Fortification of food products with chemically synthesized vitamins is a traditional way of increasing their biological value in order to prevent vitamin deficiency, which can have negative effects on the development and functioning of the body. A lack of B vitamins in humans can lead to various diseases. Folic acid deficiency can lead to osteoporosis, coronary heart disease, decreased cognitive function and Alzheimer’s disease, hearing loss [1
], and an increased risk of breast cancer [2
]. Riboflavin deficiency can lead to skin and liver damages, as well as changes in glucose metabolism in the brain [3
]. Thiamine deficiency causes beriberi disease, which can have consequences for both the cardiovascular and the nervous system. Niacin deficiency can lead to pellagra, lack of pantothenic acid to acne, pyridoxine deficiency to dermatitis and epileptic seizures, and a lack of biotin can cause growth retardation in children [4
]. Recently, the efficacy of synthetic vitamins has been in doubt and so they need to be analyzed. It is reported that their bioavailability is worse than that of natural forms of vitamins, which means they might not be as effective [5
]. Also, some studies show that chemically synthesized vitamins can even have a negative effect on human health. For example, high consumption of foods enriched with chemically synthesized folic acid, not the natural form, in some people can lead to masking vitamin B12 deficiency, leukemia, arthritis, and an increased risk of cancer [6
]. At the same time, synthetic vitamins are quite expensive due to the large number of stages of production. Finally, consumer requirements for food quality are increasing and preference is given to a healthy diet, so much attention is paid to the absence of synthetic additives in the composition.
Biofortification by fermentation of lactic acid bacteria and bifidobacteria with vitamin-producing ability is a promising approach for obtaining foods with increased concentrations of natural forms of B vitamins. Most members of the Lactobacillus
are normal representatives of the human intestinal microbiota and are generally recognized as safe (GRAS), which makes them the most suitable candidates for these purposes. Some authors insist that careful selection of starter cultures with increased vitamin B-synthesizing ability can enhance the concentration of folic acid in yogurts over 200 μg L−1
] or beverages, and thereby lead to the development of innovative functional products [10
]. Such foods will not only be healthy, but also economically beneficial. The strains of lactic acid bacteria (LAB) selected according to the specified criteria have already been used as starters for bread and pasta production enriched with riboflavin, which leads to an increase in this vitamin concentration of about 2–3-fold in the final product [11
]. It was shown in model rats with folic acid deficiency that yogurt fermented with the vitamin-producing strain L. plantarum
GSLP-7 V restores the disturbed microbiota and increases levels of serum folate and homocysteine, while folic acid aggravated intestinal dysbiosis [12
]. Moreover, vitamin production is considered a criterion in the functionality assessment in some studies—for example, on isolation of new LAB from various sources such as vegetable raw materials [13
Vitamin-producing lactic acid bacteria and bifidobacteria strains may become potential candidates for their synthesis in vivo in the intestine after colonization [14
]. It is reported that LAB synthesizing folate and riboflavin can be used as an additional treatment in people suffering from inflammatory bowel diseases [15
]. To ensure the effects, LAB have to reach the colon in a viable state and be characterized by high stress tolerance when passing through the digestive tract (tolerance to acidic pH, bile, and proteolytic enzymes). These abilities refer to probiotic properties [16
]. In addition to stress tolerance, the strains should have a high aggregation ability, which is often associated with adhesion to the intestinal epithelium and colonization properties [17
]. They will have more advantages over nonaggregated strains that can be easily removed from the intestinal environment [18
In this study, for the first time, the probiotic properties and B-vitamin-producing potential of various bifidobacteria and lactobacilli strains are evaluated in order to select the most promising candidates. Their stability under conditions that model the human gastrointestinal tract passage, autoaggregation ability, and ability to synthesize and extracellularly accumulate water-soluble B vitamins are studied. The obtained results can be used in medicine, healthcare, the food industry, and farming in order to expand the applications of LAB and bifidobacteria.
3.1. Stress Tolerance Ability
3.1.1. Tolerance of Bifidobacterium and Lactobacillus Strains to Simulated Gastric Juices
The standard conditions for incubation in real or simulated gastric juices have not been fully developed yet [31
]. However, it is known that, depending on the consumed food and beverages, incubation time in the stomach can vary from 15 to 194 min [32
]. Therefore, to simulate the presence of lactobacilli and bifidobacteria in the stomach, an incubation period of 90 min was determined. As the death rate of bifidobacteria will be greater than that of lactobacilli, the measurements for the strains of Bifidobacterium
genus were carried out more frequently. In this case, only the final results of the test (after 90 min) are applicable for intergenera comparison.
