The Application Potential of the Raw Goat Milk-Derived Strain Lactococcus lactis MK 1/3 for the Dairy Industry

Featured Application

Based on beneficial properties involving postbiotic activity, the strain Lactococcus lactis MK1/3 was indicated as a potential additive for the dairy industry.

Abstract

Raw goat milk-derived Lactococcus lactis MK1/3 (CCM 9209) was studied to show its potential for use in the dairy industry. Finding an innovative strain indicates having a new safe, original additive for functional food. The strain has been shown to be safe using a model experiment with Balb/c mice, when no mortality was noted. Its counts were increased continually during 120 days, with the highest value on day 90 (4.38 ± 1.24 colony-forming unit per gram (CFU/g, log 10). In vivo (in the experimental mice), anti-staphylococcal effect was noted with difference 1.82 log cycles. The safety of the strain MK1/3 has been also indicated by the fact that it did not produce damaging enzymes, it has been susceptible to antibiotics, and it has shown low-grade biofilm-forming ability (0.126 ± 0.35). This strain has tolerated bile, and low pH sufficiently. It produced a postbiotic active substance with inhibitory activity against cheese and milk contaminants (Enterococci), reaching antimicrobial activity up to 3200 AU/mL. The count of the strain MK1/3 was higher in yogurts from ewe goat milk (4.66 ± 0.30 CFU/g, log 10), in comparison with its count in yogurts from ewe milk (4.10 ± 0.10 CFU/g, log 10), with no influencing yogurt pH. Its use in 100% starter culture to process fresh cheese based on goat milk was revealed in the standard cheese quality with sufficient amount of lactic acid microbiota. To support the benefit of the strain MK1/3, additional human trials have been reinforced.

1. Introduction

Milk in general, goat milk included, has been considered a complete food which can be also indicated as a functional food [1,2]. Functional food means a food having functional (naturally occurring, biologically active) components, which confer health benefits far more than ordinary nutrition. The health benefits of goat milk make it a remedy for some disease conditions. It is milk with a low allergenicity and high digestibility, which makes it a popular dairy product also for immune-compromised individuals [1]. The current trend of naturally driven foods with digestibility and therapeutic benefits resulted in an increased demand for goat milk and products made from it [3]. Besides many beneficial components such as proteins, lipids, fats, vitamins, and minerals [1], raw goat milk also contains beneficial bacteria from the phylum Firmicutes [4]. Regarding, e.g., proteins, the study revealed new insights into whey proteomics at various stages of lactation with an abundance of 238 proteins [5]. The developmental changes of casein micellar structure of goat milk were also analyzed formerly. Fifteen proteins were identified during the whole lactation cycle [5]. An advantage of goat milk is also that it provides a smooth texture to milk products, due to its small-sized fat globules in comparison to other kinds of milk. It has a relatively lower amount of αs1-casein, which results in a softer gel structure with higher water-holding capacity and lower viscosity which is a desirable attribute in many fermented milk products [1].

Lactic acid genera commonly detected in raw goat milk include Lactococcus, Lactobacillus, Lactiplantibacillus, Pediococcus, Limosilactobacillus, Leuconostoc, and Lacticaseibacillus but also those novel genera mostly detected from raw cow milk such as Schleiferilactobacillus, Holzapfelia, Amylolactobacillus, Ligilactobacillus, Agrilactobacillus, etc. [6]. However, the most frequently isolated bacteria from goat milk using the standard microbiological technique represent Lactococci [7,8,9]. They are one of the most important lactic acid bacteria (LAB) used in the dairy industry [8,9], especially the species strains Lactococcus lactis. This Gram-positive bacterial species belongs to the phylum Firmicutes, the class Bacilli, the order Lactobacillales, the family Streptococcacae and, finally, to the genus Lactococcus [7,8,9]. The species Lactococcus lactis does not produce spores and is non-motile. It has generally recognized as having safe status (GRAS) [9]. The species L. lactis is one of the best characterized bacteria regarding genetics, metabolism and biodiversity [10]. In milk, it uses enzymes to produce energy molecules (ATP) from lactose. The byproduct of ATP energy production is lactic acid, which curdles milk, and curd is formed, which has been used for cheese production [11]. Moreover, certain strains of the species L. lactis can produce a multitude of different antagonistic compounds, including bacteriocins [7,12].

Bacteriocins are substances of proteinaceous character with antimicrobial effect [12]. Those substances produced by Lactococci are mostly small, thermo-stable proteins; they are most frequently recognized as inhibiting the growth of closely related bacteria, but also Gram-negative species strains [12,13]. Recently, bacteriocins have been assigned to the group of postbiotics which are defined as preparations of inanimate microbiota and/or their components, conferring a health benefit to the host [7,13]. Lactococci, with postbiotic activity from raw goat milk, were also reported in our previous study, informing the fortification of yogurts by them, to use as functional food [7]. The strains were shown to be stable in yogurts and their concentrated postbiotic substances inhibited the growth of Enterococci and Staphylococci by up to 97.8%.

The strain MK1/3 has been also isolated from raw goat milk. Firstly, it has been found with bacteriocin-like activity testing by the Skalka method [14]. Then it was tested for its technological characteristics [15]. It was shown not to produce diacetyl or CO₂, and it is lipolysis-activity negative [15]. On the other hand, it is able to grow in environments with a higher content of NaCl. This means it is able to grow even in cheeses with a higher or standard content of salt [15].

Those facts taken together encouraged us to check the strain MK1/3 for its potential use in the dairy industry as a functional strain. It is a new postbiotic active strain among the species L. lactis. Therefore, the aim of this study was to show the safety and benefit of the strain MK1/3 and its application potential.

2. Materials and Methods

2.1. The Strain Isolation and Identification

The strain was isolated using the standard dilution microbiological method (ISO, International Organization for Standardization) from 53 raw goat milk samples supplied by farmers in the central Slovakia region. Milk samples diluted in Ringer solution (pH 7, Merck, Darmstadt, Germany, 1:9 solution) were plated on M17 agar (pH 6.9, Difco-Becton Dickinson company, Sparks, MD, USA). The plates were incubated at 37° C for 48 h, to grow Lactococci. The picked colony was checked for purity by its inoculation on Brain heart infusion/agar (BHA, Difco, Sparks, MD, USA) enriched with sheep blood. The pure strain was stored using the Microbank system (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) for further testing.

