Effect of Ziziphora clinopodioides Essential Oil Stress on Viability of Lactobacillus acidophilus and Bifidobacterium bifidum Microencapsulated with Alginate-Chitosan and Physicochemical and Sensory Properties of Probiotic Yoghurt

Introduction

Yoghurt is a fermented dairy product popular among people all over the world. It is a complete source of minerals such as calcium, proteins, fats and some kinds of useful microorganisms such as Streptococcus therm-ophilus (S. thermophilus) and Lactobacillus bulgaricus (L. bulgaricus). In recent years, scientists have tried to increase the organoleptic and health properties of yoghurt using different methods (Fernandez and Marette, 2017). Incorporation of probiotic bacteria into yoghurt is one of the most effective ways to greatly facilitate the improvement of the health charac-teristics of this product (Senadeera et al., 2018; Fenster et al., 2013).

Probiotics are defined as living microrganisms, when ingested in adequate quantities in yoghurt, beneficially influence the health of the host by improving the composition of intestinal microflora. Moreover, probiotics may play a beneficial role in several medical conditions, including lactose intolerance, cancer, allergies, hepatic disease, Helicobacter pylori infections, urinary tract infections, hyperlipidemia and assimilation of cholesterol (Tasi et al., 2019). Using beneficial probiotic bacteria such as Lactobacillus acidophilus (L. acidophilus) and Bifidobacterium bifidum(B. bifidum) is a suitable way to increase nutritional, physico-chemical, sensory and rheological properties of yoghurt. L. acidophilus and B. bifidum are normal human intestinal flora with considerable probiotic properties. They are recognized for their applications in dairy products, particularly yoghurt (Evivie et al., 2017).

The results of some recent investigations on probiotic products have shown that probiotic organisms cannot resist in fermented dairy products, and also in gastrointestinal condi-tions. Furthermore, various probiotic lactobacilli and bifidobacteria have shown a decline in their viability during products shelf life (Millette et al., 2013; Pitino et al., 2012). Thus, it is essential to increase the growth, viability and survival of L. acidophilus and B. bifidum in probiotic dairy products. Using prebiotics is one of the best ways to enhance the growth, viability and survival of probiotic bacteria. Prebiotics are food ingredients that induce the growth or activity of beneficial probiotic microorganisms (Tasi et al., 2019; Evivie et al., 2017).

The genus Ziziphora belongs to the Lamiaceae family and consists of four species: Z. clinopodioides Lam, Z. persica Bunge, Z. capitata L., and Z. tenuior L. This plant is widely distributed in different parts of Iran. Fresh leaves and stems were commonly used as sedative, carminative, appetitive, antiseptic, stomach tonic, wound-healing material, bron-cho expectorant, and antiseptic. ZEO is rich in useful antioxidants such as 1, 8-cineole, pule-gone, carvacrol, thymol, limonene and cymene. Moreover, the air-dried aerial parts of the plant were traditionally used in culinary as spice in different foods such as meat, cheese and yoghurt to enhance their flavor and aroma (Shahbazi, 2017; Smejkal et al., 2016). Furthermore, ZEO contains a large variety of minerals, amino acids, lipids, vitamins and even carbohydrates. Thus, it can be used as prebiotic to improve the growth and survival of probiotic bacteria. Several documented data revealed that inoculation of ZEO into different types of probiotic products caused significant increase in growth, viability and survival of probiotic bacteria, especially L. acidophilus and B. bifidum (Mahmoudi et al., 2017; Ziaolhagh and Jalali, 2017).

Another way to increase the survival of probiotic bacteria in food matrix and also gastro-intestinal condition is microencapsulation. Microencapsulation is a novel method through which a target compound is covered by a thin layer of polymeric material. In this technique, a variety of functional agents, including flavors, EOs, enzymes, and microorganisms, are the most considered target substances. Microencapsulation technique has been investigated for enhancing the viability of probiotic microorganisms in both dairy products and gastro-intestinal tract (Sarao and Arora, 2017; Samedi. and Charles, 2019).

There is limited literature regarding the app-lication of sublethal dose of natural EOs and also microencapsulation to improve survival of probiotic bacteria in yoghurt. Thus, the present research was done to assess the effect of ZEO and microencapsulation with alginate-chitosan on viability of L. acidophilus, and B. bifidum bacteria,and sensory and physicochemical properties of probiotic yoghurt.

Materials and Methods

Preparation of Inoculum

B. bifidum (Bb-12) and L. acidophilus (La-5) were obtained from Chr. Hansen Company (Hørsholm, Denmark). Probiotics were culture-ed in de Man Rogosa Sharpe (MRS, Merck, Germany) broth at 37ºC for 24 h. Then, activated culture was diluted in fresh media (1%) and incubated at 37ºC. This procedure was performed three times in a week and the slant cultures on Brain Heart Infusion (BHI, Merck, Germany) were stored at 4ºC (Noori et al., 2017).

