Lakhtin M.V., Lakhtin V.M., Afanasiev S.S. , Aleshkin V.A.

G.N. Gabrichevsky Institute for Epidemiology & Microbiology, Moscow, Russia

SELECTED PROSPECTS FOR APPLICATION OF

SYMBIOTIC LECTIN SYSTEMS OF HUMAN MICROBIOCENOSES

Summary

Selected prospects of symbiotic lectin systems (SLS) of the human opened mucus cavities microbiocenoses investigated by us are described. SLS function as metabolomebiotics, imitators of probiotics, carriers of therapeutic and prebiotic glycometabiotics, providers of the extended spectrum of coupled biologically active substances, holders and depositors within and on the mucus, agents masking and/or realizing glycoconjugates (GC) for the local reactions of surroundings. GC- metabiotics and symbiotic lectins regulate synergistically, provide complex factors for the direct and indirect actions against relatively pathogenic microorganisms. GC can be potentially used as décor elements supporting normal survival of human cells, tissues and organs.

In addition, SLS (recognizing synthetic GC-imitators of important substances for human) serve instruments for screening probiotic-like microorganisms and their consortia possessing new potential for constructing multistrain pro-, sym- and synbiotics (also on the basis of traditional probiotics). Proposed anaerobic (active without oxidoreductase systems) synergistic combinations of probiotic lectins and other antimicrobials are perspective as multifunctional switching assistive recognition systems cofunctioning to higher hierarchic human protective systems (also in the absence of the locally available oxygen). Results support technological prospects of the protective cascades of the human intestinal indigenous SLS against pathogenic exo- and endogenic eukaryotic (yeast-like fungi) and prokaryotic (Gram-positive bacteria) microorganisms.

Keywords: symbiotic lectins, glycoconjugates, microbiocenoses, multistrain probiotics, mucosal cavities, antimicrobials, human protection.

1. Introduction

Probiotics, prebiotics and symbiotics are perspective in clinical application, medical biotechnology and human microecology [1-4]. Microbes and the human communicate each other by the way of recognition and direct binding between glycoconjugates (GC) of varying complexity and proteins (mainly adhesions and lectins) [5-9]. Lectin systems (LS) of symbiotic microorganisms (LSSM) recognizing GC represent new and multifunctional symbiotic factors [10-17]. LSSM are related to useful for human protein/ peptide-containing compounds and their complexes recognizing GC. LSSM reveal features of imitators of probiotics [11, 18]; members of new class of bacteriocins-like destructors of biofilms of yeast-like fungal and Gram-positive pathogens [19]; systems cofunctioning together with enzymes of all known classes [10, 20]; agents possessing antipathogenic synergism of different LS in antimicrobial combinations (between LS of Lactobacillus species, Bifidobacterium species, between genera Lactobacillus and Bifidobacterium, between LS of probiotic bacteria and lectins from medical plants, between LSSM and antibiotics) [11, see Table 2 below].

Any LS (as part of super LS of super organism [15]) reveals significantly higher multifunctionality (antimicrobial, cytokine-like, others) and adaptive ability in surroundings in comparison to any component of LS. Applied prospects of LSSM in microbial associations of biotopes in the human body are of promised interest. LSSM and their reactive GC support balanced functioning in organism in respect of created during evolution of organs-like infrastructures of mutual interest for human and biotope microbiocenosis [15]. Systems LSSM—GC represent important perspective ingredients of human interactome revealing themselves as important contributors into infrastructures, signalling, antimicrobial and antiviral actions, support of balanced biotope microbiocenosis and its cofunctioning together with other protective human systems [21-24].

The purpose of the review is to sum selected applied prospects of LSSM in biology and biotechnology mainly on the basis of own data. Especially, such a review is of interest for investigators in the fields of medical biotechnology and microbiology.

