Metabolite Multiprobiotic Formulas for Microbial Health

3.1 Symbiotic lectins as system regulators and delivery agents

LSSM function as metabolomebiotics regulating metabolome according to the principle “LSSM network—whole organism metabolome network or interactome” [15]. The network of LSSM is created in the following manner: lectin molecule of determined molecular weight (in the Laemmli system) is represented by LS including forms of varying charge and possessing a range of biological and physiological activities; such a minimal LS can be further transformed in a natural manner into extended network of complexes and supramolecular ensembles as a result of directional and sequential cascade binding of carbohydrates and GC. As a result of forming complexes and ensembles, lectin specificity of complexes and ensembles can be modified or changed during further development of recognition cascade network that will change the summary vector of specificity of LS. The latter will result in dynamic qualitative and quantitative changes of the local biotope surrounding. Thus, the final whole resulting network of LSSM (as metabolomebiotics) will regulate the whole metabolome and interactome of organism involving human glycome (carbohydrates and GC: glycoproteins, glycoenzymes, glycolipids, receptors, and others [16]). The metabolome possesses the ability to back direct and reversible effects of LSSM representing a part of hierarchic interactome. Multiple forms of adapted functioning LSSM microbiocenosis in the biotope will depend on the originality developed in a joint process of evolution involving host body local infrastructures for the distribution and disposition of microbiocenoses (organ-type constructions of both host and microbiocenosis interests are possible) [11, 12]. LSSM are ready to realize biologically active GC (as prebiotics, therapeutic agents, and metabiotics) in such symbiotic organs. The network of LSSM functions as noncellular simulators of symbiotics (probiotics) in the direct or indirect (through human higher hierarchic protection systems) predictable manners. For example, of communications between LSSM and own human protection systems, LSSM (as well as phytohemagglutinins from plants of medical significance) and artificial polymeric GC influenced peritoneal macrophage mobility in a coupled manner depending on doses of agents; LSSM were involved in modulation of cytokine production by human blood leukocytes [9, 17].

New useful properties of LSSM can be predicted and verified (cofunctioning to enzymes, adhesion, etc.), based on the fact that LSSM form a functional superfamily of symbiotic lectins (on example of probiotic lectins and lectins of nitrogen-fixing bacteria). In addition, LSSM are members of the new class of destructors of biofilms of yeast-like and Gram-positive pathogens that simplify antimicrobial choice of components among LSSM. Other possibilities to operate with LSSM include their potential participation in a set of hierarchic pathways of advanced human innate protective systems in biotopes for cross talks. Both types of communications allow LSSM to be a successful synergistic assistant against pathogens in biotopes together with other antimicrobials and antimicrobial physical stress factors. As a result, LSSM reveal prolonged (early and late) anti-Candida activities as cascade (coupled) actions possibly influencing microecological niches of pathogens within biotope [registered during coculturing in the first 3 days (early events; also involving probiotic-like leader strains of L. acidophilus and L. casei), 1–2 weeks, or 2–3 months (late events)] [18].

The used GC are characterized with known chemical structures simplifying interpretation and prognostics of results. Such GC reveal potential of metabiotics which may use LSSM as carriers [8, 12, 19]. As highly homogenous, synthetic GC better functionally imitate natural GC targets [bacterial proteoglycans, fungal (phosphor)mannans, others] important for antimicrobial/anti-infectious actions of LSSM. As a result, LSSM are very perspective in delivery and deposition of adequate specific GC which are locally releasing as therapeutics possessing anti-infectious, prebiotic, and/or communicative signal abilities and actions.

In biotopes LSSM participate in continuous (on-duty) support and periodical (biorhythmic) completion and exchange of normal GC décor of cells, tissues, and organs that must provide delaying or stopping further the development of the spot/island/mosaic-landscape-originated and directed/metastasized abnormal processes as well as conserve any negative currently developmental events (initiation of appearance and survival of tumor-like cells as a result of innate intercellular communications involving lectin receptor-coupled cell surface receptor mosaics).

LSSM (natural combinative sets of LSSM-GC complexes) influence all yeast-like fungal phases of life by prolonging the degradation and lysis of pathogenic microbiocenosis massifs or biofilms in human biotopes (on examples of Candida species). In respect to CFB functioning as niches, lectins of lactobacilli (LL as preferentially mucins/mucus-recognizing) and lectins of bifidobacteria (BL as preferentially mannan-recognizing) synergistically act against internal and peripheral subniche territories, respectively. The late destructive and lytic events in CFB may also take place due to LSSM cooperative complex action as metabolomebiotics together with hydrolases involved in pathogen destruction network. Synergistic actions between LSSM and other antifungals increase resulting in final (early or late [also of apoptotic origin]) antipathogenic events as shown in Table 2 [20].

