time (P<0.05). The viability of L. casei of all samples significantly decreased during 28 days of storage (P<0.05). Following 28 days of storage time, the highest survival rate of L. casei was detected for CEO (6.05±0.03 log CFU mL-1) and control (6.01±0.02 log CFU mL-1) treatments, which was significantly different from others (P<0.05). No significant difference was observed within NC and BS treatments (P>0.05). Nevertheless, the lowest survival rate of L. casei was observed in the stress condition treated with CEONCBS at all days of storage time (P<0.05).

Figure 1. Effect of Cuminum cyminum essential oil (CEO), NaCl (NC), bile salts (BS), and their combinations on the survival of L. casei ATCC-39392 (log CFU mL-1). Deviation bars designate the standard error of the method (n = 3).

pH

The data for pH (Figure 2) showed that the effect of time and type of samples were significant (P<0.05). The pH of all the yogurt samples decreased significantly during the storage period (P<0.05), and higher pH was attributed to samples under stress (except for the CEO) (P<0.05). At the end of 28 days of storage, the highest and lowest pH values for CEONCBS and the control treatments were 4.23±0.06 and 3.91±0.06, respectively (P<0.05). The comparison among all stress treatments showed the highest and lowest pH of samples were attributed to CEO and CEONCBS during storage time, respectively (P<0.05). No significant difference was observed between NC and BS treatments on the same day of storage (except on day 14) (P>0.05).

Figure 2. Effect of Cuminum cyminum essential oil (CEO), NaCl (NC), bile salts (BS), and their combinations on changes in pH of probiotic yogurt. Deviation bars designate the standard error of the method (n = 3).

Titratable acidity (TA%)

The titratable acidity (Figure 3) results showed that interaction between group and time was significant (P<0.05). The acidity of all samples significantly increased during storage time (P<0.05). The acidity of the control sample was less than those of stress treatments (P<0.05). Treatments CEO and CEONCBS had the highest and lowest acidity, respectively (P<0.05). After 28 days of the storage time, the lowest and highest acidity values were related to treatments under the stress of CEONCBS (1.19±0.008) and control (1.50±0.006), respectively. No significant difference was observed between NC and BC treatment during 28 days of storage (P>0.05).

Figure 3. Effect of Cuminum cyminum essential oil (CEO), NaCl (NC), bile salts (BS), and their combinations on acidity changes of probiotic yogurt. Deviation bars designate the standard error of the method (n = 3).

Syneresis

Results showed that exchange within-group and time was notable (P<0.05). As shown in Figure 4, the amount of syneresis significantly rose in all samples during storage time (P<0.05). The increase in intensity for control and CEO treatments was less than that for stress treatments (P<0.05). Among the stressful treatments, the treatments under stress with CEO and CEONCBS had the lowest and highest percentage of syneresis, respectively, during 28 days of storage (P<0.05). No significant difference was observed between treatments with NC and BC on the same day of storage time (P>0.05). After 28 days of storage time, the lowest and the highest percentages of syneresis were observed for control (8.30±0.09) and stress with CEONCBS (9.25±0.13) treatments, respectively.

Figure 4. Effect of Cuminum cyminum essential oil (CEO), NaCl (NC), bile salts (BS), and their combinations on syneresis changes in probiotic yogurt. Deviation bars designate the standard error of the method (n = 3).

Sensory properties

Table 2 showed the results of sensory characteristics (flavor, texture, and general acceptance) during 28 days of storage time. The interaction between group and time on sensory characteristics was significant (P<0.05). The sensory scores of examples decreased during storage time. A more severe drop in sensory score was observed for stress treatments except for the CEO (P<0.05). Flavor scores for all treatments showed there were no significant differences among all samples on the first day of storage (P>0.05). The most leading and lowest scores were attributed to control (4±0) and CEONCBS (3.11±0.33) at 28 days of storage time, respectively (P<0.05). The texture feature results showed no significant difference within samples on the first day of storage (P>0.05). The highest and lowest scores were attributed to control (4.33±0.50) and CEONCBS (3.22±0.44) at 28 days of storage time, respectively (P < 0.05). Overall acceptance of samples indicated no significant difference among all samples on the first day of storage (P > 0.05). The highest and lowest scores were attributed to control (4±0) and CEONCBS (3.220 ± 0.44) at 28 days of storage time, respectively (P < 0.05).