The taxonomy of the strain was identified using MALDI-TOF mass spectrometry, according to Bruker Daltonic [16], as previously described by Lauková et al. [17]. The strain evaluation revealed a score which responded to probable genus identification (1.700–1.999, [16]).

Moreover, its taxonomy was confirmed using sequencing analysis, as previously described by Lauková et al. [18]. DNA from the pure colony was extracted by DNAzol direct (Molecular Research Centre, Inc. Cincinnati, OH, USA), according to the manufacturer’s instruction. The 16S rRNA gene from the colony was amplified by PCR with the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) originally presented by Lane [19], as reported by Kačírová et al. [20]. OneTaq 2X Master Mix with the Standard buffer (New England Biolabs, Foster City, CA, USA) was used in this analysis. The PCR cycling conditions comprised an initial denaturation phase of 5 min at 94 °C, then 30 cycles of denaturation at 94 °C for one minute (min), annealing at 55 °C for one min, and primer extension at 72 °C for 3 min. The final primer extension step was performed at 72 °C for 10 min. The PCR was conducted in a thermal cycler (TP professional Basic, Biometra GmbH, Goettingen, Germany). The product was visualized with GelRed (Biotium Inc., Hayward, CA, USA) on 3% (w/v) agarose gel electrophoresis in Tris-acetate-EDTA buffer (pH 7.8).

The amplified products were sent for Sanger sequencing (Microsynth, Austria GmbH, Wienna, Austria). The obtained chromatogram of sequences was analyzed using Geneious 8.0.5 (Biomatters, Auckland, New Zealand). The strain was identified based on data searches comparing 16S rRNA sequences obtained from reading with sequences available in the GenBank, using BLASTn-Basic Local Alingement Search Tools nucleotide (http//www.ncbi.nlm.nih.gov/BLAST, 31 April 2022) from the National Centre for Biotechnology Information. The selected nucleotide sequence was deposited in GenBank to receive an accession number.

2.2. In Vivo Safety Evaluation of the Strain MK1/3

The experimental design followed the scheme previously reported by Dvorožnáková et al. [21]. The animal study protocol was conducted in accordance with current European and Slovak national legislative requirements for the handling of animals, optimality of animal use, and with cruelty of the procedures specified in the Slovak Law no. 377/2012 on veterinary care. It was approved by the Animal Care Ethics Committee of the Parasitological Institute of the Slovak Academy of Sciences and the State Veterinary and Food Administration of the Slovak Republic (Ro-3184/14-221). The Balb/c male mice (pathogen-free) aged 8 weeks (VELAZ, Prague, Czech Republic, n = 42) were involved in the experiment. All handling and processing with mice were arranged and managed so as to perform humane treatment. Mice weights ranged from 18 up to 20 g. They were acclimatized for 15 days after the stress of transport. When mice did not show signs of illness, they were used for the experiment. The experiment was carried out in the accredited vivarium at the Institute of Parasitology of the Slovak Academy of Sciences (Košice, Slovakia). The mice were kept in a 12 h light/dark regime at 22–24 °C, and at 56% humidity. They were kept on a commercial diet, with access to water. The animals were divided randomly into two groups, control-C (n = 21) and experimental-E (n = 21). The rifampicin-labeled variant of the strain MK1/3 (to distinguish it from another LAB) was applied in the E-group (a dose of 10⁹ CFU/mL in 100 µL of Ringer solution—Merck, Darmstadt, Germany), prepared as previously reported by Lauková et al. [22]. L. lactis MK1/3, labeled by rifampicin, was applied daily for 30 days and then on each third day, up to the end of the experiment (120 days). Sampling was provided on day 0/1 (before application), on days 15, 30, 60, 90, and final sampling was managed on day 120. The standard microbiological method (ISO) described in Section 2.1 was used to treat samples (one g). After dilution in Ringer solution (Merck, Darmstadt, Germany), individual dilutions were spread on selective media such as M17 agar with rifampicin (100 µg/mL), to count the strain MK1/3. M17 agar with maize was used to detect amylolytic cocci. Lactic acid bacteria were counted on MRS agar (Merck, Darmstadt, Germany). Enterococci were grown on M-Enterococcus agar (Difco, Sparks, MD, USA). Staphylococci (coagulase-negative, CoNS) were counted on Mannitol Salt agar (pH = 7.2, MSA, Difco, Sparks, MD, USA). Coagulase-positive Staphylococci (CoPS) were counted on Baird-Parker agar supplemented with yolk tellurite (pH = 6.9, Difco, Sparks, MD, USA). Coliforms were determined on MacConkey agar (pH= 6.9, Difco, Sparks, MD, USA). The total bacterial count was expressed in colony-forming units per gram (CFU/g) log 10 ± SD.

2.3. In Vitro Safety Testing: Hemolysis, Enzymes, Antibiotic Profile, Biofilm-Forming Ability

The method according to Semedo-Lemsaddek et al. [23] was used for hemolysis checking. The strain was inoculated onto Brain heart agar supplemented with 5% defibrinated sheep blood (Difco, Sparks, MD, USA). After cultivation at 37 °C for 18 h, the presence/absence of cleared zones around grown colonies was evaluated. It was assessed as α, β-hemolysis, and negative (no hemolysis), γ-hemolysis.

To eliminate and/or confirm safety under in vitro conditions, the API-ZYM panel enzyme system (BioMerieux, Marcy LEtoile, France) was used. This test includes these enzymes: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The method was previously described by Lauková et al. [7]. The enzyme activity was evaluated by color intensity values assessed from 0 to 5. The values checked corresponded with relevant values in nanomoles (nmol, 5–40 nmol).

Antibiotic tests include the standard disc diffusion method and E-strip method with the minimum inhibitory concentration (MIC), as well as EUCAST [24]. In the case of the agar diffusion test, 9 antibiotics were screened, such as novobiocin (5 µg), ampicillin (10 µg), erythromycin, azithromycin (15 µg), chloramphenicol, tetracycline, erythromycin, rifampicin (each in a concentration of 30 µg), and gentamicin (120 µg). BH-agar supplemented with sheep blood (Difco, Sparks, MD, USA) was inoculated with 100 µL of the overnight strain broth culture. Disks were applied onto the agar plate surface. They were supplied by Oxoid Limited (Basingstoke, Hampshire, UK). After incubation at 37 °C for 18 h, susceptibility (inhibitory-zone diameter) and/or resistance was evaluated according to EUCAST [24].

