Assessment of the Biological Activity of a Probiotic Fermented Milk Product with the Addition of Lactobacillus helveticus Cell-Free Supernatant

Abstract

Products enriched with probiotic microorganisms have proven to possess immunomodulatory, antioxidant, hypo-cholesterolemic, hypotensive, and antimicrobial properties. Biologically active substances, which are by-products of microbial fermentation, have potential applications in various industries. Cell-free supernatants, depending on the microorganisms used and production conditions, can exhibit antimicrobial, antioxidant, bifidogenic, and other biological activities. This paper presents a study on the biological activity of a probiotic fermented milk product, supplemented with 0.01% lyophilized cell-free supernatant from Lactobacillus helveticus. The fermented milk product was developed based on a composition of Lacticaseibacillus rhamnosus F, Lactococcus cremoris CR201, and Propionibacterium shermanii E2. The research evaluated antimicrobial activity, Bifidobacteria growth stimulation, and the content of organic acids, amino acids, and B vitamins. It was found that adding lyophilized cell-free supernatant to the fermented milk product enhanced its biological activity. In particular, the experimental samples showed a threefold increase in vitamin B₆ content compared to the control, reaching 22.412 μg/100 g. Additionally, the amino acid content in the experimental samples exhibited a significant increase of more than 100% in the essential amino acid tryptophan compared to the control. Notably, antimicrobial activity increased against several opportunistic strains. The experimental samples also showed a significant increase in lactic and formic acids, which may enhance the product’s inhibitory properties against pathogens. An increase in antioxidant activity was observed, potentially due to the higher content of tryptophan and vitamin B₆. The positive effect of adding cell-free supernatant on the growth of Bifidobacteria was also demonstrated. Thus, the findings suggest that this cell-free supernatant can be recommended as an additive in the production of fermented milk products, food additives, dietary supplements, and animal feed.

1. Introduction

Fermented milk products containing live probiotic lactic acid bacteria have gained popularity among consumers due to their potential health benefits. Products enriched with probiotic microorganisms have proven immunomodulatory, antioxidant, hypo-cholesterolemic, hypotensive and antimicrobial properties [1,2].

The probiotic and technological properties of fermented milk products are determined by biologically active substances synthesized by lactic acid bacteria in the food matrix [3,4,5]. These components include exopolysaccharides (EPS), organic acids, and bacteriocins [6].

Biologically active substances produced through microbial fermentation have potential applications in various industries [6]. In the literature, these components are called “metabiotics” and “cell-free supernatants” [7,8].

The process of obtaining cell-free supernatant (CFS) typically involves cultivating microorganisms in nutrient media, separating the cell biomass by centrifugation, followed by filtration and lyophilization of the cell-free supernatant, which contains microbial metabolites [9].

CFS, depending on the microorganisms used and the conditions of production, can exhibit antimicrobial, antioxidant, bifidogenic and other types of biological activity. The antimicrobial activity of CFS is attributed to the content of organic acids, bacteriocins, enzymes and other compounds that suppress the growth of gram-positive and gram-negative microorganisms [10]. CFS can also contain biologically active peptides with antioxidant properties, formed by the proteolytic system of lactic acid bacteria [3].

Currently, several medications containing CFS are available to consumers [9,11]. However, food supplements based on probiotic CFS have not yet gained widespread commercial use, although several scientific reports detail the use of CFSs as food ingredients. According to the data presented in [12], the addition of CFS from Lactobacillus acidophilus, L. casei and L. salivarius into a milk base inhibited the growth of Listeria monocytogenes. In [13], CFS from Lactobacillus gasseri, containing bacteriocins, was used as a preservative in custard. A study [14] demonstrated the antimicrobial effect of CFS from L. acidophilus, L. plantarum and B. bifidum against foodborne pathogens in milk and feta cheese. It should be noted that using lactobacillus CFSs as food ingredients requires consideration of their possible interaction with the food matrix, since they can affect the sensory properties of the final products. In addition, some components of the food matrix may inhibit CFS activity, reducing its biological activity [14,15].