Endogenous LS antimicrobial actions (e.g., intestinal probiotic bifidobacterial and lactobacillar LS against intestinal yeast-like fungi) provide more directed, sensitive, and completed resulting effectiveness against pathogens (the primary absence and further late complete destruction of residual resistant colonies in external and internal regions of CFB of Candida albicans) compared to the action of phytolectin mixture from grasses of medical significance in the same conditions. In cases of antibiotic-resistant strains, relatively sensitive to LSSM C. albicans strain 547 (less potentially pathogenic compared to the strain 515, see below), the sorbed lactobacillar cationic LS revealed their synergistic ability to “regenerate” original anti-Candida effectiveness of antibiotic (on example of nystatin) in the internal region of CFB of yeast-like fungus (appearance of pathogen-free landscape connected to the border of the disk antibiotic). The phenomenon of synergistic reparation of the original ability of the sorbed antibiotic indicates prospects of additional mechanisms of LSSM combinative actions and partially describes how to increase resulting antifungal effectiveness during prolonged contact to pathogenic CFB at fungal late steps. In the case of more resistant (potentially “more pathogenic”) fungal strains (like C. albicans strain 515), the sorbed bifidobacterial cationic LS synergistically increased anti-Candida action of the grass phytolectin mixture.

Results indicate that LSSM may also participate in temporary separation and conservation of natural infectious biofilms to prevent early visual landscape development of diseases. The latter may be perspective as the assistant factor of improving quality of life (its prolongation, mucus and skin reparations, cosmetic significance, etc.) as it can be expected using symbiotics [21].

On the whole, our experimental approach (observations of antagonistic relationships between LSSM and pathogens in prolonged stress conditions) and the data obtained are supported by the conception describing microecological stress events in organism as the normal but sensor natural reactions [22].

3.2 The choice of strains and their consortia possessing potential for constructing new multistrain pro- and symbiotics

Screening, choice, and selection of new or improved antimicrobial and other useful properties of symbiotic (probiotic) cultural properties of strains and consortia of human MB are important and strategic goals in prophylaxis and therapy of diseases, increasing resistance of organism and the acceleration of patient rehabilitation [1, 2, 3, 4, 23]. Among GC investigated, glycans of mucin type (mucosal glycans) reveal special interest [24].

On the basis of own results, we proposed new algorithm of screening adequate probiotic-like microorganisms and their consortia possessing increased directed anti-infectious LS to construct new multipro-/sym-/synbiotics. The algorithm using LSSM (involving complexes to GC) among a panel of key factors in creating multiprobiotics (MP) included (a) the choice of synthetic GC-imitating bacterial proteoglycans and (phospho)mannans of yeast-like fungi; (b) identification of different LSSM (GC-type-dependent) among proteins of cultural fluids of probiotic strain or consortium of strains; (c) comparison of (GC-type)-dependent LS (evaluation of summary LS intensity, length of LS distribution in pH-gradient tested, mosaic asymmetric configuration of distributed forms in LS, major forms as dominating in contribution to antimicrobial actions of LS, minor forms as expressing signal regulators of biorecognition in microbiocenoses, signals of communications to surrounding infrastructures, as well as additional participants of recognition of new types of biomarker GC); (d) identification of unique sets of components of LS significant for typing strains, species, or genera; (e) revealing and choice of combinative sets of potential antimicrobial forms of LS for further control and testing; and (f) control of antimicrobial activities initiated, supported, and/or influenced by LS-containing preparations in other (nondependent) methods.

The data which were useful for constructing any multiprobiotics (Acilact-like) is presented in Table 3.

In Table 3 the data needed for constructing formulas of any MP are presented on example of strain component compositions of Acilact (the well-known MP which served as a model). Algorithm for constructing formulas includes a few steps:

The first step: For formulas of any MP of category A (formulas as sum of wishful selected superiorities): the choice of all parameters of superiorities of MP (No. 4 as MP in code notes: positions 1.3, 1.4, 1.6, 2.7, 2.8, 2.13, 2.14, 2.15, and 4.3; major ingredient strain contributors are at the second position in the code sequences [from left to right] position in code).