2. Methods

Used strains of lactobacilli and bifidobacteria were from the collection of microorganisms of the normal microflora of G.N. Gabrichevsky Institute for Epidemiology & Microbiology, as well as probiotics Bifidin and Acilact were products of our Institute. Bacteria were grown in media containing casein hydrolysate. LSSM were extracted from the protein fractions 27-220 kD using isoelectrofocusing (IEF) in a plate of polyacrylamide gel (PAG) in a gradient of pH 4-8. Identification of proteins was performed by electroblotting on a hydrophobic membrane and staining with SYPRO Blot Stain (BioRad Lab.). Non-stained proteins were evaluated by other spectrophotometric methods. The distribution of LSSM among proteins was determined by treatment of blots with biotinylated GC (GC-b) containing multiple residues of sugar(s) linked to the polyacrylamide (PA) linear core (www.lectinity.com; Table 1) followed by additional treatment with Streptavidin-Peroxidase conjugate. The bound peroxidase on the blot was registered in the presence of chemiluminescent substrate in regime of real time in the system BioChemi System (UVP, Calif.). Antimicrobial activities and synergism of LSSM, antibiotics and phytolectins were tested on solid agar media during the prolonged growth and survival of communicative fungal bodies (CFB) in the presence of discs antimicrobials [25, 26]. Biosurfactants were tested using detection of sample drop activity against mineral oil film on water surface. Amino acid compositions of samples were established using standard Amino Acid Analyzer column chromatography. Oxidases-Reductases systems were detected on blots after IEF-PAG as resulting kinetic protein stain disappearance (decolorization) within pI 5,1-5,6. Hydrolases systems were visually evaluated on blots after IEF-PAG (as resulting hydrolysis of protein bands). Maillard reaction products were partially evaluated as browning in culture supernatant according to optical density at 420 nm.

Table 1. A list of synthetic GC used (in brackets – natural substances which are imitated):

1. α-D-GalNAc-PA-b (animal mucins, antigens T),

2. β-D-GalNAc-PA-b (animal mucins),

3. β-D-GlcNAc-PA-b (insect chitins and chitosans),

4. β-D-Gal-PA-b (plant or animal galactans),

5. β-D-Gal-3-sulfate-PA-b (acidic animal galactans),

6. α-D-Man-PA-b (yeast mannans),

7. α-D-Man-6P-PA-b (yeast phosphomannans),

8. α-L-Fuc-PA-b (fucans from brown algae),

9. α-L-Rha-PA-b (Gram-negative bacterial rhamnans),

10. MDP-PA-b (bacterial peptodoglycans),

11. Adi-PA-b (A-blood group substance GalNAcα1-3Galβ1-),

12. Fs-PA-b (Forsman antigen GalNAcα1-3GalNAcβ1-),

13.Tαα –PA-b (bacterial antigen Galα1-3GalNAcα1-);

3. Results and discussion

3.1. Symbiotic lectins as the system regulators and delivery agents

LSSM function as metabolomebiotics regulating metabolome according to principle "LSSM network—Metabolome network" [27]. 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 activity); 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 [28]). 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 microbiocenoses interests are possible) [14, 15]. LSSM are ready to realize biologically active GC (as prebiotics and therapeutic agents) in such symbiotic organs. The network LSSM—GC functions as non-cellular simulators of symbiotics (probiotics) in the direct or indirect (through human higher hierarchic protection systems) predictable manners. For examples of communications between LSSM and own human protection systems, LSSM (as well as phytohaemagglutinins) and artificial polymeric GC influenced peritoneal macrophage mobility in a coupled manner depending on doses of agents; LSSM also induced cytokine production by human blood lymphocytes (on example of factor-alpha of tumor necrosis) [10].

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) [16]. 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 [19]. 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 [21, 22, 23, 29]. Both types of communications allow LSSM to be successful synergistic assistant against pathogens in biotopes together with other antimicrobials and antimicrobial physical stress factors. As a result, LSSM reveal early and late anti-Candida activities as cascade (coupled) actions possibly influencing microecological niches of pathogens within biotope (registered during coculturing first 3 days [early events; also involving probiotic-like leader strains of Lactobacillus acidophilus and L.casei], 1-2 weeks or 2-3 months [late events]) [19, 25, 30, 31].

