Experimental Application of Beneficial, Freeze-Dried Strain Enterococcus durans ED 26E/7 with Postbiotic Activity in Different Yogurts, Its Survival and Stability

Abstract

Yogurt is generally defined as a cultured milk product made using some species of lactic acid bacteria. Moreover, some additive bacteria are frequently involved in yogurts to provide health benefits. The objective of this study was testing the stability and survival of a beneficial strain with postbiotic activity, Enterococcus durans ED 26E/7, in cow, goat, and ewe–goat milk yogurts. The validated methods were used in the study. Postbiotic, concentrated substance (CBs) from the strain ED 26E/7 inhibited growth of indicator bacteria by 60.5%. The strains E. hirae (96%) were susceptible to CBs (inhibitory activity from 200 to 25,600 AU/mL). The growth of staphylococci was inhibited by 79% with activity of 100 up to 25,600 AU/mL. Also, 40 out of 46 fecal E. coli were inhibited (activity 100 AU/mL). CBs was thermo-stable and remained active also after storage for 11 months at −20 °C and −80 °C. Exposing CBs to proteolytic enzymes did not lead to its complete deactivation indicating that it is probably not only a proteinaceous substance. The highest counts of the freeze-dried (encapsulated), safe ED 26E/7 strain and its stability were detected in ewe–goat milk yogurts. They reached up to 5.0 cfu/g. ED 26E/7 represents a further promising additive, although other testing will be performed.

1. Introduction

Fermented milk products such as yogurts have become a popular, nutritious food [1]. In general, fermented foods are defined as foods manufactured through controlled microbial growth and enzymatic conversion of major and minor food components [2]. The nutritional composition of yogurt has made it a widely accepted dairy product. It contains carbohydrates, proteins, minerals, and essential vitamins [3]. Yogurt is a product generally defined as a cultured milk product made using bacteria such as Streptococcus thermophilus and/or Lactobacillus delbrueckii subsp. bulgaricus. However, some additives especially beneficial/probiotic bacteria are used in yogurt production to provide health benefits; then, yogurt can be described as a functional food [1]. Functional foods are foods that have functional (naturally occurring, biologically active) components which tend to confer health benefits far more than ordinary nutrition [4]. There exists a correlation between gut microbiota composition and health, because changes in gut microbiome can cause diseases [5], and the use of functional food has potential to modulate the gut microbiota and so to prevent diseases and/or improve the gut microbiome [5]. Yogurt consumption has also been linked to health benefits, including hastening digestion attributed to its probiotic constituents. Those constituents can enhance the gut microbiota and prevent symptoms, e.g., constipation, bloating, and/or diarrhea [6]. Many of those formerly mentioned beneficial bacteria are representatives of lactic acid bacteria (LAB) genera such as Lactobacillus, Bifidobacterium, Lactococcus, and Enterococcus [1,7]. In addition, the implementation of protective cultures of LAB in diverse food products contributes to food protection and food safety [8]. Some beneficial bacteria are known to produce antimicrobial substances of a proteinaceous character—bacteriocins [9] which were included in the group of postbiotics recently [10]. A postbiotic is defined as a “preparation of inanimate microorganisms and/or their components” that confers a health benefit on the host [10]. One of the postbiotic concept interest drivers is their inherent stability, both during industrial process and storage [10]. They also represent an intense innovation in the field of functional foods (foodiceuticals) as health-promoting bioactive compounds [11].