The second step: For formulas of any MP of category B: accounting additional superiorities of MP [No. 4 as MP in code notes; selected minimal positions of parameters for No. 4 in sequence indicate maximal expression of the contrary (by implicity) parameters]. For example, in the case of No. 4 in codes 1.1 (maximal resulting hydrolytic activities in respect to acidic proteins; increased level of antimicrobial peptides), 1.2 (maximal resulting hydrolytic activities in respect to cationic proteins; increased level of antimicrobial peptides including bacteriocin-like), 1.5 (the minimal level of aggregation upon storing concentrates), 2.3 (the minimal level of fluorophores exciting at 254 nm; contribution of Tyr and Trp or their derivatives), 2.3.1 (the minimal level of fluorophores exciting at 365 nm; contribution of Trp); and 4.2 (the minimal level of colored products); the major ingredient strain contributors are accounted as the third position in code.

The third step: The final formulas (formulas of category C) include combinations of formulas A and B. Multifunctionality of parameters analyzed can be extended (as in cases of amino acids [25]).

Extended approach for constructing more adaptive mixtures of lactobacillar and bifidobacterial MP (on the basis of Acilact extended by accounting industrial bifidobacterial strains) is presented in Table 4.

As expected, taxonomically mixed probiotics (symbiotics) will possess increased survival in biotopes of human organism. The same principles and algorithm (as for formulas of Acilact variants described above) can be applied. Combinations of Acilact ingredient strains and ingredients of Bifidin (B. longum spp. adolescentis MS-42), Bioprotectin (B. bifidum No. 1), and other bifidobacterial probiotics produced in Russia are of priority interest (also due to the possibility of their usage as standard models).

Table 4 demonstrates algorithm of further passage from intra-genus cases (Lactobacillus) of Gram-positive MP to inter-genus cases of MP as combinations of probiotic lactobacilli and bifidobacteria of the human gut origin.

Selective sets of parameters (points in Table 4) indicate principle differences between lactobacilli and bifidobacteria (as blocks of lactobacillar and bifidobacterial strains in 7-mark code). Completing lactobacillar/bifidobacterial synergism is expected and predicted. Selected combinations of strains from both blocks can be used for creation of directed MP. Other more complex cases include unblocked lactobacillar and bifidobacterial sequences within the code (additional prognostic conclusions are possible). Code positions “1” and “5” (both strains produce high levels of cytoagglutinating LS) reveal adjacent/similar behavior that indicates high level of compatibility of the strains NK1 and MS42.

Further constructing other or extended multistrain symbiotics is depended on choice of important parameters of interest (to increase the number of comparable codes used in Tables 3 and 4). Important prospects in constructing taxonomically mixed symbiotic formulas are expected on the basis of identified LSSM sets of the strains as counted ingredients of multisymbiotic as well as evaluation of the relative contribution of LSSM types in resulting multifunctional activities of mixed product. For example, the general properties of LS of lactobacilli and bifidobacteria investigated by us are “recognition of mucin-type targets” more or less than “recognition of mannan-type targets” for LL or BL, respectively [8]. As a result, LSSM-dependent synergism (which can be directed and predicted using extended panel of GC for LSSM selection and choice) of new taxonomically mixed symbiotics can be achieved.

Universality of approach proposed in Table 4 means that the panel of comparative parameters of investigation is unlimited for selection. As advantages of this approach for creation of perspective formulas of multipro-/symbiotics, some properties of future combinative products can be predicted and verified.

Aforementioned codes (Tables 3 and 4) extend the potential of using traditional MP. Results open new possibilities for investigation of LSSM types among strains and constructed consortia to develop perspective GC-type-dependent LSSM combinations possessing needed actions toward human interactome. In terms of personalized medicine, individual LSSM applications are of reality. For example, LSSM could be applied as “a functional tissue biotope” or mucosal organ-specific agents and organizers of lectin-coupled reactions and activities [26, 27].

3.3 Anaerobic synergistic preparations containing LSSM for support of human protective systems

Due to high distribution in organism, oxidative stress (as the power destructive factor initiating diseases) needs the constant presence of the power protective antioxidant systems [28, 29]. Some therapeutic proteins regulating cellular consumption of oxygen can be involved into development of tumor and other side pathologies in organism. We isolated system anaerobic (without oxidases initiating of oxygen and peroxide radicals) preparations of acidic/anionic and alkaline/cationic LSSM from cultures of symbiotic (probiotic) industrial strains of human bifidobacteria and lactobacilli as consortia that were successfully applied. Such preparations devoid the ability to induce destructive oxidative stress (cross-linking and inactivation of therapeutic proteins, etc.) in respect to surrounding infrastructures.

