Introduction

The market contains a considerably large number of probiotics, which are intended for the restoration of violated intestinal biocenosis and contain live beneficial microorganisms [1]. Current probiotics are obtained by liquid-phase cultivation on nutrient media as proposed by L. Pasteur. However, in recent years, it has been determined that bacteria living in nature are greatly different by the properties from the cultures obtained in “classic” laboratory conditions [2]. In the natural environment, the dominant lifestyle of bacteria is a biofilm. The biofilm is a stress-tolerant community of microorganisms held together by a self-produced extracellular matrix, which forms on the surface of objects of the environment and the tissues of living organisms. In the view of some authors, the biofilm is a multicellular organism with its inherent development cycle, co-operative behavior of its individuals coordinated by system of quorum sensing [3]. The “natural” way of bacterial existence is actively investigated. Mainly, the biofilms of pathogenic bacteria are studied in order to learn how to effectively combat chronic infectious pathology [4]. Recently, Williams and Costerton [5] had demonstrated the unquestionable advantages of staphylococcal biofilms over their laboratory cultures in the modeling of the infection process in animals. However, biofilms on human surfaces are not always detrimental. As it is equally beneficial for the resident bacterial flora of the human body to grow in a biofilm [6], we attempted to obtain a biofilm in vitro of the probiotic Lactobacillus plantarum 8-RA-3 strain and to evaluate the possibility of its use for the restoration of the disturbed normal intestinal lactobiota by antibiotics in mice.

A variety of ways to obtain biofilms [7] are described in literature, but all of them are of little effect biotechnologically. Therefore, the technology of solid substrate cultivation was first utilized to grow a probiotic bacteria biofilm. Solid-phase bio-processes are used to obtain antibiotics, enzymes, organic acids, food and feed additives, biopharmaceutical products, etc. Solid-phase fermentation is characterized by the growth of microorganisms, mainly fungi, on the moist (water content oscillates between 30 and 75 %) solid substrates in the absence of free liquid. The water insoluble materials from agriculture or by-products from food industry are used as substrates for microbial cultivation. The dry solid carrier can be saturated with a liquid nutrient medium [8, 9].

The representatives of the Lactobacillus genus are among the dominant biofilm inhabitants of human and animal intestine [6]. Moisture of the large intestinal contents does not exceed 70–75 % [10], corresponding to the solid-phase conditions, so it could be assumed that lactobacilli are able to develop a biofilm on solid substrates with a reduced water content.

We paid a special attention to the issue of cell survival during drying of the fermentative mass, as it is of fundamental importance for technology development for the obtaining of probiotic preparations.

Materials and Methods

The air-tolerant Lact. plantarum 8-RA-3 strain, the basis of the probiotic preparation “Lactobacterin” (Russia) provided by the Collection of Normal Microbiota Microorganisms of Gabrichevsky Institute for Epidemiology and Microbiology was used. The strain was cultivated on the liquid nutrient medium MRS (Lactobacillus MRS Broth, Himedia Laboratories). Lyophilized Lact. plantarum cells with a titer of 1.2 × 1010 CFU/ml were dissolved in sterile MRS medium, and the obtained bacterial suspension was mixed with a solid substrate.

Shredded wheat bran particles were intended to represent a solid support for lactobacilli biofilm development. Bran wheat: dietary supplement of “Zdravitsa-C” (Russia) was used. The bran was dry-heat sterilized at 120 °C for 40 min. 5 g of bran and 10 ml of inoculated liquid MRS medium were inserted into each of sterile container with a volume of 50 ml for the solid substrate fermentation. Initial moisture content of the bulk was of 50.07 ± 0.89 %. The cultivation was performed at 37 °C.

In the process of cultivation, the counting of the total number of microbial cells, as well as the titer of the lactobacilli, capable of colony formation (CFU/g of dry bran) was carried out. In this order, cell desorption from the substrate was performed: 1 g of the sample was placed in 99 ml of saline solution, and after the intensive mixing on the magnetic mixer for 10 min and bran sedimentation for 1–2 min, tenfold dilutions were prepared, and cell titer was determined by inoculation onto MRS-agar. The total number of desorbed bacteria in the obtained suspension was estimated by Breed’s direct-counting method (http://www.microbiologylabs.info/water-microbiology/direct-microscopic-count-practical-test). The pH value of fermented bulk after its dilution with distilled water 1:10 was determined using a pH-meter (pH-meter S20-K, Mettler Toledo).