On one hand GC possessing known chemical structures (www.lectinity.com) can serve potential metabiotics which may use LSSM as carriers [32]. On the other hand synthetic GC better imitate important for antimicrobial LSSM action bacterial proteoglycans and fungal (phospho)mannans in comparison to low molecular mass residual heterogenic fragments from natural GC. As a result LSSM help in delivery and deposition of adequate specific GC which are locally releasing as therapeutics possessing antimicrobial, prebiotic and/or communicative signal abilities and actions. 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 further development of the spot/ “island”-originated directed abnormal processes as well as conserve any negative currently developmental events (initiation and survival of tumor-like cells, appearance of negative communications involving different altered and partially undifferentiated cells).

Systems LSSM—(natural GC) influence all yeast-like fungal phases of life by prolong degradation and lysis of pathogenic microbiocenosis massifs or biofilms in human biotopes (on examples of Candida species). In respect of communicative fungal bodies (CFB) as niches, LL (as preferentially mucins/mucus-recognizing) and BL (as preferentially mannans-recognizing) synergistically act against internal and peripheral subniche spaces, respectively [33]. The late destructive and lysis events in CFB may also take place due to LSSM cooperative complex action together with hydrolases when involving in pathogen destruction network [10]. More over, synergistic actions between LSSM and other antifungal agents increase resulting and final (early or late [also of apoptotic origin]) anti-pathogenic events as shown in Table 2 [34].

Table 2. Antipathogenic synergism of LSSM and antibiotics.

Types of synergism	Lectins^*	Distant positions, (directions Left-to-Right)	Targets (CFB)
Between LSSM
Between identical LSSM-discs	aBL and aBL	p-aBL, c-aBL (p-aBL—c-aBL)	C.albicans 991
Intra-genus (Lactobacillus)	cLL and aLL aLL and cLL	From p-cLL to CR (p-cLL—p-aLL) From p-aLL to CR (p-aLL—p-cLL)	C.albicans 515, 547 C.albicans 515, 547
Intra-genus (Bifidobacterium)	aLL and aBL	p-aLL and c-aBL (p-aLL—c-aBL)	C.albicans strains
Intra-genus (Bifidobacterium)	aBL and cBL	[PR aBL] and [CR aLL]	C.albicans strains
Inter-genera (Lactobacillus и Bifidobacterium)	aBL and aLL aLL and aBL	[PR aBL] and [CR aLL] (p-aBL—p-aLL) [PR aLL] and [CR aBL]^2* (p-aLL—p-aBL)	C.albicans strains S.aureus strains
Between LSSM and phytolectins
Between LSSM and MGBL	aLL and MGBL cBL and MGBL	from aLL to CR (p-MGBL —p-aLL) from p-cBL to CR (p-MGBL—p-cBL)	C.albicans 515 C.albicans 515
Between LSSM and	antimycotics
Between LSSM and Amphotericin B	aBL	p-Amphotericin B, c-aBL (p-Amphotericin B—c-aBL)	C.albicans strains > C.tropicalis strains
Between LSSM and Itraconazole	aBL	c-Itraconazole, p-aBL (p-Itraconazole —p-aBL)	C.albicans strains
Between LSSM and Ketoconazole	aBL	p-Ketoconazole, c-aBL (p-Ketoconazole—c-aBL)	C.albicans strains
Between LSSM and Nystatin	cLL cBL	From p-cLL to CR (p-cLL—c-Nystatin) from cBL to CR (p-cBL—c-Nystatin)	C.albicans 515, 547 C.albicans 515, 547
Between MGBL and nystatin (low level)	MGBL	From p-MGBL to CR (p-MGBL—c-Nystatin)	C.albicans 515
Multiple synergism
Between BL and LL	aBL, aLL	In triangle: p-aBL+c-aBL+p-aLL (p-aBL—c-aBL; p-aLL—c-aBL)	C.albicans strains (not for C.tropicalis)
Between LSSM and	antimycotics
Between LSSM and Amphotericin B	aLL, aBL	In triangle: p-aLL+c-aBL+p-Amphotericin B (p-aLL— c-aBL; p-Amphotericin B—c-aBL)	C.tropicalis strains> C.albicans strains
Between LSSM and (Amphotericin B and Nystatin)	aBL	In triangle: c-aBL+p-Amphotericin-B+p-Nystatin (p-Amphotericin B—c-aBL; p-Nystatin—c-aBL)	C.tropicalis strains

