**3. Results and discussion**

### **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

**75**

**Table 2.**

*\**

*Metabolite Multiprobiotic Formulas for Microbial Health DOI: http://dx.doi.org/10.5772/intechopen.86449*

**Types of synergism Lectins\* Distant positions** 

aBL

cLL and aLL aLL and cLL

aLL and aBL

aBL and cBL

aBL and aLL aLL and aBL

aLL and GL cBL and GL

Between PL and itraconazole aBL c-Itraconazole, p-aBL

cLL cBL

aLL

Between identical PL disks aBL and

Between PL

Intra-genus (*Lactobacillus*)

Intra-genus (*Bifidobacterium*)

Intra-genus (*Bifidobacterium*)

Inter-genera (*Lactobacillus* and *Bifidobacterium*)

(GL)

Between PL and phytolectins Between PL and grass lectins

Between PL and antimycotics

Between PL and amphotericin B

Between PL and ketoconazole

Between PL and nystatin

Between GL and nystatin

Multiple synergism

Between BL and LL aBL,

*Lab. Pvt. Ltd.) was used. CFB, communicative fungal bodies.*

*Antimicrobial synergism of LSSM and antibiotics.*

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

**right)**

p-aBL, c-aBL (p-aBL—c-aBL)

From p-cLL to CR (p-cLL—p-aLL) From p-aLL to CR (p-aLL—p-cLL)

p-aLL and c-aBL (p-aLL—c-aBL)

(p-aLL—p-aBL)

From aLL to CR (p-MGBL—p-aLL) From p-cBL to CR (p-MGBL—p-cBL)

aBL p-Amphotericin B, c-aBL (p-Amphotericin B—c-aBL)

aBL p-Ketoconazole, c-aBL

GL From p-MGBL to CR

*Diffusion between disks placed on Sabouraud agar in Petri dishes (disk positions: p, peripheral; c, central; CR, central region, between p-disk and the center; PR, peripheral region, between p-disk and the border of agar). Disks included anionic (a) and cationic and (c) lactobacillar and bifidobacterial LSSM (aLL, cLL, aBL, cBL). MGBL as the mixture of the grass lectins from the mill of plants of medical significance: Potentilla album and Stellaria media. Lectins were used in subhemagglutinating doses (less 1 microgram/ml). Standard panel of disk antimycotics (HiMedia* 

(p-Itraconazole—p-aBL)

(p-Ketoconazole—c-aBL)

(p-MGBL—c-nystatin)

p-aBL + c-aBL + p-aLL (p-aBL—c-aBL; p-aLL—c-aBL)

In triangle of CFB landscape:

From p-cLL to CR (p-cLL—c-nystatin) From cBL to CR (p-cBL—c-Nystatin)

[PR aBL] and [CR aLL] (p-aBL—p-aLL) [PR aLL] and [CR aBL]2

**(directions from left to**

**CBF as targets, affinity, and significance**

*C. albicans* 991

*C. albicans* 515, 547 *C. albicans* 515, 547

*C. albicans* strains

*C. albicans* strains *S. aureus* strains

*C. albicans* 515 *C. albicans* 515

*C. albicans* strains *> C. tropicalis* strains

*C. albicans* strains

*C. albicans* strains

*C. albicans* 515, 547 *C. albicans* 515, 547

*C. albicans* 515

*C. albicans* strains (Not for *C. tropicalis*)

[PR aBL] and [CR aLL] *C. albicans* strains

\*

*Oral Health by Using Probiotic Products*

1–2 weeks, or 2–3 months (late events)] [18].

prebiotic, and/or communicative signal abilities and actions.

totic origin]) antipathogenic events as shown in **Table 2** [20].

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*),

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,

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 apop-

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

**74**

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


*\* Diffusion between disks placed on Sabouraud agar in Petri dishes (disk positions: p, peripheral; c, central; CR, central region, between p-disk and the center; PR, peripheral region, between p-disk and the border of agar). Disks included anionic (a) and cationic and (c) lactobacillar and bifidobacterial LSSM (aLL, cLL, aBL, cBL). MGBL as the mixture of the grass lectins from the mill of plants of medical significance: Potentilla album and Stellaria media. Lectins were used in subhemagglutinating doses (less 1 microgram/ml). Standard panel of disk antimycotics (HiMedia Lab. Pvt. Ltd.) was used. CFB, communicative fungal bodies.*

#### **Table 2.**

*Antimicrobial synergism of LSSM and antibiotics.*

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 [1–4, 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

**77**

*Metabolite Multiprobiotic Formulas for Microbial Health DOI: http://dx.doi.org/10.5772/intechopen.86449*