The used synthetic GC in our work were characterized with antioxidant properties in respect to LS as carriers of GC (prolongation of chemiluminescence of protective complexes was observed). Similar resulting protection of LSSM was also registered in the presence of neutral and cationic bifidobacterial and lactobacillar cultural exopolymeric compounds (EPC) of nonprotein origin (as observed on the blot after IEF-PAG). Acidic and alkaline anaerobic LS of bifidobacteria and lactobacilli revealed the following general antipathogenic actions: (a) own and overlapped/synergistic; (b) toward communicative bodies of microbial massifs and biofilms of the potentially pathogenic yeast-like fungi and Gram-positive bacteria. All four types of preparations of LSSM used were characterized by own mechanisms of antimicrobial actions in comparison to action of other antimicrobial systems (antibiotics, bacteriocins, phytolectins, subisotype products of isotypes С4В and С4А of the human complement component C4) [30, 31]. LS from human probiotic bacterial cultures revealed the ability to act as cascades in such reactions as initiation/changing or switching recognition of GC of different types (imitators of mannans, mucins, components of bacterial walls, Forssman antigens, Tn, blood group substance A) using the same original pool of lectin forms of taken multistrain probiotic. The presence of cations Ru²⁺ (ingredient of SYPRO involved in photosensibilization) strongly increased discreteness and number of forms of acidic lectins—potential carriers and deliveries of GC. Stability of obtained mosaic asymmetric landscape pictures of the systems LSSM-GC as multistrain probiotic-depending and multistrain probiotic-supporting biotope balance of recognition and reversible retaining/depositing of GC (therapeutics, biomarkers, others) was observed. Combinations of anaerobic LS-containing proteins revealed themselves in respect to yeast-like and Gram-positive pathogenic targets as more selective in the choice of the adequate regional territory of massif of pathogen and limitation of early and late time (depending on localization of targeted region of communicative body of pathogen) for the mostly effective visible actions of LS, obtaining uniform pure landscapes of LSSM action on massif of Candida albicans (the absence or minimization of LSSM-resistant residual fungal colonies in the interacting intestine system “LS of human intestinal bifidobacteria and lactobacilli—human intestinalC. albicans”). Antimicrobial activities of LSSM and phytolectins (phytohemagglutinin from the kidney bean) could be realized not only directly but also through the influence (together with synthetic mannans and mucins) in respect to macrophage migration as well as through inducing production of cytokines by stimulated blood lymphocytes (on the example of tumor necrosis factor-α). Results indicate prospects of anaerobic LSSM as assistant ingredients of the possible drug forms.

3.4 Synbiotic minibioreactor using LSSM for screening GC

During the last time, synbiotics and symbiotics (as synergistic sum of probiotics and prebiotics) are of increased investigator interest due to their antimicrobial and other useful reactions [1, 2]. In this respect LSSM represent new class of antipathogenic proteins (possessing extended potential of application) which recognize different GC. LSSM represent multifunctional potential of relatively highly molecular mass polymeric metabolites of cultures of the human microbiota (microbiocenoses) and consortia (also multistrain probiotics) of human indigenous microorganisms. LSSM cofunction together with artificial and biologically active natural GC [32].

According to own results, we proposed suitable laboratory minisystem for screening prebiotic and therapeutical GP using LSSM and sterile heparinized insulin syringes of 1 ml volume. The following results were obtained: (1) LSSM-containing fraction stimulated production of both the whole and adhesive mass of bifidobacteria, (2) LiCl (15 mM and higher) increased dose depending on the number of adhesive colonies, (3) bifidobacterial LSSM (within pI 4–4.5) were characterized with strong affinity to anionic synthetic GC (possessing exposed residues of sulfated galactosides or, in a less extent of affinity, exposed residues of mannose-6-phosphates), and (4) sulfated glycosaminoglycans together with cations Li⁺ and LSSM as potential carriers of Li⁺ participated in functioning bioreactor imitating synbiotope (multiplication of bifidobacterial colonies and their survival were observed).

Proposed synbiotic system is perspective for screening prebiotic GC (as it is known for prebiotic derivatives of chitin, chitosan, fucoidan, and glycopeptides [33, 34]). Table 1 includes potential prebiotic sources such as chitin and α-L-fucan which react to LSSM (LSSM may serve carriers of metabiotic GC; the list of GC can be unlimitedly extended).

3.5 Membrane technological prospects of LSSM

Progress in membrane and solid-phased technologies using LS-GC interactions includes the potential of their application in microassays, biochips (membrane bound glycoarrays or lectin arrays), and biosensors [35, 36].