Comments: ^*Diffusion between discs placed on Sabouraud agar in Petri dishes (disc positions: p= peripheral, c= central; CR= central region: between p-disc and the center; PR= peripheral region: between p-disc and the border of agar); discs included anionic (a) and cationic (c) lactobacillar and bifidobacterial LSSM (aLL, cLL, aBL, cBL), MGBL as mixture of the grass lectins (from phytopreparation – ingredient of Endocrinol [Evalar, Altay, Russia; the grasses of medical significance: Potentilla album and Stellaria media]); lectins were used in subhaemagglutinating doses (less than 1 microgram/ml). Standard panel of disc antimycotics was used (HiMedia Lab. Pvt Ltd.). CFB= communicative fungal bodies.

Endogenic LS antimicrobial actions (for example intestinal probiotic bifidobacterial and lactobacillus LS in respect of intestinal yeast-like fungi) provide more directed, sensitive and completed resulting effectiveness against pathogen (the absence of residual resistant colonies in external and internal regions of CFB of Candida albicans) compared to the action in the same conditions in case of phytolectin mixture from grasses of medical significance [26]. 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 lactobacillus sorbed alkaline LS revealed the synergistic ability to regenerate original anti-Candida effectiveness of antibiotic (on example of Nystatin) in the internal region of CFB of yeast-like fungus (“cleaning” or making free the border of the sorbed Nistatin from massif of pathogen). Phenomenon of synergistic reparation of the original ability of the sorbed antibiotic indicates prospects how to increase resulting antifungal effectiveness during prolong contact to pathogenic CFB at fungal late or “chronic” steps). In case of more resistant (more pathogenic) C. albicans strain 515, sorbed bifidobacterial alkaline LS synergistically increased anti-Candida action of the grass phytolectin mixture [26].

Results indicate that LSSM may also participate in temporary separation and conservation of 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 [35].

In the whole, our experimental approach (observations of antagonistic relationships between LSSM and pathogens in prolonged stress conditions) and the data obtained are supported by conception describing microecological stress events in organism as the normal but sensoric (increased) natural reactions [36].

3.2. The choice of symbiotic strains and their consortia possessing new potential for constructing 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 microbiocenoses are important and strategic goals in prophylaxis of diseases, increasing whole resistance of organism, acceleration of processes of patient rehabilitation [1-4, 37, 38].

On the basis of own results we proposed new algorithm of screening adequate probiotic-like microorganisms and their consortia possessing increased directed antimicrobial LS to construct new probiotics. The algorithm using LSSM—GC as the only key factors in creating symbiotics (probiotics) 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)-depended 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; f) control of antimicrobial activities initiated, supported and/or influenced by LS-containing preparations in non-dependent methods.

Other algorithm using LSSM as the parameter coupled to other parameters for constructing symbiotics (probiotics) is presented in Table 3 [39].

Table 3. Ranged codes for constructing directed variants of multiprobiotic (on example of Acilact).