1.1 Acidic proteins pI 4–5

1.2 Cationic proteins pI 7–8

1.3 Oxidase-reductase systems pI 5–5.5

1.5 Level of aggregation upon storing concentrates

1.6 Ability in membrane ultrafiltration

2 Status of amino acids

2.1 Tyr (sites for serine proteinases, fluorophores)

2.2 Phe (sites for serine proteinases, fluorophores)

2.3 Fluorophores (Trp, Tyr) (excitation at 254 nm)

2.3.1 Fluorophores (excitation at 365 nm)

2.4 Gly (hydrophobic, anti-adhesion action)

2.5 Leu (hydrophobic, site for peptidases)

2.0 Production of amino acids

1.4 Hydrolase systems

1 Status of proteins 1.0 Content of partially hydrolyzed protein

**No. Parameters of supernatants, ranging MP and its strains, proposals (P) Code ranging MP** 

K3III24 > MP > 100 ash > NK1 P: K3III24 as the main source of active or signal oligopeptides

NK1 > 100 ash > K3III24 > MP P: NK1 as the main source of basis cytoagglutinating and adhesive agents coupled to a spectrum of biological activities

NK1 > K3III24 > 100 ash > MP P: Strains and their combinations as sources of lectins and lectin-like agents coupled to exopolymeric substances of compounds

> MP > 100 ash > K3III24 > NK1[absence] P: NK1 without extracellular major oxidoreductase systems

MP > K3III24 > 100 ash > NK1 P: NK1 without extracellular major hydrolases (caseinases, peptidases, others)

K3III24 > 100 ash > NK1 > MP[no aggregation] P: Irreversibility for K3III24 (similar to red cell biofilm storing)

MP > NK1 > 100 ash > K3III24 P: Technological advantage of MP as mixture of strains (mixture of strains is needed)

NK1 > K3III24 > MP > 100 ash P: Low antagonism between certain strains within MP can be under consideration upon strain choice

K3III24 > 100 ash > MP > NK1 P: Tyr can serve as criterion of utilization of fluorophores

NK1 > MP > K3III24 > 100 ash P: Support of point 2, revealing mechanism for point 2

100 ash > K3III24 > NK1 > MP P: Dominated contribution of Tyr and their derivatives

100 ash > K3III24 > NK1 > > MP P: Degradation/ utilization in MP (fluorophores cannot be used as targets)

NK1 > MP > K3III24 > 100 ash P: Gly as natural additive to improve signal activities and communications

> (Parameter is slightly dependent on strain origin) K3III24 > 100 ash > MP, NK1

> > fatty acids) K3III24 > 100 ash > MP > NK1

2.6 Ile (hydrophobic, participation in synthesis of biologically active volatile

**and its strains\***

3. 4. 2. 1.

1. 2. 3. 4.

1. 3. 2. 4.

4. 2. 3. 1.

4. 3. 2. 1.

3. 2. 1. 4.

4. 1. 2. 3.

1. 3. 4. 2.

3. 2. 4. 1.

1. 4. 3. 2.

2. 3. 1. 4.

2 3 1 4

1. 4. 3. 2.

3. 2. ¼\*\*. 4/1.

3. 2. 4. 1.

*Metabolite Multiprobiotic Formulas for Microbial Health DOI: http://dx.doi.org/10.5772/intechopen.86449*

*Oral Health by Using Probiotic Products*

grass phytolectin mixture.

reveal special interest [24].

presented in **Table 3**.

to right] position in code).

bifidobacterial cationic LS synergistically increased anti-*Candida* action of the

significance, etc.) as it can be expected using symbiotics [21].

**new multistrain pro- and symbiotics**

events in organism as the normal but sensor natural reactions [22].

by LS-containing preparations in other (nondependent) methods.

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

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

**3.2 The choice of strains and their consortia possessing potential for constructing** 

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 [1–4, 23]. Among GC investigated, glycans of mucin type (mucosal glycans)

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

The data which were useful for constructing any multiprobiotics (*Acilact*-like) is

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

*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

**76**



**79**

position in code.

*\*\*Alternative positions.*

*\**

**Table 3.**

*probiotic).*

(as in cases of amino acids [25]).

their usage as standard models).

bifidobacterial strains) is presented in **Table 4**.

*Metabolite Multiprobiotic Formulas for Microbial Health DOI: http://dx.doi.org/10.5772/intechopen.86449*

*4, multiprobiotic (MP) on example of variants of Acilact.*

4.2 Pigment products [optical density at 420 nm]

(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

**No. Parameters of supernatants, ranging MP and its strains, proposals (P) Code ranging MP** 

K3III24 > NK1 > 100 ash > MP P: K3III24 as the main potential source of neoglycoconjugates of nonenzymatical origin; MP as maximally decolorized product needed cofunctioning strain mixture; partial inverse correlation to point 1.3

*1, NK1 (Lactobacillus helveticus NK1); 2, 100 ash (L. helveticus 100 ash); 3, K3III24 (L. casei K3III24);* 

*Strain code ranging for multiprobiotic construction on example of Acilact (the Lactobacillus multistrain* 

4.3 Antimicrobial activity (toward a panel of diagnostic mainly Gram-negative bacteria) MP > NK1 > 100 ash > K3III24 P: Support point 3; potential of K3III24 to other targets (against Grampositive bacteria and fungi)

**and its strains\***

3. 1. 2. 4.