The following 7 antibiotics were used in the E-strip method: chloramphenicol (0.016–256 µg/mL), gentamicin (0.064–1024 µg/mL), erythromycin (0.015–256 µg/mL), vancomycin (0.016–256 µg/mL), rifampicin (0.032–32 µg/mL), tetracycline (0.016–256 µg/mL), and ampicillin (0.016–256 µg/mL). The same protocol was applied, but instead of disks, the appropriate strips were used. After overnight incubation at 37 °C, MIC (minimum inhibitory concentration) was evaluated. The strain Lactococcus lactis CCM 1998 was the positive control strain.

To test biofilm formation by the strain MK1/3, the quantitative plate assay was applied [25], as previously described by Lauková et al. [18]. Absorbance (A₅₇₀) at 570 nm of the prepared samples was assessed using a Synergy TM4 Muldi Mode Microplate reader (Biotek, USA). The strain was tested in two independent analyses, with 12 replicates. In each test, a sterile Brain heart infusion (broth) served as a negative control. As a positive control, the strain Streptococcus equi subsp. zooepidemicus CCM 7316 (provided by Dr. Eva Styková, University of Veterinary Medicine and Pharmacy, Košice, Slovakia) was used. Biofilm formation was classified as follows: highly-positive (A₅₇₀ ≥ 1), low-grade positive (0.1 ≤ A₅₇₀ < 1) and negative (A₅₇₀ < 1).

2.4. Beneficial Characteristics (Tolerance to Oxgall/Bile, Low pH, Postbiotic Activity)

For further application, the properties such as tolerance to bile and to low pH and/or postbiotic potential via bacteriocin activity, are recommended for testing. Tolerance to oxgall/bile in the medium was tested in M17 broth (Difco, Sparks, MD, USA) supplemented with 3% and 5% bile/oxgall (Difco, Sparks, MD, USA), as previously described by Lauková et al. [26]. Viable cell count was counted on M17 agar at time 0 and at 24 h after the standard dilution of samples in Ringer solution (1:9). The cell count was expressed in colony-forming units (CFUs) per mL.

Tolerance to pH 3 was controlled in simulated gastric juice containing pepsin and not containing pepsin (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany), as previously described Lauková et al. [26]. Tubes with pepsin and those not containing pepsin were inoculated with 0.1% culture of the strain MK1/3 in M17 broth. Surviving cells were counted on M17 agar (Difco, Sparks, MD, USA) at time 0 and after 180 min. After the standard microbiological dilution, the cells count was performed and expressed in CFU/mL.

Postbiotic activity (bacteriocin activity) of the strain MK1/3 was tested using the qualitative method [7] against the principal indicator (the most susceptible strain) Enterococcus avium EA5 (feces of piglet, our strain). Then, the concentrated substance (CS) was prepared as reported by Lauková et al. [7]. L. lactis MK1/3 was inoculated in 40 mL MRS broth (Merck, Darmstadt, Germany) and incubated overnight at 37 °C to achieve an absorbance of A₆₀₀ up to 1.0. The broth culture was centrifuged (10,000× g) for 30 min. The pH of the supernatant was adjusted to 5.5. The cell-free supernatant was treated by adding Chelaton III-EDTA (Sigma-Aldrich, Muenchen, Germany). This followed heating at 80 °C for 10 min, to eliminate the organic substances effect. The next step included concentration of supernatant using Concentrator Plus (Eppendorf, Hamburg, Germany), to obtain the concentrated substance (4.0 ml, CS). The inhibitory activity was analyzed by the agar spot method [27] against the EA5 strain, and expressed in arbitrary unit per ml (AU/mL). It corresponds to the highest dilution of CS (ratio 1:1 in phosphate buffer, pH 6.5) which inhibited the growth of the indicator strain. CS was stored at −20 °C in the fridge. Besides the EA5 strain, 22 different Enterococci (our isolates from fresh ewe-milk lump cheeses) were used as indicators.

2.5. Encapsulation (Freeze Drying) of MK1/3

The simplest form of encapsulation is freeze drying [7]. The strain Lactococcus lactis MK1/3 (rifampicin-labeled) was grown in 300 ml of M17 broth (pH 6.9, Merck, Darmstadt, Germany) for 24 h at 37 °C in an incubator overnight (A₆₀₀ up to 1.0). The broth culture was mixed with skim milk (Simandl company, Karviná, Czech Republic) in small flasks (in a ratio of 1:1). Flasks were placed for freezing at −80 °C. The freeze-drying process was performed using the Micro Modulyo 230 freeze dryer (Thermo-electron corporation, Asheville, NC, USA). After sufficient freeze drying, the powder was weighed. The cell count was checked as has been formerly described; part of the freeze-dried strain was diluted in Ringer solution and dilutions were spread on M17 agar (Difco, Sparks, MD, USA). Plates were incubated at 37 °C for 24 h. The control cell count was expressed in CFU/g (log 10).

2.6. Counts and Stability of Encapsulated Strain MK1/3 in Ewe-Goat Milk Yogurts

Fresh ewe-goat (75%)–milk (25%) white yogurts (145 g) for the application experiment were bought from the commercial market network. The yogurts contained commercial yoghurt cultures. They were assessed to have an energy value of 309 KJ/74 kcal and fat content of 4.6 g (of which saturated fatty acids made up 3.6 g). Carbohydrates were involved in 4.1 g, the sugar value of which was 3.4 g. Protein content formed 4.1 g, and salt 0.07 g. The encapsulated strain was controlled for cell count (10⁷–10⁹ CFU/g) before application. It was applied (0.5 g) to the experimental yogurt-E. The C-control yogurt was not enriched with the strain. Before application, yogurt samples were diluted in peptone water and spread on Mac Conkey agar (Difco, Sparks, MD, USA) to control contamination by enterobacteria. It was enterobacteria absent. Moreover, LAB counts were counted on MRS agar (Merck). The control check was provided also on M17 agar (Difco, Sparks, MD, USA) because of commercial culture Streptococcus spp. Sampling was performed after 24 h, on day 7 and 10 (10–14 days are declared as expiration time for this type of yogurt). Yogurt samples (one g) were taken and mixed (Stomacher-Masticator, IUL, Barcelona, Spain) with peptone water (Merck, ratio 1:9), diluted, and spread on the selected cultivation media (ISO) as previously reported by Lauková et al. [22]. The strain MK1/3 was selected on M17 agar enriched with rifampicin (100 µg/mL), LAB count was determined on MRS agar, and, also, amylolytic cocci were checked using M17 agar (Difco, Sparks, MD, USA). Moreover, pH was measured using a Checker-pH tester (Hanna instruments Inc., Woonsocket, RI, USA). Initial pH was 3.91. Yogurts were maintained in the fridge during the whole testing period.