No	Parameters of supernatants of lactobacilli, comments to parameters, ranging Acilact and its strains	Code as range of Acilact and its strains*
	Status of proteins
1	Content of partially hydrolyzed protein K3III24>Acilact>100аsh >NK1	3 4 2 1
1.1	Acidic proteins pI 4-5 NK1>100аsh > K3III24>Acilact	1 2 3 4
1.2	Cationic proteins pI 7-8 NK1> K3III24>100аsh >Acilact	1 3 2 4
1.3	Oxidases-Reductases systems pI 5-5.5 Acilact>100аsh > K3III24>NK1[absence]	4 2 3 1
1.4	Hydrolases systems Acilact> K3III24>1100аsh >NK1	4 3 2 1
1.5	Level of aggregation upon storing concentrates K3III24>100ash>NK1>Acilact[no aggegation]	3 2 1 4
1.6	Ability to membrane ultrafiltration Acilact>NK1>100аsh > K3III24	4 1 2 3
	Status of biosurfactants
2	Associated biosurfactants NK1>Acilact>>100аsh > K3III24	1 4 2 3
2.1	Biosurfactants active against mineral oil K3III24>Acilact>100аsh >NK1	3 4 2 1
	Status of emulsifiers
3	Emulsifiers 100аsh >NK1>Acilact>> K3III24 [absence]	2 1 4 3
	Status of Maillard products
4	Maillard products [optical dencity at 400 nm] K3III24≥NK1>100аsh >Acilact	3 1 2 4
	Status of aminoacids
5	Production of all aminoacids (absolute contents) NK1> K3III24>Acilact>100аsh	1 3 4 2
5.1	Tyr (sites for serine proteinases, fluorophore) K3III24 >1100аsh ≥Acilact>NK1	3 2 4 1
5.2	Phe (sites for serine proteinases, fluorophore) NK1>Acilact> K3III24>100аsh	1 4 3 2
5.3	Fluorophores (Trp, Tyr) (excitation at 254 nm) 100аsh > K3III24>NK1>Acilact	2 3 1 4
5.3.1	Fluorophores (excitation at 365 nm) 100аsh > K3III24>NK1>>Acilact	2 3 1 4
5.4	Gly (hydrophobic, anti-adhesion action) NK1>Acilact> K3III24>100ash	1 4 3 2
5.5	Leu (hydrophobic, site for peptidases) (parameter is slightly depended on strain origin) K3III24 >100аsh ≥Acilact=NK1	3 2 1-4 1-4
5.6	Ile (hydrophobic, participation in synthesis of biologically active volatile fatty acids) K3III24>100ash>Acilact>NK1	3 2 4 1
5.7	Ala (partially from peptidoglycans, site for exo- and ando- exopeptidases) Acilact>K3III24=100ash>NK1	4 2-3 2-3 1
5.8	Ser (site for glycosylation) Acilact> K3III24>100аsh >NK1	4 3 2 1
5.9	Thr (site for glycosylation) NK1>100аsh >Acilact> K3III24	1 2 4 3
5.10	Lys (from cationic poly[oligo]peptides, site for serine proteinases, fluorophore, participation in Maillard reaction) Acilact>100аsh > K3III24>NK1	4 2 3 1
5.11	Val (hydrophobic, participation in synthesis of biologically active volatile fatty acids) K3III24>100аsh >NK1>Acilact	3 2 1 4
5.12	Glu/Gln (site for amidases ) NK1>Acilact≥K3III24 >100аsh	1 4 3 2
5.13	Asp/Asn (sites for glycosylation, sites for amidases Acilact≥100ash>NK1> K3III24	4 2 1 3
5.14	His (participation in autooxidation of protein) [similarity to Trp], high affinity to metal cations, activation of oxidases and haem) Acilact>NK1>100аsh > K3III24	4 1 2 3
5.15	Arg (from cationic poly[oligo]peptides, destruction during pigments forming in Maillard reaction) Acilact >NK1> K3III24>100аsh	4 1 3 2
5.16	Met (antioxidant) NK1>Acilact> K3III24>100аsh	1 4 3 2
5.17	Cys2disulfide bonds (oxidation of SH-groups into Cys2) K3III24>NK1>Acilact>100ash[not]	3 1 4 2
5.18	Pro (Pro-bends in regular structures of proteins,low contents) 100аsh >Acilact> K3III24>NK1[trace]	2 4 3 1
5.19	Antimicrobial activity (panel of diagnosticum strains) Acilact>NK1>100аsh > K3III24	4 1 2 3

Comments. *1= NK1 (Lactobacillus helveticus NK1), 2= 100_аsh (Lactobacillus helveticus 100_ash),

3= К₃III₂₄ (Lactobacillus casei К₃III₂₄), 4= Acilact.