Plant Materials and Essential Oil Preparation

Fresh aerial parts of Z. clinopodioides were collected from Tehran province during full flowering period in March–July 2019. The plants were identified as Z. clinopodioides Lam. by a botanical taxonomist. Voucher specimens of plants were deposited in the botany herbarium of the Research Center of Natural Resources of Tehran, Iran. Aerial parts were carefully washed with distilled water and then air-dried indoor in a shady place at room temperature for 12 days (water content approached 75% of plant fresh weight). After that, The ZEO was obtained according to the previously method published by the European Pharmacopoeia (Counsil of Europe, 1997).The dried-sample (100 gr) was grounded and homogenized in distilled water with a ratio of 1:5 and submitted to hydro-distillation for 3.5 h using a Clevenger-type apparatus. The oil over water was recovered, dried with anhydrous sodium sulfate, sealed in brown glass bottle and stored at dark in refrigerator conditions until analysis.

Gas Chromatography–mass Spectrometry (GC–MS) Analysis of EO

Analytical gas chromatography was conducted on a Thermo Quest Finningan apparatus fitted with HP-5MS 5% phenyl methylsiloxane capillary column (30 m length × 0.25 mm i.d. and 0.25 μm film thickness). Helium (purity: 99.99%; flow rate 1.2 mL/min and split ratio 1:20) was used as a carrier gas. Column temperature was initially set at 50°C, then gradually increased to 265°C at a rate of 2.5°C/min and finally fixed at 280°C. The EO analysis was also run on Thermo Quest Finningan coupled to mass spectrometer with the same analytical conditions as indicated above. The MS was run in the electron ionization mode, using the ionization energy of 70 eV (Azizkhani et al. 2013).

Detection of Lethal and Sub-lethal Concentrations of ZEO on Probiotics

The La-5 (10⁹ colony forming units (CFU)/mL) and Bb-12 (10⁹ CFU/mL) were inoculated on tubes contained 5 mL MRS broth media with different concentrations of ZEO (0, 1500, 1750, 2000 and 2500 ppm). The La-5 and Bb-12 were incubated at 37°C for 2 hr. The culture of probiotics was carried out on time Zero (prior to incubation) and after 2 h incubation. Serial dilutions of cultures were prepared. The selected dilutions were superficially cultured on plates contained the MRS bile agar for the La-5 and MRS agar with 0.05% L-cysteine and 0.3% sodium propionate for the Bb-12. The colonies were then enumerated per each milliliter of media. The lethal dose was determined as a concentration in which at least 2 log decrease of probiotic survival found and previous concentrations were determined as sublethal doses (De Souza et al., 2016).

Probiotic and EO Exposure

The La-5 (10⁹ CFU/mL) and Bb-12 (10⁹ CFU/mL) were exposed to sublethal dose of ZEOon MRS broth for about 2 hr. The tubes were then centrifuged (4000 rpm) for about 10 min at 4°C and following washing for 3 times with PBS and centrifugation, the OD of bacterial solution was adjusted to 1 (Nasab et al., 2018).

Bacterial Encapsulation

The extrusion of encapsulation was done according to the method described by Krasaekoopt et al. (2004) (Krasaekoopt et al., 2004) as follows: Sodium alginate 4% (w/v) solution (Sigma-Aldrich, Steinheim, Germany) was prepared and sterilized at 121ºC for 15 min. For the preparation of chitosan solution, low-molecular-weight chitosan (≥75% deac-etylation, Sigma- Aldrich) (0.4 gr) was mixed with 90 mL of acidified distilled water (acidified with 0.4 ml glacial acetic acid). The pH was adjusted to 5.7–6 by adding 1 mol/L NaOH. Subsequently, chitosan solution was filtered within Whatman qualitative filter paper No. 4 and its volume was adjusted to 100 mL before being autoclaved at 121ºC for 15 min. For encapsulation, 5 mL of bacterial culture (1.5×10⁹ CFU/mL) was suspended in 10 mL of sodium alginate solution. The suspensions were extruded dropwise via a 0.11 mm needle into a sterile hardening solution (0.1 mol/L CaCl2). After 30 min of gelification in CaCl2, the beads were washed with distilled water, immersed in 100 mL of chitosan solution and then were shaken on an orbital shaker at 100 rpm for 40 min. The chitosan-coated beads were washed with distilled water and used on the same day.