2.7. Counts and Stability of Encapsulated Strain MK1/3 in Ewe Milk Yogurts

Fresh ewe-milk white yogurts (150 g) in the application experiment were bought through the commercial market network. The yogurts contained yogurt commercial cultures. They had an indicated energy of 319 KJ/76.1 kcal, and saturated fatty acids participated with 3.46 g. Carbohydrates were involved in 3.6 g, the sugar value of which was 2.9 g. Protein content formed 4.3 g, and salt 0.12 g. The encapsulated strain was checked for cell count before application and then 0.5 g was applied in the experimental yogurt-E. The C-control yogurt was not enriched with the strain. Before application, yogurt samples were diluted in peptone water and spread on their microbial background, as previously described. Sampling was performed after 24 h, on day 7 and 10. Sampling and treatment were provided as indicated in Section 2.6. The initial pH of yogurts was 3.28.

2.8. Use of the MK1/3 Strain in Fresh Goat-Milk Cheese

L. lactis MK1/3 (100%) was used for fresh ewe-milk cheesemaking. Raw goat milk pH was 6.69, and the total microbial cell count was 3. 90 CFU/mL (log 10) using Brain heart blood agar. The first step included the thermo-treatment of raw goat milk in the cheese chamber at 70 °C, with a minute of endurance. Then, pasteurized milk was heated (5 L) to 33 °C, followed by the starter-culture addition into the milk (1.5%), meaning 75 mL of the strain MK1/3 per 5 L of milk. The next steps included the following: 30 min of milk remaining at 33° C after starter-culture addition; the addition of 36% solution of calcium chloride (2 mL per 5 L) in milk; rennet addition (1 mL/5 L); milk curdling and coagulating at 33 °C for 30 min; cheese curd cutting, with 5 min of resting; the processing of cheese curd (harping, cheese grain mixing for 20 min with slight tempering, to achieve 35–36 °C); cheese curd forming—pouring into stainless steel molds, draining of whey for 1 h; cheeses fermenting in the climatic chamber at 25 °C; decrease in fermenting temperature (20 °C). Twenty h after processing, the cheese fermenting ended; cheese salting was carried out for 1 h in 20% solution of sodium chloride (NaCl, pH 5.0). The other step included cheese drying after salting and storing in the fridge. Before cheese curding, the pH value of pasteurized milk measured 6.58. Sampling was provided 1 h after whey draining, at 6 h and 20 h, then on day 7 of storage and on days 14 and 21 of storage. The pH value was measured with a Checker-pH tester (Hanna instruments Inc., Woonsocket, RI, USA). The counts of presumptive Lactococci were determined on M17 agar (Difco, Sparks, MD, USA). Before salting, the following parameters were measured in cheeses (fat, 27.75 g/100 g; dry matter, 49.47 g/100 g; fat in dry matter, 56.09%). Cheeses were assessed for flavor and texture.

2.9. Statistical Analysis

In general, statistical analysis was performed using one-way analysis of variance followed by Tukey’s post-test. The results statistically evaluated are quoted as means ± SD; the level of significance is set at p < 0.05.

3. Results

3.1. The Strain MK1/3 Taxonomy and Its In Vivo Safety

Based on MALDI-TOF mass spectrometry, the strain MK1/3 was evaluated with the score value 1.827. This score responds to probable genus identification (1.700–1.999). The strain was allotted to the species Lactococcus spp. However, after sequencing, it was definitively allotted to the species Lactococcus lactis. The accession number ON114093 was provided in GenBank for the nucleotide sequence SUB11246790 Seq1. The safety of the strain MK1/3 was confirmed using Balb/c model experiment. The strain MK1/3 increased its counts continually, with the highest value on day 90 (

Table 1. The count of the strain MK1/3, other lactic acid bacteria (LAB), and amylolytic cocci in feces of mice on days 15, 30, 60, 90, and 120 (CFU/g, log 10 ± SD).

Day	MK1/3	LAB	Amylolytic Cocci
Control 15 (n = 21)	nt	7.10 ± 0.00	7.10 ± 0.00
Experimental 15 (n = 21)	1.63 ±0.65 ab	7.10 ± 0.00 ab	7.10 ± 0.00 ab
Control 30	nt	7.59 ± 0.42	7.55 ± 0.46
Experimental 30	2.88 ± 1.02 ec	7.85 ± 0.38	7.86 ± 039 ec
Control 60	nt	nt	nt
Experimental 60	3.36 ± 0.32 bc	7.88 ± 0.10	8.71 ±0.38 ab,bc
Control 90	nt	8.10 ± 0.00	9.39 ± 0.00
Experimental 90	4.38 ± 1.24 ac	8.87 ± 0.33 ab	9.91 ± 0.27 ac,ec
Control 120	nt	nt	nt
Experimental 120	4.12 ± 0.05 ad, bd	8.25 ± 0.07 ac	10.1 ± 0.00 ad,ed,bd

n = 42 (21 + 21); C15-120, control group of mice at different days of sampling; E15-E120, experimental mice at different days of sampling; MK1/3, ^ab p < 0.001; ^ac p < 0.001; ^ad p < 0.001; ^ec p < 0.001; ^bc p < 0.01; ^bd p < 0.001; LAB, ^ab p < 0.001; ^ac p < 0.05; the other NS is not significant; Amylolytic Streptococci, ^ab p < 0.001; ^ac p < 0.001; ^ad p < 0.001;^ec p < 0.001; ^ed p < 0.001; ^bc p < 0.01; ^bd p < 0.001; The other are not significant; nt—not tested.