In the Table 3 the data needed for constructing formulas of multistrain symbiotic is

proposed on example of Acilact [the well-known multiprobiotic as a model]. Algorithm for

constructing formulas includes few steps.

The 1^st step. For formulas of Acilact of category A (formulas as sum of wishable selected

superiorities): the choice of all parameters of superiorities of multiprobiotic (No 4 as Acilact in code:

positions 1.3, 1.4, 1.6, 5.7, 5.8, 5.13, 5.14, 5.15, 5.19; major ingredient strain contributors are

can be taken into account at the 2^nd position in the code sequences [from-left-to-right] position in

code). The 2^nd step. For formulas of Acilact of category B: accounting additional superiorities of

multiprobiotic (No 4 as Acilact in code; selected minimal positions of parameters for No 4 in

sequence indicate maximal expression of the contrary parameters). For example, in case of No 4

in codes 1.1 [maximal resulting hydrolytic activities in respect of acidic proteins; increased level of

antimicrobial peptides], 1.2 [maximal resulting hydrolytic activities in respect of cationic proteins;

increased level of antimicrobial peptides including bacteriocin-like], 1.5 [the minimal level of

aggregation upon storing concentrates], 4 [the minimal level of Maillard products], 5.3 [the minimal

level of fluorophores exciting at 254 nm; contribution of Tyr and Trp], 5.3.1 [the minimal level of

fluorophores exciting at 365 nm; contribution of Trp]; major ingredient strain contributors are

accounted as the 3^rd position in code). The 3^rd 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 aminoacids [40]).

Extended approach for constructing more adaptive mixtures of lactobacillar and bifidobacterial multiprobiotics (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 in Table 3) can be applied. Combinations of Acilact ingredient strains and ingredients of Bifidin [B.longum spp.adolescentis MC-42], Bioprotectin [B.bifidum No 1] and other bifidobacterial probiotics produced in Russia are of priority interest (also due to possibility of their using as standard models) [41].

Table 4. Ranged codes for constructing new multiprobiotics including bifidobacteria and lactobacilli.

N	Parameters of concentrate (C), their ranging, prognoses (P)	Code: range of strains, Acilact
1	Antifreeze components >27 kD, pI 4-8 (against any type of crystal forming during IEF-PAG): gall>bif1>MS42>NK1>Acilact> K3III24,100аsh. P: bifidobacterial C for stabilization of K3III24 (especially) and 100аsh.	7,6,5,1,4,3-2,2-3
2	Forming organic crystals in the presence of components > 27 kD (in conditions of 7М urea, 5% saccharose; 8^оС +night [principle factor], in slab of PAG upon IEF): рI4-6: K3III24,100аsh>Acilact>NK1>MS>bif1>gall(not); pI6-8: K3III24,100аsh>Acilact>NK1>gall>bif1>MS42.	2-3,2-3,4,1,5,6,7 2-3,2-3,4,1,7,6,5
3	Complex protein C >27 kD: pI 4-6: NK1>MS42> K3III24>gall>100аsh>bif1>Acilact; pI 6-8: NK1>gall> K3III24>MS42>100аsh>Acilact>bif1. P: Donors of cationic bacteriocin-like associates with exopolymeric compounds.	1,5,3,7,2,6,4 1,7,3,5,2,4,6
4	Adhesins as colourless transparent not-water-soluble drops on polystyrene (number of drops): gall>MS42>NK1>bif1>Acilact> K3III24=100аsh(not) P: The size of drops indicate level of emulsification of C (and original supernatant).	7,5,1,6,4,2-3,2-3
5	Associated biosurfactants: bif1>gall> K3III24>Acilact>100аsh>MS42>NK1 P: C «NK1+gall» and «NK1+bif1» as synergistic antimicrobials.	6,7,3,4,2,5,1
6	LSSM as mucin–binding: pI 4-5.5: gall>Acilact>NK1>MS42>bif1> K3III24,100аsh pI 5.5-8: MS42>bif1>gall>NK1>Acilact>100аsh, K3III24 P: C for delivery into intestinal and urogenital mucosal cavities.	7,4,1,5,6,2-3,2-3 5,6,7,1,4,2-3,2-3
7	LSSM as mannan–binding: pI 4-5.5: 100аsh, K3III24>NK1>MS42>gall>Acilact>bif1 pI 5.5-8: gall>bif1>MS42>NK1>Acilact>100аsh, K3III24 P: Potential against eukaryotic (yeast and yeast-like), prokaryotic (staphylococci) and HIV/ HIV-related infections; delivery into cell and cell organelles.	2-3,2-3,1,5,7,4,6 7,6,5,1,4,2-3,2-3