Among LAB, the representatives of the genus Enterococcus are found in many food products such as animal-derived products, e.g., dairy products [12]. The most commonly occurring enterococcal species in different milks are Enterococcus faecium, E. faecalis, E. hirae, and/or E. durans [12]. In our previous study, the representative strain of the—last-mentioned species E. durans ED 26E/7 (possessing beneficial properties involving bacteriocin-postbiotic activity) was described [7]. It was isolated from Slovak local ewe milk lump cheese. The strain E. durans ED 26E/7 was taxonomically identified using MALDI-TOF spectrometry. This strain is gelatinase and hemolysis-negative, susceptible to commercial antibiotics. It produces 10 nmoL of the enzyme β-galactosidase. It is free of virulence factor genes such as Hyl (hyaluronidase), IS16 element, and GelE (gelatinase). It does not cause mortality using a model experiment with Balb/c mice [7]. To reach the beneficial effect of the additive strain, and also its stability and sufficient survival in the host/niche, an important factor is its application form. The most requested condition for applications is simplicity. In recent years, encapsulation is frequently used as suitable form for application of beneficial strains and/or postbiotics [13]. One of the most important reasons for encapsulation of active ingredients is to provide improved stability in final products and during processing [14]. Among the simplest forms of encapsulation, freeze-drying has dominated. Taking into account this information, the objective of the study was to test the stability and survival of ED 26E/7 strain in yogurts made from cow, goat, and ewe–goat milks for further yogurts serving as functional food. Functional foods offer health benefits beyond their nutritional value and they are foods fortified or enhanced with specific nutrients or substances that have a beneficial effect on health [15]. The safety approach to enterococci is even controversial (profitable vs. enterococci as indicators of hygiene conditions) [7]. Several enterococcal strains have shown their benefits in food, in dairy products as well [16]. The British Advisory Committee on Novel Foods and Processes has already accepted the use of E. faecium strain K77D as a starter culture in fermented dairy products in 1996 [7,16]. Testing of ED 26E/7 has been also had results based on our previous studies. ED 26E/7 strain was experimentally tested in broiler rabbits [17]; its antimicrobial effect was demonstrated in the caecum and appendix by a decrease in coliforms. Reduction in Eimeria oocysts was also noted on day 21 (21st day of application of the strain), when also a significant increase (p < 0.05) in phagocytic activity was registered. Application of ED 26E/7 strain did not evoke oxidative stress in rabbits. Moreover, Pogány Simonová et al. [18] reported the highest body weight gain (increase by 1.5%) in the period of ED 26E/7 application in broiler rabbits and after the strain application also a tendency of the jejunal morphology to improve was noted. The strain with its bacteriocin improved cecal enzymatic activity [18]. The novelty of the present study lies especially in using the strain with postbiotic activity ED 26E/7 (producing the bacteriocin durancin) in its encapsulated form in yogurts from different milks to indicate it as a suitable application form to produce enriched yogurts as a functional food.

2. Materials and Methods

2.1. Enterococcus durans ED 26E/7, Testing its Susceptibility to Antibiotica Using the E-Strip Method

Enterococcus durans ED 26E/7 was isolated from ewe milk lump cheese and characterized in detail as previously reported [7]. For testing its susceptibility to antibiotics, the strain was grown in Brain Heart Infusion (BHI broth, pH 6.9, Difco Becton Dickinson, Sparks, MD, USA) overnight in an incubator at 37 °C to have absorbance A₆₀₀ up to 1.0. Although susceptibility of the strain ED 26E/7 to five antibiotics has been already been partially shown in our previous study [7] using the E-strip method, in this study additive antibiotics were involved in testing and the minimum inhibitory concentration (MIC) was established using strips of 10 antibiotics: penicillin (P, 0.016–256 µg/mL), chloramphenicol (C, 0.016–256 µgm/L), vancomycin (Va, 0.016–256 µg/mL), tetracycline (TE, 0.016–256 µg/mL), kanamycin (KM, 0.016–256 µg/mL), ampicillin (Amp, 0.016–256 µg/mL), erythromycin (E, 0.016–256 µg/mL), streptomycin (S, 0.064–1024 µg/mL), rifampicin (R, 0.002–32 µg/mL), and gentamicin (CN, 0.064–1024 µg/mL). Brain heart agar (BHA, Difco Becton Dickinson, Sparks, MD, USA) plates were seeded with overnight broth culture of the strain ED 26E/7 (100 µL). The strips were placed on the surface of the plates. Enterococcus faecalis ATCC 2921 was included as the positive control strain. Results were evaluated according to the EUCAST system (European Committee on Antimicrobial Susceptibility Testing) [7].