) reaching 4.38 ± 1.24 CFU/g (log 10). Significant increases were even noted in the E mice (experimental): on day 15 and day 60 (^ab p < 0.001); on days 15 and 90 (^ac p < 0.001; on days 15 and 120 (^ad p < 0.001). A significant increase was also estimated on day 30 (^ec p < 0.001) compared with day 90; on days 60 and 90 (^bc p < 0.01), and on day 60 (^bd p < 0.001) compared with day 120, as well. The counts of LAB were high and well-balanced. The significant increase was noted in E mice on day 15 (^ab p < 0.001) compared with day 90 and also on day 15 (^ac p < 0.05) compared with day 120. The significant increase in amylolytic cocci was noted in E mice on day 15 (^ab p < 0.001) compared with days 60 and 90 (^ac p < 0.001) and on day 15 compared with day 120 (^ad p < 0.001). An increase in amylolytic cocci was also visible, with significance, on day 30 compared with day 90 (^ec p < 0.001); on days 30 and 120 (^ed p < 0.001); on days 60 and 90 (^bc p < 0.01), and on days 60 and 120 (^bd p < 0.001; Table 1).

In experimental mice, a significant increase in Enterococci was also found on day 90 compared with day 30 (^ab p < 0.01,

Table 2. The fecal count of Staphylococci, Enterococci and coliforms in mice on days 15, 30, 60, 90, and 120 (CFU/g, log 10 ± SD).

Day	Enterococci	Coagulase-Negative Staphylococci	Coagulase-Positive Staphylococci	Coliforms
C/15	4.63 ± 0.21	4.57 ± 0.01 ab	1.60 ± 0.42	4.00 ± 1.27
E/15	3.91 ± 0.67	3.10 ± 0.35 ab	2.26 ± 0.43	4.15 ± 0.26
C/30	5.20 ± 0.06	3.86 ± 0.36 cd	1.54 ± 0.08	4.00 ± 0.43
E/30	2.62 ± 1.4 ab,ac	2.04 ± 0.28 cd,ed	1.30 ± 0.56 cb	5.06 ± 0.47
C/60	nt	nt	nt	nt
E/60	3.68 ± 0.28 bd	1.92 ± 0.32 ed	1.86 ± 0.85 ab	3.75 ± 0.81
C/90	4.98 ± 0.00	3.78 ± 0.00 fg	3.00 ± 0.00	3.30 ± 0.00
E/90	7.10 ± 0.00 ab,bd	2.63 ± 0.10 fg	3.63 ± 0.76	3.59 ± 0.30
C/120	nt	nt	nt	nt
E/120	6.90 ± 0. 57 ac	3.63 ± 0.21	4.28 ± 0.18 ab	5.23 ± 0.95

n = 42; C/15-C/120, control group of mice at different days of sampling; E/15-E/120, experimental mice at different days of sampling; Coagulase-positive cocci, ^ab p < 0.05; ^cb p < 0.01; Coagulase-negative cocci, not significant (NS); ^ab—mathematical difference 1.47 log cycle; ^cd,ed—difference 1.82 log cycle; ^fg—difference 1.15 log cycle; Coliforms, not significant differences, not influenced; Enterococci, ^ab p < 0.01; ^ac p < 0.01; ^bd p < 0.05; nt—not tested.

). The increase in Enterococci was also found on day 120 compared with day 30 (^ac p < 0.01), and on day 90 compared with day 60 (^bd p < 0.05). The coliforms were not influenced; they were increased mathematically on day 120 (Table 2). CoNS were not significantly influenced. However, their counts were lower in experimental mice on day 15 compared with control mice on day 15 (mathematical difference 1.47 log cycle, Table 2). The difference of 1.82 log cycle was also noted on day 30 in experimental mice compared with control mice on day 30. CoNS still remained reduced in experimental mice on day 60, with mathematical difference of 1.15 log cycle. The same trend was noticed in experimental mice on day 90 compared with control mice (difference of 1.15 log cycle). It looks as if the anti-staphylococcal (CoNS) effect was noted in the decrease in experimental mice of up to 1.82 log cycles. The counts of CoPS were low; the reducing effect was stable and visible on day 60 compared with day 120 (^ab p < 0.05) and on day 30 compared with day 120 (^cb p < 0.01).

3.2. In Vitro Safety Testing of the Strain MK1/3

The MK1/3 strain is non-hemolytic (γ-hemolysis); it does not produce damaging enzymes such as β-glucuronidase (0 nmol), trypsin (0 nmol), or α-chymotrypsin (0 nmol). The value 5 nmol was measured for the enzyme esterase, esterase lipase, lipase, β-glucosidase, α-mannosidase, α-fucosidase, and α-galactosidase. The measured value for alkalic phosphatase was 10 nmol and for acidic phosphatase 30 nmol. Naphtol-AS-BI phosphatase reached 10 nmol. However, useful enzyme β-galactosidase was found in an amount of 5 nmol. The other enzymes were not produced (0 nmol).

Using the disk method for testing the antibiotic phenotype profile, the strain MK1/3 was susceptible, with an inhibitory-zone diameter size of 29 mm when measured for gentamicin, 33 mm for rifampicin, and 30 mm for erythromycin and azithromycin. The zone 25 mm was measured for tetracycline, 22 mm for novobiocin and chloramphenicol, 12 mm for ampicillin, and, finally, 11 mm in the case of vancomycin. Susceptibility to antibiotics of the strain MK1/3 was also confirmed using the E-strip method with the following MICs: 2 µg for vancomycin and chloramphenicol, 0.006 µg for rifampicin, 0.06 µg for erythromycin, 0.12 µg for tetracycline, 0.5 µg for ampicillin, and MIC 1.0 µg for gentamicin. The strain MK1/3 was shown to have low-grade biofilm-forming ability (0.126 ± 0.35).