The samples were dried in a shelf-drying unit at 45 °C for 45–60 min up to a water content not exceeding 7 %.

The viability of the desorbed from the substrate cells of lactobacilli was determined using Live/Dead BacLight Bacterial Viability Kit, L-7012 (Molecular Probes Eugene, USA) and fluorescent microscope MIKMED 2 LYUMAM (Russia).

For a statistical data processing and chart constructions, Microsoft Office Excel 2007 and OriginPro 8 were used. The reliability of differences was assessed by the Student’s t distribution.

The electron microscopy of the native and moist fermented bran samples was carried out using a scanning electron microscope CamScan MB2300 (Czech Republic).

The ability of the Lact. plantarum 8-RA-3 biofilm to restore intestinal microbiota was evaluated in the in vivo experiments. 30 mice (10 in each group) of the CBA line, males weighing 22–22.5 g were used for that purpose. Intestinal dysbiosis in mice was caused daily by intragastric injection of amikacin antibiotic of 5 mg/mouse for 7 days. Amikacin causes a reversible decrease in the level of lactobacilli in the large intestine [11]. After completion of antimicrobial therapy course, that is, starting from the 8th day of the experiment, the animals of the experimental group were kept for 15 days on a diet with the introduction of 0.05 % of the dry bulk of 72-h fermented wheat bran into the sterile feed and then, for the next 5 days, on a diet without the supplement. Mice of the control group 1 also received amikacin for 7 days, and then a sterile feed containing 0.05 % bran, not fermented by lactobacilli for 15 days. Animals of the control group 2 received sterile saline instead of amikacin. On 8th, 17th, 22nd and 27th days after the experiment beginning, the lactobacilli titer and the presence of Lact. plantarum strain 8-RA-3 were determined in feces of experimental and control groups of the animals. All of the animal studies were approved by the Ethics Committee of the Institute of Immunological Engineering.

The identification of Lactobacillus isolates was carried out using biochemical API-test (API 50 CH, BioMerieux, France), as well as using the method of comparative analysis of nucleotide sequences of genes encoding 16S rRNA. For the polymerase chain reaction (PCR) and further sequencing of the PCR fragments of 16S rRNA genes, the universal primer system was used [12]. The sequencing of the obtained PCR fragments of genes encoding 16S rRNA was performed by the method of Sanger et al. [13] using a set of reagents Big Dye Terminator v.3.1 (Applied Biosystems, Inc., USA) on ABI PRIZM 3730 automatic sequencer (Applied Biosystems, Inc., USA). The primary analysis of the nucleotide sequences similarity of genes of 16S rRNA of the studied strains had been carried out using the software package BLAST [14]. Strain-specific differences of Lact. plantarum isolates from experimental and control 1, 2 groups of mice and of Lact. plantarum 8-RA-3 strain were studied by genomic fingerprinting using primers KRPN1, KRPN4 and KRP8 based on the divergent inverted repeats (DIR) [15]. Primers sequences: KRPN1 5′-tciiaagcttca-3′; KRPN4 5′-gccgcciicgcc-3′; KRPN8 5′-gciicgccgccg-3′, with i as inosine. The molecular investigations were performed at the Core Research Facility of the “Bioengineering” Centre, Moscow.

Results and Discussion

Shredded wheat bran represented porous particles with developed surfaces (Fig. 1), easily absorbing the liquid nutrient medium. After 24 h of Lact. plantarum 8-RA-3 solid substrate cultivation on the bran impregnated with MRS a reliable (P < 0.001) increase in the microbial cell number from 5.0 × 108 up to 23.0 × 108 CFU/g (Table 1) was revealed. The results obtained demonstrate the ability of the given strain to proliferate in the solid-phase conditions. The process of bacterial growth was accompanied by some pH decrease of mixture from 5.50 to 4.45. Further cultivation on the background of practically constant moisture content the fermentative mass led to the attenuation of metabolic processes and the reduction in the active acid formation. pH decreased to 4.05 in the 48-h culture and remained without change in the 72-h culture. The tendency had been observed to reduce the number of cells, desorbed from the surface of the solid substrate, from 26.9 ± 14.4 × 108 CFU/g in 24 h to 18.1 ± 7.2 × 108 CFU/g in 72 h of cultivation (Table 1; Fig. 2).