Table 2. Sensory properties of probiotic yogurt samples with Cuminum cyminum essential oil (CEO), NaCl (NC), bile salts (BS), and their combinations.

Sensory properties Yogurt samples Storage time (Days)
0 7 14 21 28
Flavor Control 5 ± 0a 5 ± 0a 5 ± 0a 4.67 ± 0.50a 4 ± 0a
  CEO 5 ± 0a 5 ± 0a 5 ± 0a 4.75 ± 0.44a 4 ± 0a
  BS 5 ± 0a 4 ± 0b 4 ± 0b 4 ± 0b 3.33 ± 0.50b
  NC 5 ± 0a 4 ± 0b 4±0b 3.78 ± 0.44b 3.33 ± 0.50b
  CEOBS 5 ± 0a 4 ± 0b 4 ± 0b 3.78 ± 0.44b 3.33 ± 0.50b
  CEONC 5 ± 0a 4.33 ± 0.50b 3.78 ± 0.44b 3.78 ± 0.44b 3.33 ± 0.50b
  NCBS 4.67 ± 0.50a 4 ± 0b 3.78 ± 0.44b 3.22 ± 0.44c 3.11 ± 0.33b
  CEONSBS 4.75 ± 0.44a 4.33 ± 0.50b 4.33 ± 0.50b 3.22 ± 0.44c 3.11 ± 0.33b
Texture Control 5 ± 0a 5 ± 0a 5 ± 0a 4.67 ± 0.23a 4.33 ± 0.50a
  CEO 5 ± 0a 5 ± 0a 4.56 ± 0.53b 4.33 ± 0.50bc 4.33 ± 0.50a
  BS 5 ± 0a 4 ± 0b 4.33 ± 0.50bc 4.33 ± 0.50bc 3.44 ± 0.53b
  NC 5 ± 0a 4.33 ± 0.50b 4.33 ± 0.50bc 4.33 ± 0.50bc 3.22 ± 0.44b
  CEOBS 4.56 ± 0.53a 4 ± 0b 4 ± 0c 4 ± 0c 3.44 ± 0.55b
  CEONC 5 ± 0a 4 ± 0b 4 ± 0c 4 ± 0c 3.44 ± 0.44b
  NCBS 4.67 ± 0.50a 4.33 ± 0.50b 4 ± 0c 3.22 ± 0.44d 3.44 ± 0.44b
  CEONSBS 4.67 ± 0.50a 4 ± 0.70b 3.78 ± 0.44b 3.22 ± 0.44d 3.22 ± 0.44b
Overall acceptability Control 5 ± 0a 5 ± 0a 4.89 ± 0.33a 4.67 ± 0.50a 4 ± 0a
  CEO 5 ± 0a 5 ± 0a 4.56 ± 0.53a 4.33 ± 0.50ab 3.78 ± 0.44ab
  BS 5 ± 0a 4 ± 0b 4 ± 0b 4 ± 0.70b 3.22 ± 0.44b
  NC 5 ± 0a 4 ± 0.70b 4 ± 0b 4 ± 0b 3.22 ± 0.44b
  CEOBS 4.75 ± 0.44a 4 ± 0b 4 ± 0b 4 ± 0b 3.22 ± 0.44b
  CEONC 5 ± 0a 4 ± 0b 4 ± 0b 4 ± 0b 3.22 ± 0.44b
  NCBS 4.67 ± 0.50a 4.33 ± 0.50b 4 ± 0b 3.56 ± 0.53c 3.22 ± 0.44b
  CEONSBS 4.67 ± 0.50a 4 ± 0.70b 4.33 ± 0.50b 3.22 ± 0.44c 3.22 ± 0.44b

Each observation is a mean ± SD of three replications. In each column and row, means with the same letters had no significant difference at P > 0.05.

Discussion and Conclusion

The effect of MIC of the essential oils and salts in return to the survival of L. casei was quite high; the inhibitory effect of concentrations less than MIC was also seen on pathogenic bacteria. Hence, the usage of concentrations of essential oils, NaCl, and bile salts less than MIC may also eliminate pathogens without harm to probiotics (Calsamiglia et al., 2007).