4. 1. 2. 3.

*The third step*: The final formulas (formulas of category C) include combinations of formulas A and B. Multifunctionality of parameters analyzed can be extended

Extended approach for constructing more adaptive mixtures of lactobacillar and bifidobacterial MP (on the basis of *Acilact* extended by accounting industrial

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

**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

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

probiotic lactobacilli and bifidobacteria of the human gut origin.

level of compatibility of the strains NK1 and MS42.


#### **Table 3.**

*Oral Health by Using Probiotic Products*

**No. Parameters of supernatants, ranging MP and its strains, proposals (P) Code ranging MP** 

MP > K3III24, 100 ash > NK1 P: Antagonism between strains results in partial cell wall degradation in MP

> MP > K3III24 > 100 ash > NK1 P: Support of point 1.4

NK1 > 100 ash > MP > K3III24 P: Thr as criterion for evaluation of metabolism of cluster square positions for recognition in proteins (in comparison to point 2.8)

> proteinases, participation in *Maillard* reaction) MP > 100 ash > K3III24 > NK1 P: Support of point 1.4

fatty acids) K3III24 > 100 ash > NK1 > MP P: Mostly important criterion concerning volatile fatty acids producing

NK1 > MP, K3III24 > 100 ash

MP, 100 ash > NK1 > K3III24 P: Partial support of point 1.4

cations, activation of oxidases and heme) MP > NK1 > 100 ash > K3III24 P: Potential for metal chelate affinity chromatography and immobilization (for microassays)

> during pigments forming in *Maillard* reaction) MP > NK1 > K3III24 > 100 ash P: One of the way of decolorization of MP

> > NK1 > MP > K3III24 > 100 ash

K3III24 > NK1 > MP > 100 ash[not observed]

100 ash > MP > K3III24 > NK1[traces]

NK1 > MP > > 100 ash > K3III24 P: NK1 as the main source of movable permeable (detergent like) complex effectors; support point 4.3

K3III24 > MP > 100 ash > NK1

100 ash > NK1 > MP > > K3III24 [absence] P: K3III24 needs combination to any other strain to increase cultural components (>27 kD) to be emulsified

2.11 Val (hydrophobic, participation in synthesis of biologically active volatile

2.7 Ala (partially from peptidoglycans, site for exopeptidases)

2.8 Ser (sites for hydrolase splitting and O-glycosylation)

2.10 Lys (from cationic poly/oligopeptides, site for serine

2.12 Glu/Gln (also as sites for amidases)

2.13 Asp/Asn (sites for amidases and N-glycosylation)

2.15 Arg (from cationic poly[oligo]peptides, destruction

2.17 Cys2 disulfide bonds (oxidation of SH-groups into Cys2)

2.18 Pro (Pro-bends in regular structures of proteins)

3 Status of biosurfactants 3.0 Associated biosurfactants in complexes >27 kD

3.1 Biosurfactants active against mineral oil

4 Other parameters 4.1 Emulsifiers

2.16 Met (antioxidant)

2.14 His (participation in auto-oxidation of protein, high affinity to metal

2.9 Thr (site for O-glycosylation)

**and its strains\***

4. 2/3. 3/2. 1.

4. 3. 2. 1.

1. 2. 4. 3.

4. 2. 3. 1.

3. 2. 1. 4.

1. 4. 3. 2.

4. 2. 1. 3.

4. 1. 2. 3.

4. 1. 3. 2.

1. 4. 3. 2.

3. 1. 4. 2.

2. 4. 3. 1.

1. 4. 2. 3.

3. 4. 2. 1.

2. 1. 4. 3.

**78**

*Strain code ranging for multiprobiotic construction on example of Acilact (the Lactobacillus multistrain probiotic).*

(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.


*1, NK1 (L. helveticus NK1); 2, 100 ash (L. helveticus 100 ash); 3, K3III24 (L. casei K3III24); 4, MP (Acilact); 5, MS42 (B. adolescentis МS42); 6, bif1 (B. bifidum No. 1); 7, gall (B. gallinarum GB); C, concentrate (>27 kD) of cultural fluid supernatant; IEF, isoelectrofocusing; PAG, polyacrylamide gel; P, prognostic proposals; \* Alternative position. Blocks of lactobacillar (boldface) and bifidobacterial (italic font) strains are shown.*

#### **Table 4.**

*Strain code ranging for multiprobiotic construction on example of new multiprobiotics including bifidobacteria and lactobacilli.*

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, LSSMdependent 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.