Comments. 1= NK1 (Lactobacillus helveticus NK1), 2= 100_аsh (Lactobacillus helveticus 100_ash), 3=

К₃III₂₄ (Lactobacillus casei К₃III₂₄), 4= Acilact, 5= MS42 (Bifidobacterium adolescentis МS-42), 6 =

bif1 (Bifidobacterium bifidum No 1), 7= gall (Bifidobacterium gallinarum GB). IEF=

isoelectrofocusing, PAG= polyacrilamide gel. P= prognoses.

The Table 4 indicates potential of extending Acilact and its ingredient industrial strains (code figures in bold) in direction of lactobacillar combinations together with industrial symbiotic (probiotic) strains of bifidobacteria of human origin. Some cases indicate principle differences between lactobacilli and bifidobacteria (blocks of lactobacillar and bifidobacterial strains in 7-marks-code; completing lactobacillar-bifidobacterial synergism is especially expected; selected combinations of strains from both blocks can be used for creating directed multiprobiotics). Other cases (as more complex) include unblocked sequences of the code; additional prognostic conclusions are possible). Positions No 1 and No 5 (both strains produce high levels of cytoagglutinating LS) in code reveal adjacent (similar) positions according to all parameters tested that indicates high level of compatibility of the strains NK1 and MS-42.

Further constructing development of multistrain symbiotics is depended on extent 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 [11]. As a result, LSSM-depended 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.

It is clear that the panel of comparative parameters of investigation is unlimited. Among advantages of the universal approach for creation of perspective formulas of multisymbiotics, some properties of future combinative products can be predicted and verified.

Aforementioned algorithms (Tables 3, 4) extend potential of using traditional multistrain

probiotics [39, 41]. Results open new possibilities for investigation of LSSM types among strains

and constructed consortia to develop perspective LSSM combinations possessing prognostic actions

towards human interactome [23]. Aspects of individual LSSM applications in term of personalized

medicine are of reality. For example, LSMM could be applied as “a functional tissue

biotope”- or tissue-specific agents and organizers of lectin-coupled reactions and activities [42, 43].

3.3. Anaerobic synergistic biopreparations containing LSSM, for supporting human protective systems

Due to high distribution in organism, oxidative stress (as power destructive factor initiating deseases) needs the constant presence of the power protective antioxidant systems [44-46]. Some therapeutic proteins regulating cellular consumption of oxygen can be involved into development of tumor and other side pathologies in organism [47]. 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 [19]. Such preparations devoid the ability to induce destructive oxidative stress (cross-linking and inactivation of therapeutic proteins, etc.) in respect of surrounding infrastructures.