Fig. 1
figure 1

Non-inoculated wheat bran particles

Full size image
Table 1 Indicators of solid-phase cultivation of Lact. plantarum 8-RA-3 on the wheat bran
Full size table
Fig. 2
figure 2

Indicators of Lact. plantarum 8-RA-3 development at solid-phase cultivation on wheat bran

Full size image

In the electronic microscopy photographs of 24-h culture, lactobacilli cells were seen actively dividing on bran particle surface (Fig. 3). By 48 h of growth, the formation of biofilm was traced: bacteria immersed in the intercellular matrix were observed (Fig. 4). The biofilm fully formed by 72 h of solid-phase cultivation (Fig. 5).

Fig. 3
figure 3

Inoculated wheat bran, 24-h cultivation, ×10,000. Bacillary Lact. plantarum 8-RA-3 cells in short chains dominate

Full size image
Fig. 4
figure 4

Inoculated wheat bran, 48-h cultivation, ×20,000. Visible biofilm of Lact. plantarum 8-RA-3

Full size image
Fig. 5
figure 5

Lact. plantarum 8-RA-3 mature biofilm, 72-h cultivation, ×10,000

Full size image

In the process of the biofilm maturation, intercellular interaction increases [3] that stipulate the observed decline in the number of bacteria desorbed from the phyto-substrate surface.

Starting with 48 h of cultivation, a significant decrease in the share of colony-forming units among the desorbed cells was noted. Thus, after 48 h, 84 % of the cells were cultured; in 72 h—only 8.8 %, and after 96 h—less than 1 % of the cells were able to create CFU (Table 1; Fig. 2). In literature, there is information about the fact that the microbial cells composing the mature biofilm lose their ability to reproduce on the artificial nutrient media [4]. Non-cultivated forms appear to be more resistant to adverse external influences [16], and this is of fundamental importance for the development of probiotic preparation technology. Probiotics, in particular, must preserve the viability after their drying.

To study the dehydration impact on the viability of lactobacilli cells in the biofilm, biomass samples were subjected to drying at 45 °C, in 0, 24, 48 and 72 h of the cultivation. The drying of the preparations of solid-phase cultures led to a decrease in the number of lactobacilli, capable of forming colonies from 23.0 × 108 up to 6.9 × 105 CFU/g in 24 h biomass samples, and in 48- and 72-h samples, they generally retained less than 104 CFU/g (Table 1). However, according to the fluorescent microscopy data of the preparations stained with live/dead bacterial viability kit, about half of the cells desorbed from the samples of the dried biomass were viable (Table 2; Fig. 6). It should also be noted that in 72-h samples, when biofilms were already fully formed, 75.2 % of the living, but not cultivated lactobacilli, had coccobacillus-like form (Fig. 7). This number was authentically higher than the share of coccobacillus-like cells in the control (P ≤ 0.05) and in 24-h biofilm (P < 0.001).

Table 2 The results of the experiment on determination of the live and the dead cells of Lact. plantarum 8-RA-3 in 1 g of the dried fermentative mass
Full size table
Fig. 6
figure 6

The change of the total cell number of Lact. plantarum 8-RA-3 and the number of viable cells desorbed from the dried fermented wheat bran

Full size image
Fig. 7
figure 7

Gram-stained Lact. plantarum 8-RA-3 cells desorbed from dried 72-h fermented bran. Coccobacillar cells dominate

Full size image

The obtained data indicate that the solid-phase cultivation of lactobacilli with the subsequent drying of biomass leads to the formation of a significant number of uncultivated, but viable cells, mainly, of coccobacillus-like form. The transition of probiotics to the non-cultivated condition could have a positive impact on the bacterial survival in passing the aggressive environment of the stomach and small intestine. Could the non-cultured cells restore the replicative function and probiotic activity? It is known that the passage of resting bacterial forms through the sensitive host organism could lead to induction of the cultivated state [17].

For the study of this matter, the influence of fermented wheat bran on the efficiency of the recovery of the lactobacilli level in the feces of mouse received and not received antibiotic had been investigated. The results are summarized in Table 3. The animals fed with 72-h fermented bran demonstrated more effectively the recovery of the number of lactobacilli in comparison with the group of mice with the ration of non-fermented bran. By the 27th day of studies, the level of lactobacilli in mice of the test group corresponded to the normal, while in the control group it was below the initial level by 2 orders of magnitude.