The use of affine pore hydrophobic (uncharged) membranes predictably covered with mosaics of multifunctional sets of LSSM (additional significant purification of LSSM on membranes is reached) allows prolonged storing LSSM without decreasing samples in activities. The following prospects of LSSM-GC combinations may be of practical interest: (a) antifungal covers of prolonged action in combination with antibiotics and physical factors of stress [radiation (ultraviolet and ultrasonic), light (biorhythm “day-night”), temperature, pH, oxygen, season changes (also biorhythmic), others] and (b) chemiluminescent systems cofunctioning in regime of real time for medical and industrial biotechnology and bionanotechnologies [our results include the following coupled systems: “low acidic LSSM, low acidic oxidoreductases of lactobacilli”; “alkaline bifidobacterial LS, alkaline bifidobacterial exopolymeric compounds”; “neutral lactobacillar/bifidobacterial LS, neutral lactobacillar/bifidobacterial biosurfactants”; “LS, strongly acidic (pI 3–4) serial phyto-oxidoreductases/phyto[glycosyl]oxidases”].

Membrane technologies using separated proteins, oligopeptides, and their complexes (especially) together with intrinsic or exogenic (SYPRO dye) fluorescence registered in live bioimagination are especially sensitive and perspective (protein band discreteness using fluorescence technique was better compared to the chemiluminescence technique). The latter allows identification stabile boundaries of the whole protein massifs for further establishment of LSSM and other biologically and physiologically active components among protein mosaics. Bioluminescence (fluorescent technique in combination with chemiluminescence technique) in optimal (depending on the object and the goal of study) conditions allows express-ranging cultural fluid groups of proteins and LS according to molecular mass (for additional standardization and typing of strains), evaluation of interstrain synergism and contribution of protease and oxidoreductase systems of mono- and multistrain probiotics (symbiotics) and other type consortia, and identification of mosaics of complexes containing fluorochromes in extended interval of pI/pH (complexes and cell wall fragments as carriers of visible energy which is ready for energetic exchange with surrounding infrastructures as well as for monitoring directed supramolecular assembling and their reorganization).

Aforementioned data presented develop other possible important prognostic approaches and proposals. The choice of wished prognostic (LSSM-selected-type and GC-type interactions)-directed events in biotope can be determined and regulated by involving GC types needed (panels of natural and artificial GC used in biotope; extending the list of GC indicated in Table 1 is possible). The use of artificial polymeric GC with established chemical structures allows receiving adequate, understandable, and reliable results.

According to the multifunctional potential of artificial GC used, resulting events in biotope can be the following ones: antipathogenic actions (the use of GC-imitating pathogenic cell surface structures), prebiotic and symbiotic (synbiotic) actions (the use of GC-imitating prebiotic structures), increasing own human organism protective systems (the use of GC-interacting and GC-regulating macrophages and macrophage-like lymphocytes through their systemic receptor lectins), antitumor actions (potential using antigenic GC together with LSSM as antagonists and synergists in intercellular antitumor and anti-infectious communications [37]), and cytokine lectin/lectin-like cascades initiated/regulated with LSSM potentially influencing redistribution of cytokine network in organism.

3.6 Prospects of LSSM for prophylaxis and therapy of infectious diseases

Innate immunity plays an important role especially when antibody immunity is under development, suppression, or alteration. So the search of new natural anti-infectious agents is of actuality. In contrast to probiotic cells, LSSM (as systems of LL or LB) are not sensitive to the presence of antibiotics and chemotherapeutics as well as do not need special conditions for survival. LSSM function as metabolomebiotics according to the principle “the network in the network”; participation of major lectins in supporting physiologically significant glycodecor of biotope mucosal probiotic compartments, and minor lectins—in signal communications; support of such compartments (compensation of the absence of probiotic cells in MB upon therapy with antibiotics, delivery of fucosylated and galactosylated prebiotics of mutual supporting influence between populations of bifidobacteria and lactobacilli, cofunctioning to anti-infectious agents (antibiotics, metabiotics, GC) and cells of organism protective systems (macrophages and leukocytes).

We characterize LSSM (acidic, low acidic, cationic: aLL, laLL, cLL, aLB, and cLB) of domestic probiotics which recognize polymeric artificial GC mucin-like analogs of antigens and EPC (fungal and bacterial mannans, bifidobacterial α-L-Fuc-containing, sulfated and phosphorylated).

Antimicrobial directions of LSSM action were established [26, 31, 38].

3.7 Potential of results for innovations

Results obtained by us in a study of probiotics can be useful as a set of approaches in study of the following aspects of clinical microbiology and medical biotechnology (in brackets—significance and prospects of results for infectology) [40, 41].