The results of the research on the survival of six bifidobacteria strains and 10 lactobacilli strains during the incubation in simulated gastric juices at pH 2.0 for 1.5 h are shown in Table 2
and Table 3
, respectively. Each tested strain of bifidobacteria demonstrated a considerable decrease in the viable cell count during the first 30 min of incubation. The strains of B. adolescentis
VKPM AC-1662, B. pseudolongum
VKPM AC-1785, and B. breve
VKPM AC-1911 showed the highest tolerance to the effects of simulated gastric juices. The marked decrease in the population was observed in the B. longum
VKPM A C-1912, B. longum
VKPM AC-1665, and B. bifidum
VKPM AC-1779 strains. The strain B. bifidum
VKPM AC-1779 was characterized by the decay of viable cells after 40 min of exposure.
All the studied lactobacillus strains also demonstrated a decrease in the viable cell count under the model conditions of simulated gastric juices (Table 3
). The strain L. sakei
VKPM B-8936 demonstrated the greatest resistance to low pH values, while L. salivarius
VKPM B-2214 had the lowest. Total inactivation of the lactobacillus population was not observed for any strain after 1.5 h of incubation.
3.1.2. Tolerance of Bifidobacterium and Lactobacillus Strains to the Simulated Duodenal Environment
The time of food transit via the small intestine is usually 1 to 4 h, which gives a total transit time in the upper gastrointestinal tract of 3 to 8 h [33
]. Therefore, in order to simulate the passing of lactobacilli and bifidobacteria in the intestine, the incubation period of 3 h was established. The viability of lactobacilli after 3 h in the duodenum-simulating conditions was comparatively higher than in bifidobacteria (Table 4
and Table 5
). At the same time, the survival rate of both lactobacilli and bifidobacteria was strain-specific and differed between the cultures.
The strain B. adolescentis VKPM AC-1662 had the highest survival rate, almost equal to 100%. The greatest sensitivity to the conditions of the intestinal environment was demonstrated by the bifidobacteria B. pseudolongum subsp. pseudolongum VKPM AC-1785 and B. breve VKPM AC-1911 strains, which had the survival percentage of 80% after 3 h of incubation.
Among lactobacilli, the lowest decrease in population was observed in the L. plantarum VKPM B-11007 and L. acidophilus VKPM B-2213 strains. The survival rate of the L. salivarius VKPM B-2214 strain was the lowest.
3.1.3. Tolerance of Bifidobacterium and Lactobacillus Strains to Digestive Enzymes
To clarify the effect of enzymes as principal components of digestive juices, the strains were tested separately for pepsin and pancreatin action. A study of the effect of the proteolytic enzymes, such as pepsin and pancreatin, on the survival of bifidobacteria showed that they had an equally strong effect on every considered strain. Total death of microorganisms occurred after 1 h of incubation The results of the research on the sensitivity of lactobacilli to pepsin and pancreatin are shown in Table 6
and Table 7
The inactivation effect of pepsin on the lactobacilli viability was higher than that of pancreatin. If the survival rate of lactobacilli after 3 h of incubation with pancreatin was about 90% for all the cultures, the sensitivity to pepsin greatly varied between the strains, reaching 60% in some cases. Thus, the strain L. paracasei subsp. paracasei VKPM B-4079 demonstrated the lowest tolerance to pepsin, and L. acidophilus VKPM B-2213 the greatest.
3.1.4. Tolerance of Bifidobacterium and Lactobacillus Strains to Bile
The results of the study on bile’s effect on the viability of bifidobacteria and lactic acid bacteria strains are shown in Table 8
and Table 9
. Almost every strain considered showed a decrease in the population of viable cells. The most sensitive to the presence of bile in the medium was the B. breve
VKPM AC-1911 strain (the survival rate was 70%). The B. bifidum
VKPM AC-1779 strain was characterized by the greatest resistance to the effects of bile; the survival rate hardly changed (about 99%).
Among the Lactobacillus strains considered, the L. acidophilus VKPM B-2213 strain demonstrated the greatest sensitivity to bile, and L. acidophilus VKPM B-2105 the lowest.
3.2. Autoaggregation Assays of Bifidobacterium and Lactobacillus Strains
The results of the study on the autoaggregation of bifidobacteria strains are shown in Table 10
. The highest percentage of autoaggregation was demonstrated by the strain B. adolescentis
VKPM AC-1662: 69% after 24 h of incubation. B. longum
VKPM AC-1665 aggregation was a little lower. The other strains demonstrated low autoaggregation ability.
The results of the Lactobacillus
strains autoaggregation test are shown in Table 11
. The greatest autoaggregation was observed in the L. salivarius
VKPM B-2214 strain: 56% after 24 h of incubation. Also, a high autoaggregation ability was observed in the L. acidophilus
VKPM B-6551, L. acidophilus
VKPM B-2213, and L. rhamnosus
VKPM B-8238 strains. The aggregation percentages of the other lactobacilli strains considered were lower.