2.2. Antimicrobial Activity, Storage Stability, and Character of Postbiotic Substance (Concentrated Bacteriocin Substance) ED 26E/7

Following our previous study [7], more indicator bacteria were used to show the spectrum of inhibitory activity of CBs ED 26E/7. There were included fecal staphylococci from sheep (36 strains), 7 strains of the species Staphylococcus epidermidis, 7 strains of S. equorum, 7 strains of S. pseudintermedius, one strain of S. haemolyticus, two strains of S. saprophyticus and 12 strains of Staphylococcus spp. as well as 16 strains of fecal methicillin-resistant staphylococci (MRS) from horses, dogs, and roe deer; altogether, 52 various staphylococcal strain species. Moreover, 54 fecal Enterococcus hirae strains were involved as indicators (30 fecal canine strains, 7 strains from ducks, 6 strains from rabbits, 4 strains from hens, 3 strains from horses, two strains from beavers, one strain from serval, and one strain from fox). In addition, 46 fecal strains of E. coli from cats were tested (kindly provided by our colleague from Natural Sciences Faculty in Košice, Slovakia). Altogether, 152 indicator strains were used; 106 out of 152 strains were Gram-positive bacteria (staphylococci and enterococci), and 46 strains were Gram-negative E. coli. Partial purification of bacteriocin ED 26E/7 has been already reported in our previous study [7]. Analysis of concentrated bacteriocin substance (CBs)’s character was continued by testing its storage stability and by treatment with proteolytic enzymes. The CBs was previously tested against different Gram-positive enterococci, staphylococci, and listeriae which all were inhibited with antimicrobial (inhibitory) activity up to 25,600 AU/mL. At that time, the growth of Gram-negative indicators (Escherichia coli strains) was not inhibited [7].

To test storage stability of CBs, samples of CBs were exposed to heat under different temperatures (37 °C for 1 h and 2 h, 60 °C for 1 h, 2 h, 100 °C for 10 min) as previously described by Todorov et al. [19]. CBs was also stored at different temperatures (4, −20, −80 °C for one month, and 11 months). After each tested time period, inhibitory activity was tested using an agar spot test [20] against the principal indicator strain (the most sensitive) Enterococcus avium EA5. Moreover, enzymes (proteinase, trypsin, α-chymotrypsin, and pepsin, SERVA Sigma, Germany) were added to the CBs (500 µL) in the final concentration 0.5 mg/mL. Samples with and without enzymes were incubated at 37 °C for 60 min before the remaining activities were determined as formerly indicated.

2.3. Testing Safety and Stability of ED 26E/7 at Days 15, 30, 60, 90, and 120 Using Balb/c Mice

The testing was performed on 8-week-old male Balb/c mice (n = 42), free of pathogens, weighing 18–20 g (VELAZ, Prague, Czech Republic) as previously described by Vargová et al. [21] after the required ethical approval. Mice housing and experimentation were conducted in strict accordance with current Slovak ethical rules, the Guidelines for Care and Use of Laboratory Animals of the Parasitological Institute of the Slovak Academy of Sciences and the State Veterinary and Food Administration of the Slovak Republic (no. Ro-1604/19-221/3). The mice were maintained on a commercial diet. A 12 h light/dark regimen at room temperature was applied (22–24 °C) and 56% humidity. The animals were grouped into a control group (C, n = 21) and experimental group (E, n = 21). The mice in the experimental group were administered daily per os with 100 µL of ED 26E/7 cells (10⁹ cfu/mL) prepared as previously described [19]. Fecal samples (the small intestine) were obtained from mice (3 mice per group) at day 0, 15, 30, 60, 90, and 120. They were treated according to the standard microbiological dilution method (ISO). Dilutions of feces in Ringer solution after treatment in a Stomacher-Masticator (Spain) (pH 7.0, Merck, Darmstadt, Germany) were spread on the selective agar media. M-Enterococcus agar (pH 6.9 ± 0.2, Difco, Sparks, MD, USA) enriched with rifampicin was used to count the ED 26E/7 strain. The total enterococcal count was detected on M-Enterococcus agar (Difco). The count of LAB was determined on de Man–Rogosa–Sharpe agar (pH 6.2, Difco) and coliforms were enumerated using MacConkey agar (Difco). The ED 26E/7 strain was administered from day 0 up to day 30. Then, it was administered 3 times per week up to the end of testing (day 120, meaning 90 days’ duration).