3.3. Tolerance to Oxgall/Bile and Low pH of the Strain MK1/3, Postbiotic Activity

L. lactis MK1/3 has grown sufficiently in BHI broth supplemented with 5% oxgall/bile. Its count reached 5.00 ± 0.00 CFU/mL (log10) at 24 h compared with its counts at time 0 (1.76 ± 0.02 CFU/mL). Using 3% oxgall/bile concentration, the growth was higher at time 0 than in broth with 5% oxgall. It increased at 24 h (7.09 ± 0.09 CFU/mL, log 10). This means that the strain MK1/3 counts were increased up to 24 h in both oxgall % concentrations. Similarly, the strain MK1/3 tolerates pH 3; only a 21% decrease in cell count was noted compared with counts at time 0 (5.54 ± 0.06 CFU/mL), and after 180 min (1.15 ± 0.15 CFU/mL, log 10).

Using the qualitative method, we measured the postbiotic activity of the strain MK1/3 against the principal indicator Enterococcus avium EA5 released in the 9 mm-diameter inhibitory zone. Evaluated inhibitory (postbiotic) activity of concentrated substance (CS) reached 1600 AU/mL against the principal indicator strain EA5. Moreover, the other indicator bacteria were tested, such as 22 different enterococcal species strains isolated from ewe-milk lump cheeses, including 6 strains Enterococcus faecium, 11 E. faecalis and 5 Enterococcus spp. The growth of all strains was inhibited with inhibitory activity in the range from 100 up to 3200 AU/mL. The species strains E. faecalis were more susceptible to CS from the strain MK1/3 than E. faecium. In the case of six E. faecium strains, inhibitory activity was measured from 100 up to 200 AU/mL. The strains E. faecium EF5/2, EF1/1, EF8B/1, and EF13A were inhibited with activity of 100 AU/mL. The strain EF 4/2 was inhibited with inhibitory activity of 200 AU/mL. Inhibitory activity against E. faecalis strains reached 100, up to 3200 AU/mL. The indicator strain EE21E1 was inhibited with activity of 3200 AU/mL. Seven strains (EE31E2, EE33E4, E23E9, EE36E4, EE10E4, EE25E1, EE22E5) were inhibited with activity of 1600 AU/mL. The inhibitory activity 800 AU/mL was measured against the strains EE5E4 and EE11E4. Only indicator strain EE2/3 was inhibited with activity of 100 AU/mL. The strains Enterococcus spp. 21B/2, 22B/2, 23B/1, and 9A/1 were also inhibited with activity of 100 AU/mL.

3.4. The MK1/3 as Starter Culture in Fresh Cheesemaking from Goat Milk

The parameters achieved in fresh cheese with the strain MK1/3 responded to the quality for this type of cheeses. The pH value was not negatively influenced. To measure it at 1 h of whey draining, the value 6.34 was measured and presumptive Lactococci counts reached 8.58 CFU/g (log 10). The counts of Lactococci at 6 h of whey draining reached 9.75 CFU/g, and pH measured 5.36. The pH value dropped (5.01) at time 20 h of whey draining, with almost the same lactococcal count (9.65 CFU/g) as at 6 h. The counts of presumptive Lactococci on day 7 of cheeses storage were the highest (10.10 CFU/g), with a slight pH decrease (4.75). A slight decrease in Lactococci was noted on day 14 of cheeses storage compared with day 7 (9.71 CFU/g, log 10), but still gave a high count, which was almost the same as on day 21 of storage (9.41 CFU/g, log 10). The pH value was stable at both those times (4.82). Cheeses before salting (1–3 weeks) were assessed as possessing lactic flavor similar to goat milk, with hard cheese texture continually changed into soft, crumbly texture after one week, and more crumbly texture up to the third week.

3.5. Survival of the Strain MK1/3 in Ewe-Goat Milk Yogurts and in Goat-Milk Yogurts

Yogurt microbial backround was analyzed to eliminate contamination. The pH values in yogurts made from ewe-goat milk reached 3.91 ± 0.12. During 10 days it was almost stable—not influenced (

Table 3. The counts of encapsulated strain MK1/3, lactic acid bacteria and amylolytic cocci in yogurts processed from ewe-goat milk (CFU/g, log 10 ± SD) and pH values through 10 days’ storage.

n = 2	pH	MK1/3	Lactic Acid Bacteria	Amylolytic Cocci
E/24	3.91 ± 0.12	4.61 ± 0.20	5.10 ± 0.00 ab	5.10 ± 0.00
C/24	3.93 ± 0.10	nt	4.41 ± 0.30 ab	4.41 ± 0.30
E/7	4.10 ± 0.50	4.43 ± 0.30	5.10 ± 0.00 cd	5.10 ± 0.00
C/7	4.09 ± 0.20	nt	4.62 ± 0.30 cd	5.30 ± 0.30
E/10	3.90 ± 0.10	4.66 ± 0.30	6.10 ± 0.50	6.10 ± 0.50
C/10	3.85 ± 0.60	nt	6.41 ± 0.30	6.10 ± 0.30

E/24—the experimental yogurts at 24 h, C24—the control yogurts at 24 h; E/7—the experimental yogurts at day 7, C/7—the control yogurts at day 7; E/10— the experimental yogurts at day 10, C/10—the control yogurts at day 10; nt—not tested; Lactococcus actis MK1/3-rifampicin labeled, encapsulated (freeze-dried); lactic acid bacteria; amylolytic cocci; NS; lactic acid bacteria, ^ab, ^cd (mathematical difference 0.69 and 0.48 log cycle).

,

Table 4. The counts of encapsulated strain MK1/3, lactic acid bacteria and amylolytic cocci in yogurts processed from ewe milk (CFU/g, log 10 ± SD) and pH values throughout 10 days’ storage.

n = 2	pH	MK1/3	Lactic Acid Bacteria	Amylolytic Cocci
E/24	3.28 ± 0.20	1.95 ± 0.10 ac	9.95 ± 0.90	10.10 ± 0.00
C/24	3.38 ± 0.83	nt	9.95 ± 0.97	10.10 ± 0.00
E/ 7	3.30 ± 0.20	1.95 ± 0.10 bc	7.50 ± 0.69	9.25 ± 0.39
C/7	3.35 ± 0.54	nt	9.89 ± 0.94	9.85 ± 0.92
E/10	3.31± 0.10	4.10 ± 0.20 ac,bc	7.00 ± 0.00	9.82 ± 0.91
C/10	3.40 ± 0.60	nt	8.79 ± 0.89	10.10 ± 0.00