Thus, the strains B. adolescentis VKPM AC-1662, B. longum subsp. longum ВКПМ AC-1665, L. salivarius VKPM B-2214, L. acidophilus VKPM B-6551, L. acidophilus VKPM B-2213, and L. rhamnosus VKPM B-8238 have a high potential ability to attach to epithelial cells and mucosal surfaces.
3.3. The Study of Lactic Acid Bacteria’s and Bifidobacteria’s Ability to Accumulate B Vitamins Extracellularly
While studying the ability of lactic acid bacteria and bifidobacteria to produce B vitamins, it was assumed that, if a specific vitamin was found in the supernatant, the considered strain was able not only to synthesize, but also to excrete vitamins simultaneously with their accumulation in the medium. The results of the studies are presented in Table 12
and Table 13
Based on the results, each bifidobacteria culture considered consumed nicotinamide (its concentration was higher in the medium before fermentation). The riboflavin that was detected in the nutrient medium was consumed by all cultures except B. adolescentis AC1662 and B. longum subsp. infantis AC-1912, which excrete it in the medium, such that an increase in concentration was observed. The ability to accumulate pyridoxine extracellularly was found in B. adolescentis VKPM AC-1662, B. pseudolongum subsp. pseudolongum VKPM AC-1785, B. longum subsp. infantis VKPM AC-1912, and B. longum VKPM AC-1665, among which the maximum amount was detected for the last one. Thiamine, pantothenic acid, and folic acid were not detected in the supernatant of the fermentation broths.
Each Lactobacillus strain also consumed nicotinamide. The ability to produce thiamine in the medium was found in L. casei VKPM B-2873 and L. rhamnosus VKPM B-8238; pantothenic acid in L. acidophilus VKPM B-2105, L. salivarius VKPM B-2214, L. casei VKPM B-2873, and L. rhamnosus VKPM B-8238; and pyridoxine in L. acidophilus VKPM B-2105, L. plantarum VKPM B-11007, L. acidophilus VKPM B-6551, L. casei VKPM B-2873, L. rhamnosus VKPM B-8238, and L. sakei VKPM B-8936. Folic acid was detected in the fermentation broths of L. salivarius VKPM B-2214 and L. acidophilus VKPM B-6553.
Among the Lactobacillus strains considered, L. salivarius VKPM B-2214 is of the greatest interest since the pantothenic acid concentration reached 138.600 mg L−1 in the fermentation broth.
Carrying out proper in vitro studies to screen potential probiotic strains is a necessary first step before in vivo experiments to establish the possible health benefits of probiotics [34
]. An important evaluation criterion during the selection is stress tolerance to passing through the digestive tract, which characterizes the resistance of the probiotics to acid and bile, as well as the production of antimicrobial compounds and the ability to aggregate with intestinal cells [34
]. The acidic gastric juices and bile salts are the main obstacle to the survival of probiotic bacteria and reaching the distal part of the intestine [36
The results obtained in our research show that the stress tolerance of bifidobacteria and lactobacilli to the effects of low pH, proteolytic enzymes, and bile varies greatly, both between species and between strains of the given species. The effect of artificial gastric juices on bifidobacteria strains led to the death of B. longum
VKPM AC-1912, B. longum
VKPM AC-1665, and B. bifidum
VKPM AC-1779. Similar results were reported by Charteris et al. [37
], who showed a lack of resistance to simulated gastric conditions (pH 2.0 for 90 min) in the B. bifidum
, B. animalis
, B. infantis
, B. breve
, and B. adolescentis
strains. Strong differences in resistance to low pH between strains within the species B. longum
were observed by Izquierdo et al. [38
]. In our study, the B. adolescentis
VKPM AC-1662 strain, which was isolated from the human intestine, was characterized by the greatest acid resistance. The effect of the model duodenum conditions on the bifidobacteria survival was less harmful, and the survival rate for all strains was above 80%. The strain B. adolescentis
VKPM AC-1662 also showed the highest stability.
When comparing the tolerance of the tested lactobacilli and bifidobacteria to the gastrointestinal tract model conditions, the higher resistance of the former was anticipated. They were resistant to both simulated gastric juices and bile. Acid resistance is a characteristic of most lactobacillus species due to their ability to control intracellular pH [39
]. Some of them (e.g., Lactobacillus casei
, Lactobacillus reuteri
, Lactobacillus vaginalis
, Lactobacillus fermentum
, and Lactobacillus casei
) may be members of the gastric microbial community [40
]. In our research, the strain L. sakei
VKPM B-8936 showed the greatest resistance to low pH. Similar results were reported by Song et al. [41
], who showed the acid resistance of the Lactobacillus
sp. NN 8829, L. casei
MB3, L. sakei
MA9, and L. sakei
CH8 strains under incubation conditions at pH 2.5. Thus, the obtained data are generally consistent with the results presented earlier. On the other hand, taking into account the confirmed strain-specific tolerance, the accumulation of information on new strains is theoretically and practically valuable. The experiment dedicated to the treatment of bifidobacteria populations separately with pepsin and pancreatin led to the death of all tested strains. Lactobacilli were more resistant to the effects of proteolytic enzymes. The greater sensitivity of lactobacilli was marked in the experiments with pepsin.