2.4. ED26E/7 Strain Freeze-Drying, Its Preparation for Application into Yogurts, and Its Stability and Survival

For application, the ED 26E/7 strain was freeze-dried as previously described by Lauková et al. [22]. The strain ED 26E/7 was marked by rifampicin to differentiate it from the total LAB count according to the protocol by Nemcová et al. [23]. The strain count was controlled after cell dilutions in Ringer solution (pH 7.0, Merck, Darmstadt, Germany) to a concentration up to 10⁷–10⁹ cfu/mL on M-Enterococcus agar (Difco, Sparks, MD, USA) with rifampicin (100 µL/L). Fresh white yogurts produced by Slovak producers (145–150 g) used in the experiment were bought from the market network. According to the product label information for the goat milk yogurt of commercial yogurt culture, it contained 3.5% fat, with the parameters: 254 kJ/61 kcaL, fat 3.7 g of which saturated fatty acids formed 2.2 g, carbohydrates 3.9 g, the sugar value was 2.0 g, protein content formed 3.7 g, and salt 0.26 g for 100 g of the product altogether [24]. The components of cow milk yogurt were declared for 100 g of product to be: energy 500 KJ, fat 9.0 g of which saturated fatty acids formed 5.2 g, carbohydrate 4.5 g, the sugar value of which was 4.4 g. In cow milk yogurts, protein content formed 3.4 g and salt 0.1 g [7]. According to the product label regarding fresh ewe (75%)–goat (25%) milk white yogurt from commercial yogurt culture, it contained energy 309 KJ/74 kcaL, fat 4.6 g of which saturated fatty acids formed 3.6 g, carbohydrates 4.1 g, the sugar value was 3.4 g, proteins 4.4 g, and salt 0.07 g [24]. Yogurt samples were diluted in Ringer solution according to the standard microbiological dilution method (ISO). Dilutions were spread on MacConkey agar to control enterobacteria. Yogurts have no traces of enterobacteria. Then, 0.5 g of freeze-dried strain ED 26E/7 was applied in the experimental yogurts. ED 26E/7 strain was absent from the control yogurt. The standard microbiological dilution method (ISO) was also used for ED 26E/7 strain checking in yogurts and LAB as well using M-Enterococcus agar (Difco) enriched with rifampicin and MRS agar (Merck, Germany). For sample treatment, a Stomacher-Masticator (Barcelona, Spain) was used. The pH values of yogurts were checked using a Checker pH tester (Hanna Inst. Inc., Woonsocket, RI, USA). The initial pH values of yogurts reached values 3.90, 3.40, and 4.70. Yogurts were placed in the fridge during the checking period [24].

2.5. Statistical Analysis

For statistical analysis, the Tukey post hoc test was used. Data for safety and stability of the strain ED 26E/7 in mice and in yogurts are expressed as the mean and standard deviation (SD) of the mean with significant difference (p < 0.05). Statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad Software Incorporation, San Diego, CA, USA).

3. Results

3.1. E-Strip Susceptibility Test, Antimicrobial Activity of Postbiotic Substance, and Its Character

Before using the strain ED 26E/7, its susceptibility to antibiotics was repeatedly checked using the E-strip method. The strain was assessed as susceptible to recommended antibiotics. Kanamycin resistance is chromosomally encoded in most enterococcal species.

The growth of 92 out of 152 (52 Gram-positive strains and 40 Gram-negative E. coli) was inhibited after treatment with CBs ED 26E/7 (Durancin-like) meaning 60.5% (

Table 1. Postbiotic (bacteriocin) activity of CBs ED26E/7 against indicator bacteria.

Indicator Strains	No. Tested/No. Inhibited	Activity (AU/mL)
E. hirae (canine feces)	30/30	200–25,600
E. hirae (rabbits, hen feces)	10/10	1600–6400
E. hirae (horse feces, serval, fog, beaver, duck)	12/12	1600–6400
MRS Staphylococcus spp. (horses, dogs, roe deer)	16/16	1600–3200
MRS S. epidermidis (feces of sheep)	7/3	400–12,800
MRS S. equorum (feces of sheep)	7/5	400–1600
MRS S. pseudintermedius (feces of sheep)	7/7	100–25,600
MRS S. saprophyticus (feces of sheep)	2/0	-
MRS S. haemolyticus (feces of sheep)	1/1	3200
MRS Staphylococcus spp.	12/9	200–25,600
Escherichia coli (feces of cats)	46/40	100

E., Enterococcus; MRS, methicillin-resistant staphylococci.