E/24- the experimental yogurts at 24 h, C24—the control yogurts at 24 h; E/7—the experimental yogurts at day 7, C/7—the control yogurts at day 7; E/10—the experimental yogurts at day 10, C/10—the control yogurts at day 10; nt—not tested; Lactococcus lactis MK1/3, rifampicin labeled, encapsulated (freeze-dried); the strain count of MK1/3 increased (^ac,bc) with 2.15 log cycle difference in E/24, E/7 compared with the experimental yogurts on day 10 (E/10); lactic acid bacteria, amylolytic cocci—not influenced, not significant difference; their counts were higher in controls on day 7 and 10, which indicates competitive interaction. The same situation was found in amylolytic cocci on day 10, when comparing experimental and control yogurts.

and

Table 5. The counts of encapsulated MK1/3 strain in yogurts from ewe-goat milk and ewe milk (CFU/g, log 10 ± SD) and pH values at different days of storage.

	pH/Ewe	pH/Combinative	MK1/3 #Ewe + Goat	MK1/3 #Ewe
E/24	3.28 ± 0.20	3.91 ± 0.10	4.61 ± 0.20	1.95 ± 0.25 ac
C/24	3.38 ± 0.83	3.93 ± 0.10	nt	nt
E/7	3.30 ± 0.20	4.10 ± 0.50	4.43 ± 0.30	1.95 ± 0.25 bc
C/7	3.35 ± 0.54	4.09 ± 0.20	nt	nt
E/10	3.31± 0.10	3.90 ± 0.10	4.66 ± 0.30	4.10 ± 0.10 ac,bc
C/10	3.40 ± 0.60	3.85 ± 0.10	nt	nt

E/24—the experimental yogurts at 24 h, C24—the control yogurts at 24 h; E/7—the experimental yogurts at day 7, C/7—the control yogurts at day 7; E/10—the experimental yogurts at day 10, C/10—the control yogurts at day 10; nt—not tested; Lactococcus lactis MK1/3-rifampicin labeled, encapsulated (freeze-dried); pH/combinative, pH in yogurts from ewe ± goat milks; pH/ewe, pH in yogurt from ewe milk. The strain count of MK1/3 increased (^ac,bc) with 2.15 log cycle difference in E/24, E/7 compared with the experimental yogurts on day 10 (E/10).

). In yogurts made only from ewe milk, the pH values were very similar and they continued to be almost stable during the whole period (Table 3 and Table 4). The count of the strain MK1/3 in ewe-goat milk yogurts was high (the initial value of the encapsulated, rifampicin-labeled strain MK1/3 was 10⁹ CFU/mL). The counts were well-balanced during the whole checking period. The counts of MK1/3 in ewe-milk yogurts were low at the start of addition (Table 3), with mathematical increase on day 10 (difference of 2.15 log cycle). However, in ewe-milk yogurts we counted high values of LAB and amylolytic cocci (up to 10.0 CFU/g log 10, Table 4), while in ewe-goat milk yogurts the counts of LAB and amylolytic cocci were lower but they were well-balanced (6.10 ± 0.50 CFU/g log 10). As mentioned, mathematical difference was noted, not a statistically significant result.

4. Discussion

Currently, more and more new techniques have been developed for bacterial identification. Although MALDI-TOF MS has become a widely used technique for the rapid, accurate, durable, economical, and trustworthy method of bacteria identification [28], 16S rRNA gene sequencing, as an amplicon-based method that can identify and classify bacteria, has been used simultaneously with the next-generation sequencing technique (NGS). BLAST (basic local alignment search tool) finds regions of local similarity between sequences. The program compares nucleotide or protein-sequences databases and calculates the statistical significance of matches. In our case, sequencing led to the taxonomic allotment of the raw goat milk-derived strain MK1/3 as Lactococcus lactis. The species Lactococcus lactis is characterized as not exhibiting β and/or α—hemolysis [29], which was also shown in the MK1/3 strain.

Regarding the enzyme production, β-galactosidase is important for an organism, as a key provider in the production of energy and as a source of carbons through the breakdown of lactose to galactose and glucose. It is important for lactose-intolerant people, as it is responsible for making lactose-free milk and other dairy products. In the case of lactase lack (which has the same function as β-galactosidase), a human is not able to properly digest dairy products. Recently, β-galactosidase has been researched as a potential treatment for lactose-intolerance through gene replacement therapy; genes can be placed into human DNA and individuals can break down lactose on their own [30]. The damaging enzyme α-chymotrypsin serves as a disease marker and β-glucuronidase can even be detected in the case of colon cancer.

If the selected strain is to be used as beneficial additive, it has to fulfill some safety criteria. Among them is included the absence of antibiotic-resistance genes. In our study, an antibiotic phenotype was evaluated (not genes), but the strain MK1/3 showed antibiotic susceptibility, when testing it by two approaches. In the other study, Flórez et al. [31] described MIC for L. lactis strains isolated from dairy and animal sources using the E-test. They found those strains susceptible to vancomycin, erythromycin, streptomycin, chloramphenicol, tetracycline, and clindamycin.

The most important point for a further functional strain is its in vivo safety. In this study, in vivo safety of the strain MK1/3 was tested using the Balb/c mice model. No mortality was noted. The strain MK1/3 was sufficiently established in the mice GIT, with the highest value on day 90 (4.38 ± 1.24 CFU/g) and with significant increases during whole testing. A significant increase in Enterococci was also found (in E90 compared with E30, and 120). Moreover, LAB counts and amylolytic cocci, as well, were well-balanced. Coliforms were not influenced, and reduction in Staphylococci (mathematical difference up to significant difference) was noted. An in vivo anti-staphylococcal effect was previously most frequently noted using other types of bacteriocin-producing strains and/or their bacteriocins, such as, e.g., Enterocin M produced by Enterococcus faecium AL41 = CCM8558 in broiler rabbits [32]. There also, immune parameter–phagocytic activity was increased.