The bacterial cell aggregation of probiotic strains is often associated with potential ability to adhere to the intestinal epithelium and mucous membranes [17
]. In most cases, bacterial autoaggregation is mediated by homotypic interactions between surface proteins [42
]. The tested cultures of lactic acid bacteria and bifidobacteria demonstrated some degree of autoaggregation, which differed greatly between the strains. The strains B. adolescentis
VKPM AC-1662, B. longum
VKPM AC-1665, and B. bifidum
VKPM AC-1779, isolated from the intestines of adults, showed strong autoaggregation ability. Among lactobacilli, the highest percentage of autoaggregation was observed in L. salivarius
VKPM B-2214, isolated from human saliva. It was also characterized by the strong dependence of bacterial cells’ aggregation on exposure time, which was greatest after 24 h. It was reported previously that L. acidophilus
is characterized by high values of autoaggregation [43
]. In our study, high autoaggregation ability was also observed in L. acidophilus
VKPM B-6551, L. acidophilus
VKPM B-2213, and L. rhamnosus
The reference probiotic bacteria, e.g., L. rhamnosus
GG or L. casei
Shirota, can be applied in research on probiotic properties to improve the reliability of the data comparison [44
]. The lack of a reference probiotic can be considered a limitation of our study. However, it should be noted that the standards were not used in the numerous works mentioned above. The FAO/WHO guidelines [47
] also do not establish strict requirements. Moreover, discrepancies in the data on the stress tolerance of the same reference strain and in the same tests were noted between studies.
The analysis of the obtained results on lactic acid bacteria’s and bifidobacteria’s ability to extracellularly production of water-soluble B vitamins showed that the consumption of some vitamins detected in the nutrient medium before fermentation and the excretion of others into fermentation broths occurred. So, riboflavin was not consumed by B. adolescentis
VKPM AC-1662 and B. longum
VKPM AC-1912. Hou et al. [48
] showed that fermentation of soy milk with Bifidobacterium longum
B6 and B. infantis
CCRC 14633 cultures increases the content of riboflavin. Pyridoxine accumulation was observed in the strains of B. adolescentis
VKPM AC-1662, B. pseudolongum
VKPM AC-1785, B. longum
VKPM AC-1912, and B. longum
VKPM AC-1665. The obtained results are consistent with the data of Deguchi, Morishita and Mutai [49
], who linked the ability to synthesize certain vitamins with the species and strain. The extracellular synthesis of folic acid was demonstrated in the studies of Deguchi, Morishita and Mutai [50
] and Pompei et al. [50
], but it was not detected for the considered strains. Although Bifidobacterium bifidum
and Bifidobacterium longum
are classified as species/strains with a high level of biosynthetic properties of folic acid [50
The B vitamins’ extracellular synthesis by lactobacilli was poor, especially when modern techniques such as capillary electrophoresis were applied. In our study, the ability to excrete thiamine, folic acid, and pantothenic acid was considered for some strains of lactobacilli. L. salivarius VKPM B-2214 is of interest due to its ability to produce up to 138.600 mg L−1 of pantothenic acid.
Thus, B. adolescentis
VKPM AC-1662 can be considered the best candidate for probiotics, since it is characterized by the greatest stress tolerance and a high autoaggregation percentage, as well as the ability to accumulate riboflavin and pyridoxine extracellularly. The application of folate-producing bifidobacteria has already been shown in rats [51
] and humans [52
]. On the other hand, data on the correlation of the synthesis of B vitamins in vitro and in the intestine are poor; additional research is required, which may involve significant methodological difficulties. Among the lactic acid bacteria, the greatest tolerance to low pH values was demonstrated by L. sakei
VKPM B-8936; L. plantarum
VKPM B-11007 showed tolerance to duodenal conditions, L. acidophilus
VKPM B-2213 to pepsin, and L. salivarius
VKPM B-2214 to pancreatin. The highest autoaggregation percentage was observed in L. salivarius
VKPM B-2214, which also accumulated the most pantothenic acid of the studied strains. L. casei
VKPM B-2873 and L. rhamnosus
VKPM B-8238 was also characterized by the accumulation of thiamine and pyridoxine in addition to pantothenic acid. These strains of lactobacilli have the potential to be used as starter cultures in functional product manufacturing.