Table 3 The influence of fermented wheat bran on the recovery efficiency of lactobacilli in mouse feces after administration of amikacin
Full size table

It should be noted that 0.05 % fermentative mass with a titer of colony-forming cells 3 × 103 CFU/g was included in the feed of mice. The calculation demonstrates that if one mouse of the average weight of 25 g consumes about 9 g of fodder a day, it receives 0.0045 g of the fermented bran and, respectively, only 13–14 CFU of Lact. plantarum 8-RA-3. Therefore, the detection of the given strain in the microbiota of the feces in 5 days after the last feeding mice with Lact. plantarum 8-RA-3 had to testify the colonization of the animal intestine with this strain and its cultivability.

Five days after the end of the 15-day feeding of the test mice with the feed containing 72-h fermented bran, the strain identified in the API test as Lact. plantarum was isolated from the animal feces. Practically complete sequences of the genes encoding 16S rRNA were determined in the newly isolated strain Lact. plantarum and strain Lact. plantarum 8-RA-3 which were found to be identical. According to the results of BLAST analysis of 16S rRNA genes, Lact. plantarum WCFS1 (AL935263) and Lact. plantarum AF1 (FJ386491) were the most phylogenetically close to the strain isolated from mouse feces of the test group and to the original Lact. plantarum 8-RA-3 strain. The level of sequence similarity of the studied isolate with Lact. plantarum WCFS1 and Lact. plantarum AF1 strains amounted to 100 %. The level of similarity sequences of the isolate under study and the type strain Lact. arizonensis (synonym plantarum) (AJ965482) amounted to 99.9 %. According to existing present time standards [18], the observed level of similarity of gene sequences of 16S rRNA testified to the fact that the studied strains belong to Lact. plantarum species.

Since the analysis technique of the nucleotide sequences of 16S rRNA genes due to the conservatism of this molecule does not possess sufficient resolution, strain-specific differences of Lact. plantarum were studied using DIR-PCR. According to DIR-PCR data, the strain isolated from the mouse test group, and the initial Lact. plantarum 8-RA-3 strain used for the fermentation of wheat bran included in the diet of these animals had the identical number and size of the fragments, which was an indicator of genomic identity of the studied strains (Fig. 8). The isolates of Lact. plantarum identical to the 8-RA-3 strain had not been detected in the mice control groups 1 or 2.

Fig. 8
figure 8

The DIR-PCR results. Fingerprints of the studied strains with the use of KRPN1, KRPN4, and KRP8 primers. M molecular weight marker, 100 bp (SM 0321, Fermentas), k control (water), 1 Lact. plantarum 8-RA-3 strain, 2 the Lact. plantarum strain isolated from the mouse feces of the test group

Full size image

So, the data obtained indicate the ability of Lact. plantarum 8-RA-3 strain to reproduce and allow for biofilm formation in solid substrate cultivation conditions. Biofilms can contribute to human and animal health. Probiotic bacteria biofilms are capable of releasing factors that confer specific health benefits such as immunomodulation and outcompeting or inhibiting pathogens [19, 20]. Bacteria grown as a biofilm produce antimicrobial compounds that they do not produce when they grow in planktonic cultures [21].

There are numerous benefits that a bacterial community might obtain from the formation of biofilms. Bacterial cells in biofilm are immersed in the formed by them exopolysaccharide (EPS) matrix. The EPS performs an important function contributing to the persistence of commensal bacteria in the host. Obducing microbial cells, EPS mask the surface antigen determinants, and thereby, protect the bacteria from the destructive effect of the host immune response [22].

In unfavorable conditions caused by a high cell density or exhaustion of the nutrition sources, lactobacilli form rounded cyst-like cells with all the features of resting forms: stress resistance and lack of ability to grow on nutrient media. Their colony-forming activity could be restored in specially arranged conditions [16, 23]. We had also demonstrated that by drying Lact. plantarum 8-RA-3 biofilms about half of the bacteria remained viable, but stayed in non-cultured state. Non-cultured (mature, dormant) forms in the Lact. plantarum 8-RA-3 biofilm had a view of coccobacilli and, presumably, could effectively overcome the gastro-intestinal barrier, and then restore the ability for culturing and other biological functions, contributing to the balancing of the resident microbiota of the large bowel disturbed under the influence of antibiotics. The mechanisms of this phenomenon are not clear, and they are going to be studied further. Nevertheless, the results of our studies indicate the possibility of biofilm application for the development of medical and veterinary probiotic preparations and products of functional nutrition of the new generation.

The method of solid substrate cultivation could be used with the purpose of obtaining of biofilms of different microorganisms: pathogenic, probiotic, industrially important, etc., as well as the resting cyst-like forms, which, apparently, is being one of the phases of the natural cycle of bacterial development [16].