Viability of Encapsulated Probiotics in Simulated Gastrointestinal Conditions

The simulated gastric juice (SGJ) comprised of 9 g/L NaCl (Merck, Darmstadt, Germany) and 3 g/Lpepsin (Sigma-Aldrich) was adjusted to pH 2 with HCl. The aliquots of 0.1 g of encapsulated bacteria or 0.1 mL of free cell suspensions were blended with 5 mL SGJ and incubated for 30 and 60 min at 37ºC with persistent agitation at 50 rpm. To prepare the simulated intestinal juice (SIJ), a solution of 3 g/L ox gall (Merck, Germany) and 1 g/L pancreatin (Sigma-Aldrich) were provided. Sterilization of the solutions was done at 121ºC for 15 min. The aliquots of 0.1 gr of beads or 0.1 mL of cell suspensions were integrated to 5 mL SIJ and incubated for 60 min at 37ºC with the same persistent agitation as for SGJ. After incubation, the beads were disintegrated in sodium citrate solution and the cell count was done using the surface plate technique. The measurement of survival percentage of free and encapsulated La-5 and Bb-12 was done with the following equation (Sultana et al., 2000):

Survival (%) = (number of viable cells after exposure to gastrointestinal conditions/number of viable cells before exposure to gastro-intestinal conditions)× 100.

Yoghurt Preparation

Low fat milk (1.5%) was obtained from the Kalleh Company (Amol, Iran). Dry matter of milk was adjusted to 12 to 15% using skimmed milk powder. The mix was then pasteurized at 85ºC for 30 min and cooled up to 45ºC. Afterward, yoghurt starter, 10⁹ CFU/g of free and encapsulated of La-5 and Bb-12 bacteria, exposed and unexposed to EO were added to the mixture and incubated up to pH 4.6. Then, the prepared yoghurt samples were cooled up to 4ºC and then stored for about 28 days. All analysis was performed on days 1, 7, 14, 21 and 28 (Bertrand- Harb et al., 2003).

Enumeration of Free and Encapsulated Probiotics in Yoghurt

For the enumeration of free and encapsulated probiotics in samples, theyoghurts (10 gr) were re-suspended in 90 ml 0.1% (w/v) peptone water and 90 ml sodium citrate solution, respect-ively.Serial dilutions were prepared (up to 10^-6) and 1 mL of selected dilutions of the La-5 and Bb-12 were cultured on MRS bile agar and MRS agar with 0.05% L-cysteine and 0.3% sodium propionate, respectively using pour plate technique. The La-5 and Bb-12 were incubated in aerobic and anaerobic conditions at 37ºC for 48 h, respectively (Van de Casteele et al., 2006; Vinderola and Reinheimer, 1999).

Measurement of Syneresis of Yoghurt Samples

The yoghurt samples (20 gr) were subjected to centrifugation at 4ºC (4000 rpm for about 20 min). The supernatant was evacuated and weighted. The syneresis percent was measured according to the relation of the supernatant weight to the primary yoghurt weight (Sahan et al., 2008).

pH Measurement

The pH of yoghurts was determined during the storage time. Each yoghurt sample (1 g) was mixed with distilled water (1:1), and pH was measured using a pH meter (Jenway, UK), calibrated routinely with fresh pH 4.0 and 7.0 standard buffers (Zainoldin and Baba, 2009).

Sensory Evaluation

The taste, texture, appearance and overall acceptance of yoghurt samples were analyzed during the storage time. Sensory analysis was performed using 7 panelists familiar with the sensory properties of yoghurt using 5-point hedonic scale (Hamedi et al., 2014).

Statistical Analysis

All tests were performed in triplicate. The collected data were analyzed using SPSS for Windows Version 21.0 (SPSS Inc., Chicago, IL, USA) and the results were expressed as mean ± standard deviation (SD). The differences in parameters among groups were evaluated using One-Way Analysis of Variance (ANOVA). Duncan was performed as post-hoc multiple comparison test. Statistical significance was set at P<0.05.

Results

Chemical Components of ZEO

Table 1 represents the chemical components of ZEO. A total of 50 chemical components (98.15%) were detected in the ZEO. The most commonly detected chemical components in the ZEO were thymol (41.70%), alpha-terpineol (7.31%), carvacrol (5.39%), linalool (4.12%) and gamma-terpinene (4.10%).

Table 1. Chemical components of Z. clinopodioides EO.

Determination of Lethal and Sub-lethal Doses of EO on Probiotic Bacteria

Figure 1 and 2 represent the survival of Bb-12 and La-5 exposed to different concentrations of ZEO, respectively. The lethal doses of ZEO for La-5 and Bb-12 were obtained 1750 and 1500 ppm, respectively.