Current knowledge suggests that bacterial viability is not mandatory for all probiotic effects, as not all mechanisms and clinical benefits are directly related to viable bacteria. In addition, an important advantage of CFS is stability in industrial production processes and in storage.

Previously, we studied the composition and probiotic potential of the CFS from L. helveticus [16]. CFS from L. helveticus contains essential and non-essential amino acids, lactic acid and acetic acid. It has been confirmed that CFS exhibits antimicrobial, antioxidant and bifidogenic activities [17]. One potential application of CFS is its use in the production of fermented milk products. Several scientists suggest that the combination of live probiotic cells and CFS can enhance the biological activity of the final product [5,6,7,8].

Previously, we developed a fermented milk product with probiotic cultures Lacticaseibacillus rhamnosus, Lactococcus cremoris, and Propionibacterium shermanii [18]. The product obtained using this combined starter culture exhibited a strong antagonistic effect against pathogenic and opportunistic microorganisms, as well as spoilage agents, and demonstrated bifidogenic effects.

The aim of our work was to develop an optimal technology for producing a fermented milk product with Lactobacillus helveticus CFS and probiotic cultures Lacticaseibacillus rhamnosus, Lactococcus cremoris, and Propionibacterium shermanii, as well as to study the biological activity of this product.

2. Materials and Methods

2.1. Probiotic Association Used

The strains used to obtain the probiotic association were stored in the microorganism collection of FGANU ‘VNIMI’. The association included the strains Lacticaseibacillus rhamnosus F, Lactococcus cremoris CR 201, and Propionibacterium shermanii E2 in a ratio of 1:2:6. To prepare the inoculum, skim sterilized milk (SSM) and hydrolysate-milk complex medium (GMK-2) (Russia) were used for L. rhamnosus F, L. cremoris and for P. shermanii E2, respectively. Cultivation was carried out at 30 °C for P. shermanii E2 and L. cremoris CR 201, and at 37 °C for L. rhamnosus F.

2.2. Preparation of Cell-Free Supernatant

The culture of Lactobacillus helveticus H9, used to obtain the CFS, was isolated from the gastrointestinal tract of a healthy individual and stored in the microorganism collection of FGANU ‘VNIMI’. Before use, the strain was preserved in Man, Rogosa, and Sharp (MRS) (Merck, Darmstadt, Germany) broth supplemented with 20% glycerol at −80 °C. The strain was then cultured in MRS-broth medium at 37 °C. CFS was obtained as described in [16]. To obtain CFS, the L. helveticus H9 strain was added to the MRS broth at a concentration of 3% and cultivated at 37 °C for 24 h. The final cell concentration was 2.2 × 10⁸ CFU/mL at pH 3.90. The cell biomass was separated by centrifugation at 4 °C for 15 min at 6000 rpm (RCF = 4025 g) using a Rotanta 46 R centrifuge (Beverly, MA, USA). The supernatant was then filtered through a 0.22 μm MF-Millipore^® membrane filter (Sigma-Aldrich, St. Louis, MO, USA). The resulting CFSs were poured into sterile trays, frozen at −40 °C, and dried to a moisture content of 4.5–4.7% using a freeze dryer (Labconco, Kansas City, MO, USA). For experimental use, the lyophilized CFS (LCFS) was dissolved at a ratio of 1:10 in SSM and added to the probiotic fermented milk product (see Section 2.3 below) at a concentration of 0.01%.

2.3. Obtaining Control and Experimental Samples of Probiotic Fermented Milk Product

To obtain the control samples of the probiotic fermented milk product, 5% of the probiotic association (from 2.1) was added to 100 cm³ of SSM and cultured at 30 °C until the pH value reached 4.5 ± 0.2. The experimental samples were prepared similarly, but with the simultaneous addition of CFS from 2.2 at a concentration of 0.01%. Specifically, the lyophilized CFS was pre-dissolved at a 1:10 ratio in SSM. The choice of this LCFS concentration was based on the results of sensory assessment conducted by experts from the accredited testing laboratory IL ‘MOLOKO’ of FGANU ‘VNIMI’. The assessment was performed using a five-point scale, evaluated the following criteria: taste, color, smell, and consistency. The samples with an added LCFS concentration of 0.01% received the highest score [19].