). Regarding the strains of E. hirae (54), only two strains (from beaver) were not inhibited (almost 4%) and 96% of E. hirae strains were susceptible to CBs ED 26E/7 (inhibitory activity ranged from 200–25,600 AU/mL). Forty-one (41) out of 52 staphylococcal indicators were inhibited (almost 79%) with inhibitory activity from 100 to 25,600 AU/mL, Table 1). Inhibited strains belonged to the species S. epidermidis, S. equorum, S. haemolyticus, meaning CoNS species; two S. saprophyticus were not inhibited and the CoPS strains of S. pseudintermedius were also inhibited using CBs ED 26E/7. Moreover, the growth of 40 out of 46 fecal E. coli was inhibited (100 AU/mL). Exposure to proteolytic enzymes did not lead to complete deactivation of CBs ED 26E/7 against the principal indicator E. avium EA5 indicating that it could have probably also an other than only proteinaceous character. However, inhibitory activity remained after heat treatment at 37 °C for 1 and 2 h; inhibitory activity of 51,200 AU/mL was noted. In the case of heat treatment at 60 °C for 1 or 2 h, remaining activity reached also 51,200 AU/mL. Activity of 1600 AU/mL was noted after treatment at 100 °C for 10 min. Using long storage at 4 °C, an activity of 51,200 AU/mL of CBs ED 26E/7 was measured after one month; inhibitory activity of 1600 AU/mL was measured after 11 months’ storage at 4 °C. At −20 °C storage, inhibitory activity remained at 51,200 AU/mL up to one month; after 11 months activity remained at 6400 AU/mL. Inhibitory activity remained at 51,200 AU/mL after one month’s storage at −80 °C, and at the same condition activity 3200 AU/mL remained after 11 months’ storage. At laboratory temperature up to two weeks storage activity remained 51,200 AU/mL. After 4 months’ storage, activity of 12,800 AU/mL at laboratory temperature remained.

3.2. Safety and Stability of ED26E/7 at Days 15, 30, 60, 90 and 120 Using Balb/c Mice

The count of ED 26E/7 strain culminated at day 60 (4.77 ± 0.40 cfu/g log 10,

Table 2. Safety and stability of ED 26E/7 at days 15, 30, 60, 90, and 120 using Balb/c mice (in cfu/g log 10) ± SD.

	ED26E/7	Enterococci	Lactic Acid Bacteria	Coliforms
Day 0 (n = 6)	nt	4.79 ± 0.21	6.10 ± 0.00	2.98 ± 0.43
Day 15/E (n = 3)	2.68 ± 0.28 b	3.58 ± 0.25 a	5.51 ± 0.44 a	3.80 ± 0.03 a
Day 15/C	nt	2.83 ± 0.46	7.10 ± 0.00	4.41 ± 0.10
Day 30/E	2.15 ± 0.22 c	3.56 ± 1.25 c	6.51 ± 0.15 b	3.93 ± 0.12 c
Day 30/C	nt	3.72 ± 0.08	6.98 ± 0.70	5.24 ± 0.49
Day 60/E	4.77 ± 0.40 a	5.69 ± 1.44 d	7.71 ± 0.37 c	3.42 ± 0.09
Day 60/C	nt	7.28 ± 0.71	7.06 ± 0.84	4.71 ± 0.22
Day90/E	3.60 ± 0.00 e	8.73 ± 0.01 b	9.10 ± 0.00 d	2.17 ± 0.22 b
Day 90/C	nt	8.63 ± 0.44	8.52 ± 0.38	3.28 ± 0.00
Day120/E	0.90 ± 0.00 d	5.54 ± 1.33 e	6.95 ± 0.27 e	3.19 ± 0.42
Day 120/C	nt	6.94 ± 0.23	7.06 ± 0.08	4.42 ± 0.44