Based on previous [15] technological tests (e.g., no diacetyl production) of the strain MK/3, we decided to test its beneficial parameters, such as postbiotic (bacteriocin) potential. The strain MK1/3 was found to be bacteriocin (postbiotic) active. This testing is still in processing. Postbiotic substance from the strain MK1/3 showed in vitro anti-enterococcal effect. Moreover, in vivo, an anti-staphylococcal effect was also noted. Akbar and Anal [33] described the strain L. lactis from fermented milk with anti-staphylococcal activity against the target of S. aureus bacteria. As formerly indicated, in this study, in vivo anti-staphylococcal effect was noted using a mice model studying safety of the MK1/3. Akbar et al. [34] even reported L. lactis subsp. lactis from fermented milk with a broad antimicrobial spectrum including S. aureus, E. coli, and S. typhimurium. This strain was similar to ours, tolerating 3% bile and pH 3, which are properties improving the functionality of the selected strain. Anti-staphylococcal and anti-listerial effects of bacteriocin produced by L. lactis 808 from fermented food products and fruits were reported by Choi et al. [35]. In general, bacteriocins produced by Lactococci (postbiotic active) usually exhibit a narrow inhibitory spectrum [36]. As reported, in the case of the strain MK1/3, in vitro postbiotic activity testing is still in processing. That is, we cannot recognize at this time if it is a substance with a narrow or a broad antimicrobial spectrum. Other characteristics of the bacteriocin (postbiotic) substance are being processed, hoping to achieve more adequate responses regarding its antimicrobial activity.

When fresh goat cheese was made with the strain MK1/3, this type of cheese quality remained. The pH value was not negatively influenced (6.34–4.82). The total counts of presumptive Lactococci reached a high level during processing (from 8.58, through 9.75 CFU/g to 9.41 CFU/g log 10), with the highest count on day 7 (10.10 CFU/g, log 10). The fresh cheese possessed a typical lactic flavor and texture, as well. Similarly, in making the Eidam cheese with the strain MK1/3, the texture and other parameters remained [14]. But growth inhibition of the MK1/3 strain was noted, with increased salt concentration in the medium through 30 h of processing [14,15]. Using 2% concentration of NaCl in the medium does not negatively influence the growth of the strain MK1/3; its growth is still sufficient (A₆₀₀/30 h-2% = 2.157). This indicates the ability of L. lactis MK1/3 to grow in cheeses with the standard and/or higher NaCl content [15,37]. The acceptable salt content, e.g., in Cheddar cheeses, ranged from a normal (1.7%) to high (2.3%) salt content [37]. González et al. [36] reported that the species strains represent a culture widely used in salt-containing dairy products. They used the strain L. lactis R-604 to show it to be salt-tolerant in M17 broth, even with 5% NaCl concentration, which is beneficial for its use in salt-containing dairy products. The strain MK1/3’s salt tolerance is also beneficial when compared to that for L. lactis R-604. It showed its advantage among the others, e.g., LAB.

Temperature is one of the most crucial factors in cheesemaking. It affects all steps of cheesemaking, from curd formation to the final texture and cheese flavor. Cheese ripening or affinage is also the phase where temperature control is vital. Most cheeses are aged at 10–15 °C with control of humidity. This ensures proper microbial activity leading to flavor and texture development. If the temperature is too warm, cheeses may spoil or develop off-flavors. On the other hand, if it is too cold, the ripening process slows down, resulting in bland cheese. However, the MK1/3 is able to grow sufficiently at 20 °C. This is another attribute for its use in cheeses, when 20° C is temperature still acceptable for this cheese-type ripening [15].

The use of the strain MK1/3 to supplement yogurts revealed its remaining standard status; no pH influence and higher incorporation of MK1/3 in yogurts from ewe-goat milk compared with those from ewe milk. However, the sustainability of the strain MK1/3 remained. Similarly, postbiotic active Lactococci isolated from raw goat milk (MK2/7 and MK2/8) survived sufficiently in goat-milk yogurts (8.1 CFU/g log 10) [7]. One of our aims has involved using dairy products with a beneficial strain as functional food; e.g., Munis Campos et al. [38] described the L. lactis strain as an interleukin delivery system for prophylaxis and the treatment of inflammatory and auto-immune diseases. We also know that the first recognized lacticin (lacticin 3147-bacteriocin) produced by the strain L. lactis subsp. lactis DPC3147, isolated from the Irish kefir-like grain, cannot be used in cheesemaking, due to a strain-associated off-flavor in the final product [39]; however, is has been effective in mastitis control. Regarding the dairy products, popularity of the fermented/cultured foods has been related especially to fermented milks, and the most common example of fermented milk are yogurts [40,41]. Therefore, to use the postbiotic active strain MK1/3 with beneficial properties (technological properties included) in dairy products can serve as a functional food which covers the strategy of ”One Health”. However, at this moment, its effect was confirmed in situ and in vivo using an animal model. To support its claim as a functional food, human clinical trials have been requested.

5. Conclusions

The species strain Lactococcus lactis MK1/3 was assessed as safe because its in vivo application using hybrid mice Balb/c did not cause mortality, and even revealed an anti-staphylococcal effect. This strain was found sustainable in yogurts processed from ewe-goat and ewe milks. However, it was found in higher counts in yogurts from ewe-goat milks than in yogurts from ewe milk, without influencing yogurts’ pH. Its use in 100% starter culture to produce fresh cheese from goat milk was revealed in the standard cheese quality, with a sufficient amount of lactic acid microbiota. The strain MK1/3 possesses postbiotic activity, due to antimicrobial substance (raw bacteriocin), with activity up to 3 200 AU/mL. The presented results and those from previous studies indicate the strain MK1/3 as promising additive in the dairy industry, which is supported also by its acceptance in the patent no. 289266 (Slovak Republic) and deposition in CCM 9209 (Czech Culture Collection, Brno, Czech Republic).

6. Patents

Patent application: PP50021-2022 filed 14 April 2022, acceptable for public in Vestník ÚPV SR no. 21/2023, 8 November 2023. Date informing: 18.12.2024 in Vestník ÚPV SR no. 24/2024, Date informing: 18.12.2024. Patent no. 289266, “The strain of lactic acid bacteria Lactococcus lactis subsp. lactis MK1/3, the method of fermented goat milk processing and the product made in this way” (in Slovak: Kmeň kyslomliečnych baktérií Lactococcus lactis subsp. lactis MK1/3, spôsob výroby fermentovaného kozieho mlieka a výrobok vyrobený týmto spôsobom) declared by the Industrial Property Office of the Slovak Republic (Banská Bystrica, Slovakia).