The control and experimental samples were also compared with the fermented milk product obtained by adding 0.01% of MRS-broth powder to the control sample.

2.4. Determination of the Biological Activity of Probiotic Fermented Milk Product

2.4.1. Determination of Antimicrobial Activity

To determine the antimicrobial activity of control and experimental samples against opportunistic gram-negative and gram-positive bacteria, standard test strains were used: Escherichia coli ATCC 25922, Salmonella enterica Typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 25668, Staphylococcus aureus ATCC 6538, obtained from the State Collection of Pathogenic Microorganisms and Cell Cultures ‘GKPM-Obolensk’. Test cultures were cultivated on agar slants using SPA medium (‘Dry Nutrient Agar’) (Obolensk, Russia). For this study, 24-h cultures were used. The antimicrobial activity of the experimental and control samples was determined by the method of co-cultivating mixed populations and comparing the growth of test strains with their monocultures. For this, 1 mL of control or experimental samples and 1 mL of microbial suspension of the test strains, adjusted to the optical turbidity standard 5ME at a concentration of approximately 1.0 × 10⁸ CFU/mL, were added to 20 mL of SSM. The results were evaluated after 24 and 48 h of co-cultivation at 37 °C. To control the growth of test strains, their monocultures were grown under the same conditions. The results were recorded by plating on selective nutrient media (Baird-Parker agar, XLD agar, Endo medium, cetrimide agar) produced by the Research and Production Center OOO ‘Biokompas-S’ (Uglich, Russia), using successive decimal dilutions in triplicate. The number of test strain cells was then counted.

2.4.2. Determination of Organic Acids, Amino Acids and Vitamins

The content of organic acids in experimental and control samples was determined by capillary electrophoresis using a Kapel 205 device (Lumex Ltd., St. Petersburg, Russia) equipped with a spectrophotometric detector and a quartz capillary with an internal diameter of 75 μm and a total length of 60 cm. The samples were pre-diluted with distilled water. The buffer electrolyte was prepared based on benzoic acid, diethanolamine, cetyltrimethylammonium bromide and Trilon B. Separation was carried out at a voltage of 20 kV with ultraviolet detection at 254 nm. Electropherograms were processed using Elforan ^® 205 software (S-Pb, St. Petersburg, Russia).

To assess the amino acid composition, the samples were first subjected to acid and alkaline (for tryptophan) hydrolysis to convert protein-bound amino acids into free ones. For all amino acids except tryptophan, phenylisothiocarbamyl derivatives were obtained, which were separated and quantified by capillary electrophoresis. Tryptophan was determined directly, without obtaining a TPA derivative. For tryptophan, a borate buffer solution was used, with a voltage of +25 kV and ultraviolet detection at 219 nm. Glutamic acid, aspartic acid and cystine were determined in a phosphate buffer solution with the addition of β-cyclodextrin, at a voltage of +25 kV, a pressure of 50 mbar, and ultraviolet detection at 254 nm. The remaining amino acids (arginine, lysine, tyrosine, phenylalanine, histidine, leucine + isoleucine, methionine, valine, hydroxyproline, proline, threonine, serine, alanine, glycine) were determined using a similar method but without pressure. Electropherograms were processed using Elforan software.

Vitamins of the B group, particularly B₆ and B₁₂, were determined by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II high-performance liquid chromatograph with an Ultivo Triple Quad LC/MS mod. 6465, Singapore, Agilent Technologies and an Agilent 1260 Infinity II high-performance liquid chromatograph with a diode array detector mod. G7115A. Chromatographic separation was carried out using an Agilent InfinityLab 120 Poroshell 120 Phenyl-Hexyl column (3.0 × 100 mm, 2.7 μm).