The viability of probiotic strains during production, food storage, and passage through the gastrointestinal tract is a major challenge in fermented dairy products. Researchers reported that enumeration of probiotic strains for beneficial and therapeutic effects should be at least 106 to 107 CFU g-1 or mL in products (Azizkhani and Parsaeimehr, 2018). Treatments with stresses of NC, BS, CEONC, CEOBS, and CEONCBS maintained viability until 21 days of storage. However, control with CEO sample-maintained viability up to 28 days of storage time. The survival rate of L. casei in control and stress treatments with the CEO increased at 7 days of storage. A rise in the survival rate of L. casei was observed to reduce pH at 7 days of storage. The increased survival rate of Lactobacillus acidophilus LA5, Lactobacillus fermentum, and Bifidobacterium Bb-12 in yogurt at 7 days of storage was reported by Azizkhani and Parsaeimehr . The bacterial population decline was attributed to the accumulation of organic acid during growth and fermentation. The main reasons for reducing pH are converting lactose to lactic acid, type of starter culture, duration of storage, and fermentation temperature (Singh et al., 2011). Several studies reported a decline in probiotic strains’ survival rate during storage time (Azizkhani and Parsaeimehr, 2018; Yangilar and Yildiz, 2018). Indeed, decreased pH of products during storage was due to activation of the beta-galactosidase enzyme at 0–5°C, as well as post-acidification. pH values decreased to 4.2. The viability of probiotic bacteria is affected by increased hydrogen ions compared to lactate ions (Kailasapathy, 2006). Compared to treatments with stress, treatment with CEO had higher population viability of L. casei than others during storage time (P < 0.05). The susceptibility of microorganisms to essential oils depends on the details of essential oil and the type of microorganisms. The antimicrobial activity of essential oils is complicated due to their volatility, insolubility in water, and complex chemical structure (Calsamiglia et al., 2007). The antibacterial effects of Cuminum cyminum Boiss. essential oil on different microorganisms depends on the concentration and composition of nutrients, storage temperature, and nature of the organism’s metabolites (Mahmoudi, 2013). The survival rate of Lactobacillus acidophilus in bioyogurt containing different concentrations of the essential oils, Mentha piperita and Ziziphoraclinopodioides, was significantly reduced 7 days of storage time at 4°C. One of the main criteria for selecting probiotic bacteria is resistance to NaCl and bile salts (Sarabi Jamab and Niazmand, 2009). According to the obtained results, treatments under stress with 0.15% BS were to be effective in the food at end of the 21st day. However, the survival rate of L. casei in BS stress treatment was significantly decreased from the 21st to the 28th day of storage time. It was less than the acceptable limit (P < 0.05). Probiotic bacteria have different mechanisms of protection against stress, one of which is the bile hydrolysis system. The resistance of some strains to bile salts is associated with the bile salt hydrolysis activity. Therefore, the hydrolysis of the bile salts will reduce their toxicity and side effects (Sahadeva et al., 2011). According to Taranto et al. (2006), Lactobacillus delbrucium subsp. Bulgaricus treated with different concentrations of thiorodoxylate (one of the bile salts) showed different levels of activity of the hydrolysis system (Taranto et al., 2006). The researchers reported that in some bacterial cells, the bile hydrolysis system’s activity was significantly stronger than others, which resulted in cells showing greater resistance to higher concentrations and longer exposure times to these bile salts (Taranto et al., 2006). When probiotic bacteria are exposed to bile salts, cellular homeostasis disorders occur. Destruction of lipid membranes and cell membrane proteins leads to bacteria’s death (Sahadeva et al., 2011). The survival rate of L. casei with considering salt stress was within acceptable limit until the end of 21 days of storage, and these findings are following other research that studied the viability of probiotic strain more than 2% of concentration (Fortin et al., 2011). Other researchers reported that probiotic strains’ viability decreased in samples with high concentrations of salt (4%) (Hekmat et al., 2009).

The suitable pH for commercial yogurt is 4.5. pH enhanced shelf life of the yogurt maintained mild taste and optimum appearance. Undesirable pH (less than 4), which had been resulted by Lactobacillus bulgaricus, produced large amounts of lactic acid, acetaldehyde, and byproducts from proteolytic activity (Mahmoudi et al., 2014). The main reason for the lower pH of CEO, BS, and NC salts compared to the control sample was due to the presence of some phenolic compounds in the C. cyminum Boiss essential oil, which may have inhibitory effects on the growth of L. casei and changed pH values (Mahmoudi, 2013). Salt also affected pH and acidity values by influencing the growth of microorganisms, which may cause lactic acid production during storage time (Fortin et al., 2013). Some studies reported that probiotic yogurt’s pH decreased during storage time (Mahmoudi, 2013 ; Azizkhani and Pasaeimehr, 2018).