**81**

*Metabolite Multiprobiotic Formulas for Microbial Health DOI: http://dx.doi.org/10.5772/intechopen.86449*

**protective systems**

infrastructures.

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

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

**3.3 Anaerobic synergistic preparations containing LSSM for support of human** 

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 (crosslinking and inactivation of therapeutic proteins, etc.) in respect to surrounding

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 probioticdepending 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

properties of future combinative products can be predicted and verified.

agents and organizers of lectin-coupled reactions and activities [26, 27].

*Oral Health by Using Probiotic Products*

**80**

*\**

**Table 4.**

*and lactobacilli.*

achieved.

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, LSSMdependent synergism (which can be directed and predicted using extended panel of GC for LSSM selection and choice) of new taxonomically mixed symbiotics can be

*Strain code ranging for multiprobiotic construction on example of new multiprobiotics including bifidobacteria* 

**No. Parameters of concentrate (C), their ranging, proposals (P) Code** 

(against any type of crystal forming during IEF-PAG): gall > bif1 > MS42 > NK1 > MP > K3III24, 100 аsh P: bifidobacterial C for stabilization of K3III24 and 100 аsh

(in conditions of 7М urea, 5% saccharose, 8°С, night, IEF in slab of PAG): рI 4–6: 3III24, 100 аsh > MP > NK1 > MS > bif1 > gall (not) pI 6–8: K3III24, 100 аsh > MP > NK1 > gall>bif1 > MS42 P: Potential approach to micro- and nanoassembling effectors

pI 4–6: NK1 > MS42 > K3III24 > gall> 100 аsh > bif1 > MP pI 6–8: NK1 > gall > K3III24 > MS42 > 100 аsh > MP > bif1 P: Donors of cationic bacteriocin-like associates with exopolymeric compounds

polystyrene (number of drops): gall > MS42 > NK1 > bif1 > MP > K3III24 = 100 аsh(not) P: The size and numbers of drops indicate level of emulsification of C compared to original supernatant

> bif1 > gall > K3III24 > MP > 100аsh > MS42 > NK1 P: C "NK1 + gall" and "NK1 + bif1" as synergistic antimicrobials

> pI 4–5.5: gall > MP > NK1 > MS42 > bif1 > K3III24, 100 аsh pI 5.5–8: MS42 > bif1 > gall > NK1 > MP > 100 аsh, K3III24 P: C for delivery into intestinal and urogenital mucosal cavities

pI 4–5.5: 100 аsh, K3III24 > NK1 > MS42 > gall > MP > bif1 pI 5.5–8: gall > bif1 > MS42 > NK1 > MP > 100 аsh, K3III24 P: Potential against eukaryotic (yeast and yeast-like), prokaryotic (staphylococci), and HIV/HIV-related infections; delivery into cell and cell organelles

*1, NK1 (L. helveticus NK1); 2, 100 ash (L. helveticus 100 ash); 3, K3III24 (L. casei K3III24); 4, MP (Acilact); 5, MS42 (B. adolescentis МS42); 6, bif1 (B. bifidum No. 1); 7, gall (B. gallinarum GB); C, concentrate (>27 kD) of cultural fluid supernatant; IEF, isoelectrofocusing; PAG, polyacrylamide gel; P, prognostic proposals;*

*Alternative position. Blocks of lactobacillar (boldface) and bifidobacterial (italic font) strains are shown.*

1 Antifreeze components >27 kD, pI 4–8

3 Complex protein C > 27 kD:

5 Associated biosurfactants:

6 LSSM as mucin-binding:

7 LSSM as mannan-binding:

2 Formation of organic crystals in the presence of components >27 kD

4 Adhesins as colorless transparent not water-soluble drops on

**ranging strains and MP**

*7. 6. 5.* **1. 4. 3/2\*. 2/3.**

**2/3. 3/2. 4. 1.** *5. 6. 7.* **2/3. 3/2. 4. 1.** *7. 6. 5.*

1. 5. 3. 7. 2. 6. 4. 1. 7. 3. 5. 2. 4. 6.

7. 5. 1. 6. 4. 2/3. 3/2.

6. 7. 3. 4. 2. 5. 1.

7. 4. 1. 5. 6. 2/3. 3/2. *5. 6. 7.* **1. 4. 2/3. 3/2.**

2/3. 3/2. 1. 5. 7. 4. 6. *7. 6. 5.* **1. 4. 2/3. 3/2.**

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].