Samples preparation for the determination of B vitamins was as follows: 1 g of the sample was mixed with 4 cm³ of deionized water, vortexed, then 5 cm³ of acetonitrile and 0.1 g of ascorbic acid were added. The mixture was vortexed again and then treated with ultrasound for 30 min, followed by centrifugation at 3500 RPM for 10 min. The sample was then frozen at −4 to −6 °C and filtered through a 0.22 μm filter.

The determination of vitamin B₁₂ (cyanocobalamin) was carried out using reverse-phase HPLC according to GOST ISO 20634-2018 [20]. Vitamin B₁₂ was measured as the aggregate of cyanocobalamin and other cobalt-containing corrinoids with biological activity, including aquo-cobalamin, hydroxocobalamin, methyl-cobalamin and adenosyl-cobalamin, converted to cyanocobalamin. The determination of vitamin B₆ (including glycosylated forms) was carried out by HPLC according to GOST EN 14663-2014 [21].

2.4.3. Determination of Antioxidant Activity

To measure antioxidant activity, water-soluble extracts (WSE) of the control and experimental samples were prepared by centrifugation at 10,000× g for 20 min at 4 °C. The supernatant was filtered through a 0.45 μm syringe filter and stored at −80 °C until further analysis.

Antioxidant activity in WSEs was determined in vitro using the ABTS radical cation (ABTS•+). The ABTS radical cation was prepared according to the method of Re et al. [22], with Trolox solution used as a standard antioxidant. For the test, 20 μL of WSE and 180 μL of ABTS radical cation solution were added to the wells of a 96-well, non-absorbing, flat-bottomed polystyrene microplate. As a blank, 180 μL of ABTS radical cation solution and 20 μL of 50 mM phosphate-buffered saline solution (pH 7.40) were used. Antioxidant activity was measured by the decrease in OD734 over 40.5 min. Measurements were taken every 60 s at 25 °C using a Synergy 2 photometer-fluorimeter (BioTek, Seattle, WA, USA).

To plot a calibration curve of decrease in optical density as a function of Trolox concentration, the Trolox concentration in the reaction medium was varied within the range of 1–10 μM. Based on the decrease in the optical density in the presence of the test compounds, the equivalent concentrations of antioxidants in the samples were determined. The antioxidant activity of the samples was expressed in μm TE.

2.4.4. Determination of the Ability of Experimental Samples to Stimulate the Growth of Bifidobacteria

To study the ability of the experimental and control samples to stimulate the growth of Bifidobacteria, the Bifidobacterium adolescentis strain MS-42 from the FGANU ‘VNIMI’ microorganism collection was used as a control culture. GMK-2 medium (NPO OOO “Biokompas S”, Russia) was used to restore the culture of Bifidobacterium adolescentis MS-42. Identification and enumeration of Bifidobacteria were performed according to ISO 29981-2013 [23].

During the experiment, 3% of the prepared association of lactic acid microorganisms, 3% of a 16-h culture of B. adolescentis MS-42, and LCFS at a concentration of 0.01% were added to the SSM. Samples without the addition of LCFS were used as control. The resulting samples were incubated at 37 °C, and the number of Bifidobacteria in experimental samples was counted after clot formation (after 8 h of incubation).

2.4.5. Processing of Experimental Data

Primary processing of the experimental data was performed using Microsoft Office 10. The dynamics of changes in studied indicators were analyzed with TableCurve 2.0 software. Functional dependencies were used during processing, where the Student’s t-test criterion corresponded to values of no more than 0.005, and the coefficients of determination were within the range of 0.99–0.95. The deviation from the mean value ranged from 0.05 to 0.1.

3. Results and Discussion

3.1. Determination of Antimicrobial Activity

The results of the antimicrobial activity test of the control and experimental samples in relation to opportunistic test strains are presented in Figure 1.

Data analysis showed that the suppression of E. coli growth during co-cultivation with control (probiotic composition) and experimental (probiotic composition with LCFS) samples was similar after 12 and 24 h, reaching approximately 30% and 60% compared to the monoculture titer. By 48 h, the inhibitory effect increased for both samples, and the antimicrobial activity of the experimental samples was more pronounced at 85% compared to 72% for the control.