Day 15/E, sampling at day 15 of experiment, experimental group; Day 15/C, sampling at day 15 of experiment, control group; Day 30/E, sampling at day 30 of experiment, experimental group; Day 30/C, sampling at day 30 of experiment, control group; Day 60 /E, sampling at day 60 of experiment, experimental group; Day 60/C, sampling at day 60 of experiment, control group; Day 90/E, sampling at day 90 of experiment, experimental group; Day 90/C, sampling at day 90 of experiment, control group; Day 120/E, sampling at day 120 of experiment, experimental group; Day 120/C, sampling at day 120 of experiment, control group; nt—not tested. The strain ED 26E/7, Day 60/E: Day 15E, ^ab p < 0.01, Day 60/E: Day 30/E, ^ac p < 0.001; Day 60/E/:Day120/E, ^ad p < 0.001, day 90/E:Day120/E, ^ed p < 0.01; coliforms, Day15/E:Day 90/E, ^ab p < 0.01; Day 30/E:Day 90/E, ^cb p < 0.01; enterococci, Day 90/E:Day15/E, ^ba p < 0.001, Day 90/E:Day 30/E ^bc p < 0.001, Day 90/E:Day 60/E, Day 90/E:Day 120/E ^bd p < 0.01; lactic acid bacteria, Day 30/E:Day 15/E ^ba p < 0.05, Day 60/E:Day 15/E ^ca p < 0.001, Day 90/E:Day 15/E ^da p < 0.001, Day 60/E:Day15/E ^ea p < 0.01, Day 90/E:Day 30/E ^bd p < 0.001, Day90/E:Day 60/E ^dc p < 0.001.

). It indicated its sufficient stability because its count was increased from day 30 although later (from day 30) it was not administered daily. The strain ED 26E/7 significantly increased at day 60 compared with day 15 (^ab p < 0.01), and also at day 30 (^ac p < 0.001). A significant increase in ED 26E/7 was also noted at day 60 compared with day 120 (^ad p < 0.001) and comparing day 90 with day 120 (^ed p < 0.01). The total enterococci count in mice feces of the E group continually increased up to day 90 (Table 2), then it decreased. A significant difference was found comparing E mice at day 90 with day 15 (^ba p < 0.001), day 90 and day 30 (^bc p < 0.001), day 90 to day 60, (^bd p < 0.01), and day 90 to day 120 (^be p < 0.01). The LAB count also increased up to day 90 and then a decrease was noted (Table 2). A significant increase in LAB was noted in the E group of mice at day 30 compared with day 15 (^ba p < 0.05), at day 60 and day 15 (^ca p < 0.001), at day 90 and day 15 (^da p < 0.001), at day 120 and at day 15 (^ea p < 0.01). A significant difference in LAB was also found at day 30 and day 90 (^bd p < 0.001), at day 60 and day 90 (^cd p < 0.001), and comparing day 90 and 120 when a decrease was found (^de p < 0.001). At day 90, even coliforms were significantly decreased in the E group of mice (^ab p < 0.01) compared with their count in the E group at day 15. A significant decrease in coliforms (^cb p < 0.01) was also noted in the E group of mice at day 30 compared with the E group at day 90 (Table 2). Coliforms were also decreased at day 15 (mathematical difference 0.61 log cycle), at day 30 (difference 1.31 log cycle), at day 60 (1.29 log cycle), at day 90 (1.11 log cycle) and at day 120 (1.23 log cycle) comparing the E and C groups.

3.3. Stability and Survival of Postbiotic Active Strain ED 26E/7 Strain in Freeze-Dried Form in Yogurts

The strain ED 26E/7 colonized cow and goat yogurts only in low counts (up to 1.90 ± 0.09 cfu/g) during the recommended assessment period (expiration date especially for goat yogurts is declared to be 10–14 days mostly,

Table 3. The stability of Enterococcus durans ED 26E/7 and lactic acid bacteria counts in cow milk, goat milk, and ewe–goat milk yogurts expressed in colony-forming units per gram (cfu/g log 10) ± SD.