The used synthetic GC in our work were characterized with antioxidant properties in respect of 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 of non-protein 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) towards communicative bodies of microbial massives 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) [19, 33, 35]. LS from human probiotic bacterial cultures revealed ability to act as cascades of such reactions as initiation/ changing or switching recognition of GC of different types (imitators of mannans, mucins, components of bacterial walls, antigens Forsman, Tn, blood group substance A) using the same original pool of lectin forms of taken strain/ multistrain probiotic. The presence of cations Ru²⁺ (ingredient of SYPRO involving in photosensibilization) strongly increased discreteness and number of forms of acidic lectins – potential carriers and deliveries of GC. It was observed stability of obtained mosaic asymmetric landscape pictures of the systems LSSM—GC as (multi)probiotic-depending and supporting biotope balance of recognition and reversible retaining/ depositing of GC (therapeutics, biomarkers, others). Combinations of anaerobic LS-containing proteins revealed themselves in respect of yeast-like and Gram-positive pathogenic targets as more selective in choice of the adequate regional territory of massive of pathogen, 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 massive 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 intestinal C. albicans”). Antimicrobial activities of LSSM and phytolectins (phytohaemagglutinin from kidney bean) could be realized not only directly, but also through the influence (together with synthetic mannans and mucins) in respect of macrophage migration as well as through inducing production of cytokines by stimulated blood lymphocytes (on the example of factor-alpha of tumor necrosis). Results indicate prospects of anaerobic LSSM as assistant ingredients of the possible drug forms.

3.4. Synbiotic minibioreactor using LSSM for screening GC

During 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 human microbiota (microbiocenoses), consortia (also multistrain probiotics) of human indigenous microorganisms. LSSM cofunction together with a set of biologically active natural and artificial GC [48].

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-depended 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). 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 known for prebiotic derivatives of chitin, chitosan, fucoidan and glycopeptides [49, 50]. Table 1 include potential prebiotic sources such as chitin and 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 include potential of their application in microassays, biochips (glycoarrays and lectin arrays) and biosensors [51-53].

Additional prospects in developing affine membrane technologies can be realized using membrane preparations of human LSSM, LS of human protein hormone (erythropoietin) and medical plant LS for study and constructing drug forms and new functional reagents [54, 55]. 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 combinations with antibiotics and physical factors of stress (radiation [ultraviolet and ultrasonic], light [biorhythm “Day-Night”], temperature, pH, oxygen, season changes [also biorhythmic], others); 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 phytooxidoreductases/ phyto[glycosyl]oxidases”, “Erythropoietin LS—Immune sandwich/ monoclonal antibodies/ synthetic GP imitating mucins and antigens”).

Membrane technologies of 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 massifes for further establishment of LSSM and other biologically and physiologically active components among protein mosaics. Bioluminescence (fluorescent technique in combination with chemiluminescent 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, 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. The choice of wished prognostic (LSSM-type—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 list of GC indicated in Table 1 is possible). The use of artificial polymeric GC with established chemical structures allow adequate and reliable results. Accordingly to the 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 protective systems (the use of GC interacting and regulating macrophages and macrophage-like lymphocytes through their systemic lectin receptors), antitumors actions (the use of antigenic GC together with LSSM); cytokine lectin cascades initiated or regulated with LSSM (as cytokines-like) inducing lymphocyte production of cytokine cascades (for example, tumor necrosis factors possessing coupled lectin properties) [23].

Conclusion

Results indicate that symbiotic lectins possess prospects to be applied as assistant coordinated metabolomic system; carriers for delivery and releasing GC, metabiotics (including simulators of cell surface patterns of opportunistic microorganisms), prebiotics, therapeutic agents, antigens; reserves for decoration elements that support stabile functioning landscapes of the human cell surfaces. Symbiotic lectins (interacting to synthetic GC-simulators) are important for screening, typing and selection of useful strains and their consortia supporting organs. The proposed anaerobic LSSM-containing preparations promise LSSM using as a soft adaptive multidirected network system supporting other protective human systems (also in conditions of the lack of local oxygen sources).

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