In the case of S. typhimurium, results showed no significant increase in the inhibitory effect of the experimental samples compared to the control after 24 h. However, by 48 h, a slight increase in the inhibitory effect of the experimental samples was noted. The degree of suppression of test culture growth by the control samples was 60%, while for the experimental ones it was about 68%.

Studies of Lactobacillus strains from the FGANU ‘VNIMI’ microorganism collection confirmed their antimicrobial properties against opportunistic enterobacteria, including E. coli and S. typhimurium [24]. The inhibitory effect varied among different strains [25]. The addition of LCFS is likely to increase the production of organic acids, enhancing the inhibitory effect on enterobacteria, which aligns with data on the synergistic antimicrobial effects of lactic and acetic acids [26].

E. coli and S. typhimurium exhibit high genomic and metabolic similarity. It is known that lactic acid bacteria produce a wide range of pathogen-inhibiting compounds, including bacteriocins, which are effective against Gram-negative and Gram-positive bacteria. Lactobacillus bacteriocins can act in a wide pH range [25,26]. In the presence of pathogens, lactic acid bacteria not only produce organic acids but may also increase bacteriocin production in response to stress, particularly after 48 h, which is consistent with previous studies [27,28,29,30].

The inhibitory effect of the samples against S. aureus was noted after 12 h, with its further intensification. The enhancement of antimicrobial activity in the experimental samples compared to the control after 24 h was significant, with suppression rates of 25% and 15%, respectively. After 48 h, the suppression increased to 48% and 20%, respectively. The increased content of lactic and formic acids in the experimental samples (

Table 1. Content of organic acids in experimental and control samples.

Organic Acids	Control	Experimental
	mg/kg ± σ
Formic acid	31.8 ± 20%	60.3 ± 20%
Citric acid	177.8 ± 20%	161.5 ± 20%
Acetic acid	1071.5 ± 20%	1106.7 ± 20%
Propionic acid	1333 ± 20%	947.7 ± 20%
Succinic acid	51.2 ± 20%	41.8 ± 20%
Lactic acid	5892.3 ± 20%	6980 ± 20%

Note: σ—relative accuracy of the indication.

) may be one factor contributing to its enhanced antimicrobial properties.

Analysis of the data obtained from co-cultivation the experimental samples with P. aeruginosa ATCC 25668 showed no difference in the effects of the experimental and control samples on the test culture’s growth, nor was there any inhibitory effect. Moreover, the titer of the culture increased by one order of magnitude during co-cultivation with the samples, compared to the monoculture.

A previous study of the antimicrobial activity of kefir starter grains and two strains of lactobacilli L. rhamnosus F and L. helveticus NK1 against P. aeruginosa ATCC 25668 showed an inhibitory effect on the growth of the test strain of all three samples [30], though the effect of L. rhamnosus F was less pronounced and only appeared after 48 h of co-cultivation. The lack of an inhibitory effect on P. aeruginosa by L. rhamnosus F in the association may be due to specific microbial interactions within the association or the presence of P. shermanii E2 and its metabolites, which P. aeruginosa may utilize as additional nutrients, stimulating its growth.

According to the literature, a study of 57 strains of Lactobacilli made it possible to isolate only two strains identified as Lactobacillus fermentum, with a strong inhibitory effect on the growth of Pseudomonas aeruginosa strains [31]. This highlights the selective action of lactic acid bacteria as antimicrobial agents against P. aeruginosa.

3.2. Determination of Organic Acids, Amino Acids and Vitamins

Organic acids content in the experimental and control samples is presented in Table 1.

In the experimental samples, compared to the control, approximately similar amounts of acetic, succinic, and citric acids were observed, along with a slight decrease in propionic acid and a significant increase in lactic and formic acids.

The degree of changes in organic acids content in the experimental samples compared to the control is shown in Figure 2.