	CMY/pH	ED 26E/7	LAB	GMY/pH	ED 26E/7	LAB	EGMY/pH	ED 26E/7	LAB
0/1 C	3.90	nt	5.10 ± 0.50	3.40	nt	10.85 ± 1.5	4.70	nt	4.99 ± 0.70
0/1 E	3.90	nt	5.10 ± 0.50	3.40	nt	10.10 ± 1.00	4.70	nt	4.99 ± 0.80
24 h C	3.90	nt	5.10 ± 0.50	3.40	nt	10.10 ± 0.90	4.89	nt	4.70 ± 0.70
24 h E	3.84	1.90 ± 0.09	3.70 ± 0.80	3.40	1.00 ± 0.00	10.10 ± 0.90	4.75	4.08 ± 0.90	6.01 ± 0.80
7/C	4.06	nt	6.10 ± 0.80	3.29	nt	9.98 ± 0.80	4.89	nt	6.10 ± 0.80
7/E	4.01	0.90 ± 0.09	6.10 ± 0.80	3.20	0.90 ± 0.00	9.17 ± 0.70	3.75	3.69 ± 0.60	6.10 ± 0.80
10/C	3.78	nt	5.20 ± 0.60	3.15	nt	9.88 ± 1.6	nt	nt	nt
10/E	4.53	0.90 ± 0.09	5.10 ± 0.50	3.05	0.90 ± 0.00	9.10 ± 0.70	nt	nt	nt
14/C	3.24	nt	5.10 ± 0.50	3.13	nt	9.74 ± 0.70	3.75	nt	6.10 ± 0.80
14/E	3.59	1.60 ± 0.09	5.10 ± 0.50	3.01	0.90 ± 0.00	10.1 ± 1.00	3.72	2.99 ± 0.50	6.10 ± 0.80

CMY/pH, cow milk yogurt and pH value; GMY/pH, goat milk yogurt and pH value; EGMY/pH, ewe–goat milk yogurts and pH value; LAB, lactic acid bacteria; ED 26E/7, Enterococcus durans; SD, standard deviation; nt, not tested; NS, not significant.

and

Table 4. The counts of Enterococcus durans ED26E/7 in yogurts (in freeze-dried form), cfu/g log 10.

ED 26E/7	Cow Milk Yogurt	Goat Milk Yogurt	Ewe–Goat Milk Yogurt
24h	1.90 ± 0.09	1.10 ± 0.09	4.08 ± 0.90
Day 7	0.90 ± 0.00	0.90 ± 0.00	3.69 ± 0.60
Day 10	0.90 ± 0.00	0.90 ± 0.00	nt
Day 14	1.60 ± 0.09	0.90 ± 0.00	2.99 ± 0.50

Testing after 24 h, at day 7, at day 10, and at day 14 of application. ED 26E/7—Enterococcus durans, nt—not tested, NS—not significant.

). The highest counts of the strain ED 26E/7 were noted in yogurts made from combined ewe–goat milk. They reached up to 5.0 cfu/g (Table 3 and Table 4) with a decrease to day 14 (3.0 cfu/g). Regarding the ED 26E/7 counts comparing three types of yogurts, there is seen immediately the highest establishment of the strain in yogurt produced from ewe–goat milk (Table 4). The counts of the total LAB were the highest in goat milk yogurts (up to 10.85 cfu/g log 10, Table 3); however, they were not significantly increased. In the case of cow milk yogurts and ewe–goat milk yogurts, LAB counts were well-balanced during the whole assessment period reaching up to 6.0–7.0 cfu/g log 10 (Table 3 and Table 4). The pH values were balanced in the yogurts produced from all types of milk; however, the highest pH value was noted in ewe–goat milk yogurts.