The acidification degree of probiotic yogurt is a crucial process control model that directly impacts the gel intensity and the commercially available fermentation period (Alizadeh Khaledabad et al., 2020). The production of organic acids by lactic acid bacteria was the main reason for increased acidity during storage time (Mahmoudi et al., 2014). Salt also influenced the acidity of samples because it impacted the growth of microorganisms, which influenced lactic acid production during storage time (Fortin et al., 2013). The consumption of lactose by lactic acid bacteria led to lactic acid production, and then the acidity of samples increased. The production of lactic acid in yogurt is the main factor in producing a unique flavor. Due to casein instability, conversion of the colloidal calcium phosphate complex to soluble calcium phosphate, calcium excretion, and casein coagulation take place at pH 4_4.6 (Ramasubramanian et al., 2008).

Significant factors such as heterogeneity, high acidity, storage, breakage of protein strand, and structural rearrangement that will induce yogurt’s whey to leakage are recognized as substantial defects (Yildiz and Ozcan, 2020). The main reason for increased syneresis in probiotic yogurt during storage might be the activation of microorganisms of starter culture and their effects on long-chain biopolymers, which could be an important factor in reducing the softness and enhancing the syneresis of the yogurt during storage (Kailasapathy, 2006). At the same time, Akgun (2018) found that the syneresis rate in probiotic yogurt samples increased during storage at 4°C. The consistency of yogurt increased by stabilizers, an increase in the amount of milk casein concentration, and a reduction of acidification rate (Everett and Mcleod, 2005). Samples with stress under essential oil had a lower syneresis percentage than others. The main reason for lower syneresis was acidification of yogurt containing herbal essential oils, which might cause high-strength gels, low permeability, a fine protein mesh, and higher water uptake. Consequently, the syneresis of probiotic yogurt was decreased (Ozer et al., 2007).

In the overall acceptability of food products by consumers, sensory characteristics represent a vital role. Researches affirm that flavor is the first criterion for food acceptance, followed by health considerations as the second rule (Alizadeh Khaledabad et al., 2020). The reduction of flavor scores in all samples during storage might be related to increasing acidity and reduced starter bacteria activity, which induced flavor components (Yangilar and Yildiz, 2018). The percentage of syneresis causes a change in the firmness of the sample. The firmness of yogurt decreased with increasing of syneresis. Indeed, the percentage of syneresis was inversely correlated to yogurt’s firmness (Ayar and Gurlin, 2014). Treatments that contained more probiotic counts had better flavor and texture than treatments with less probiotic counts, so the overall acceptance scores of yogurts with more probiotic counts were higher than the yogurt with less probiotic counts (under stress). Proteolytic strains of Lactobacillus could produce taste through carbohydrate metabolism, proteolysis, and low lipolysis processes. Enzymes of Lactobacillus hydrolyzed casein and produced large and medium bioactive peptides. Proteolytic enzymes may subsequently degrade these peptides from starter bacteria, non-acidic lactic acid probiotic bacteria to small peptides and free amino acids, which are the major contributors to taste in dairy products (Everett and Mcleod, 2005; Grom et al, 2020). All in all, the highest and lowest sensory scores were attributed to control and CEONCBS at 28 days of storage time, respectively. According to Silva et al. (2018), salt is also expressed as a factor affecting the formation and development of aroma compounds and impacts the dairy product’s sensory properties. Won-Young et al. (2020) described that olive leaf extract to yogurt reduces the yogurt’s sensory score.

According to the results, the pH and population of L. casei decreased for all the treatments during the storage time, while their acidity and percentage of syneresis increased. The enumeration of L. casei was in the range of the recommended amount (106_107 CFU mL-1) for probiotic yogurt with stresses less than MIC during 28 days of storage. Therefore, it is possible to produce novel functional products such as probiotic yogurt containing herbal essential oil. The usage of combined C. cyminum Boiss, NaCl, and bile salts in cool conditions and MIC up to 50% during 21 days of storage is recommended for probiotic yogurt containing L. casei.

Acknowledgments

This work was supported by the Department of Food Science and Technology, Islamic Azad University, Science and Research Branch, Tehran, Iran.

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