Data on the amino acid content in the experimental samples relative to the control are presented in Figure 3.

The results of studying the amino acids content in samples showed a significant increase—over 100%—in the essential amino acid tryptophan in experimental samples compared to the control (Figure 3). Amino acid tryptophan in experimental sample (41.1 mg/100 g) compared to the control (19.1 mg/100 g). The addition of 0.01% of the MRS medium powder to the control sample slightly increased the tryptophan content to 22.51 mg/100 g It is known that melatonin, a powerful antioxidant involved in many biological processes, is synthesized directly from tryptophan [32]. Previous studies have shown that LCFS contains several amino acids, which, under the influence of microorganisms in the association, may be utilized as additional nutrients, leading to increased tryptophan production. According to several researchers [33], tryptophan and its metabolic products, particularly melatonin, exhibit antioxidant activity, which is confirmed by the antioxidant activity results of the experimental samples compared to the control. In addition, tryptophan, especially in combination with B vitamins, has a positive effect on the nervous system, improving the psycho-emotional state and supporting the body during periods of high physical and emotional stress [34,35].

Analysis of the B vitamin content in the samples revealed the presence of vitamin B₆. Its amount in the experimental samples increased threefold compared to the control, reaching 22.412 μg/100 g (Figure 4). When 0.01% of the MRS nutrient medium powder was added to the control sample, the vitamin B₆ content was 8.6 μg/100 g Therefore, the increase in tryptophan and vitamin B₆ content enhances the biological activity of the experimental samples. The amount of vitamin B₁₂ was negligible.

3.3. Determination of Antioxidant Activity

Data on changes in the antioxidant activity of the experimental samples compared to the control are presented in Figure 5.

The presented data show that the antioxidant activity of the experimental sample was higher than that of the control, reaching 1300 TEAC TE µmol/L compared to 1150 TEAC TE µmol/L, respectively. The increase in antioxidant activity of the experimental samples, as mentioned earlier, can be attributed to the higher content of tryptophan and vitamin B₆. Although vitamin B₆ is not an antioxidant itself, it can enhance the antioxidant activity of the probiotic cultures in the studied association. At the same time, tryptophan and its metabolic products have inherent antioxidant activity.

3.4. Determination of the Ability of Control and Experimental Samples to Stimulate the Growth of Bifidobacteria

Data on the ability of the experimental samples with added LCFS to stimulate the growth of Bifidobacteria compared to the control presented in

Table 2. Stimulation of Bifidobacteria growth by the experimental samples with LCFS compared to the control.

Sample Type	Number of Bifidobacteria Cells CFU/mL	pH Unit
Control	2.0 × 107 CFU/mL	4.7 ± 0.1
Experimental	1.4 × 108 CFU/mL	4.5 ± 0.2

.

During the assessment of Bifidobacteria number in the experimental and control samples, the control samples contained 2.0 × 10⁷ CFU/mL (with the initial addition of 1.7 × 10⁷ CFU/mL), while the experimental samples showed a tenfold increase in Bifidobacteria number [36]. This indicates that the addition of LCFS had a stimulating effect on Bifidobacteria growth.

4. Conclusions

In this study, we assessed the biological activity of a probiotic fermented milk product using an association of strains L. cremoris CR 201, L. rhamnosus F, P. shermanii E2 with the addition L. helveticus H9 LCFS. It was found that the addition of 0.01% LCFS to the fermented milk product enhances its biological activity. In particular, antimicrobial activity increased against several opportunistic strains. The experimental samples showed a significant increase in lactic and formic acids, which may contribute to its enhanced inhibitory properties against pathogens. The observed increase in antioxidant activity of the fermented milk product is possibly due to elevated tryptophan and vitamin B₆ levels. Furthermore, the addition of LCFS positively stimulated Bifidobacteria growth, potentially due to additional nutritional sources in the LCFS [16,37].

Based on these findings, the use of 0.01% LCFS can be recommended as an additive in the production of fermented milk products, food additives, dietary supplements, and animal feed.