4. Discussion

Additives incorporated in yogurts can be natural or modified to improve the yogurt’s functionality and nutraceutical properties [25]. Yogurts enriched with functional additives, especially natural additives, have been reported to possess an improved nutritional quality and impart several health benefits to consumers [25]. Several studies illustrated the potential of natural additives (e.g., probiotic strains with postbiotic activity) in providing healthy yogurts for consumers [1]. In further studies, it is necessary to optimize the proportions of various additives incorporated in yogurts’ formulation to enhance their quality. It is also required to investigate continually advanced delivery systems of additives (e.g., encapsulation) to improve their bioavailability and stability in yogurts [25]. Freeze-drying is the most extensively applied encapsulation technique in the food industry because it is flexible and continuous. At present, probiotic bacteria are the driving force in the design of functional foods especially in dairy products maintaining their functional effect for supporting human health [14]. In our case, the ED 26E/7 strain was the most stable and detected in the highest counts in ewe–goat milk yogurts. This could be explained probably by its source, from ewe milk lump cheese, although in the application of other non-autochthonous strains in yogurts, high colonization was found [7,24]. On the other hand, e.g., lactococci from goat milk grew in yogurts made from different milks with high stability [24]. Our strain was shown as safe by using it in the experimental application in Balb/c mice. The same strain, the CBs-producing (with postbiotic activity) E. durans ED 26E/7, was found to stimulate phagocytosis in Balb/c mice with experimental trichinellosis. The stimulation of phagocytosis could contribute to decreased larval migration and reduced parasite burden in the host [21]. Vargová et al. [21] also reported that enterocin-producing enterococci could represent a prospective strategy for the prevention and control of Trichinella spiralis infection based on several results and also involving the fact that Durancin-like (CBs) ED26E/7 increased the sIgA expression during the migratory phase of trichinellosis (18–25 days post infection). That is, it positively activated the mucosal immune response of the host, the expression of mucin and sIgA, and thus protected intestinal mucosa from the parasite invasion, inhibited worm development, and reduced female fecundity.

Most thermo-stable CBs produced by enterococci are able to inhibit also the growth of Gram-negative bacteria under in vitro and in vivo conditions as well [26,27]. E.g., CBs from E. asini EAs1/11D27 strain isolated from horses of the Norik of Muráň breed inhibited in vitro the growth of nine out of 10 fecal horse-derived Gram-negative bacteria such as Acinetobacter lwofii, Pantoea agglomerans, Serratia liquefaciens, and E. coli. Antimicrobial (bacteriocin) substances from E. lactis (isolated from fresh shrimps) which generates the enterocins A, B, and/or P inhibited the growth of Pseudomonas aeruginosa [26]. In some cases, the inhibitory spectrum of CBs can depend on the source of indicator bacteria but, e.g., in case of CBs produced by lactococci, this ability was not confirmed [24]. CBs from dairy lactococci inhibited, e.g., fecal animal bacteria—staphylococci and/or enterococci. Regarding in vivo conditions, fecal coliforms (p < 0.001) and pseudomonads were reduced in rabbits by Durancin substance after 21 weeks’ application [28]. In addition, most enterococcal CBs are substances retaining sufficient activity after long storage under different conditions, i.e., Ent 4231 produced by ruminal strain E. faecium CCM4231 [9]. Rahmeh et al. [29] reported enterocins from E. faecium isolated from camel milk with remaining activity after long storage (90 days at 4 °C) and with anti-listerial dominance.

To confirm the protein nature of the bacteriocin (postbiotic) substance, its exposure to proteolytic enzymes has necessarily been performed. Confirmation of a fully protein nature means complete deactivation of CBs’s inhibitory activity by proteolytic enzymes (such as proteinase K, pronase, etc.); however, e.g., exposure to α-amylase, lipase, and others can lead to inhibitory activity remaining [19] meaning that CBs can possess also an other than complete proteinaceous character. This requires additional studies.

However, the aim at the start of evaluation was achieved and we clearly showed the stability of the encapsulated strain with postbiotic activity especially in ewe–goat milk yogurts.

5. Conclusions

The safe strain ED 26E/7 with a broad postbiotic activity (inhibitory activity reaching up to 25,600 AU/mL) produces a thermo-stable substance with inhibitory activity also remaining after long storage at −20 and/or −80 °C. The highest count of the freeze-dried (encapsulated) ED 26E/7 strain and also its stability was detected in ewe–goat milk yogurts. Based on these results, the beneficial strain ED 26E/7 in freeze-dried form can be suitable for delivery into, e.g., yogurts.