Promising Probiotic Food Supplement Based on Combination of Yeast Beta-Glucan, Bioselenium and Lactoferrin for Animal Health

A high producer β-glucans strain of Saccharomyces cerevisiae was selected from our culture collection to evaluate its ability to assimilate selenium by growing it in YPD medium supplemented with inorganic sodium selenite. This strain was also used as a host to express the murine lactoferrin gene under the control of the promoter of the S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. The yeast strain was cultivated to obtain biomass made up to of high β-glucans levels, the incorporated selenium and recombinant murine lactoferrin. This biomass was harvested and dried to obtain probiotic supplements T1 and T2. The amount of bioselenium and murine lactoferrin were determined in the resulting product and used to feed BALB/c mice for 30 days. Several parameters served to monitor evaluate the immune stimulatory effect and the physiological state of the animals during the test. Measurements were carried out at 0, 15th and 30th days. The results showed the composite supplement improves the physiological and immunological conditions of the tested animals compared to the control group. The results obtained pave the way for developing food supplements with similar characteristics for economically important species.


Introduction
Probiotics are live microorganisms that confer health benefits to the host when administrated in adequate amounts [1,2]. Yeasts are eukaryotic unicellular microorganisms belong to the fungus kingdom [3]. Saccharomyces cerevisiae metabolizes carbohydrates to carbon dioxide and alcohols in a process known as fermentation which is extensively known and used by humans in food and alcoholic beverages production since the beginning of human civilization [4]. S. cerevisiae has also been used as a model organism for biological research and biotechnological processes. In general, there are a great number of yeast species, widely distributed in the nature and its ecological role has been matter of extensive studies.
Yeasts, particularly S. cerevisiae and S. boulardii, are used as a nutritional supplement for direct consumption in different forms.
Yeast strains are used as probiotic fungi in order to improve health and physiological conditions in many living organisms including humans as well as other vertebrates [5]. They could act in many Stimulation of the innate immune system and the antiinflammatory, anti-stress and hepatoprotective processes, among other benefits for the better physiological functioning of the entire organism [6][7][8].
In addition yeasts are a rich source of numerous important nutriments as proteins, carbohydrates, vitamins and minerals.
Many of these probiotic characteristics are due to the components of the yeast cell wall. The yeast cell wall has a complex structure composed by lipids, glycoproteins and covalently interconnected polysaccharides that comprise water-and alkali soluble fractions of alpha mannans, mannoproteins and β-glucans [9]. In general, β-glucans are one of the most important biopolymers in living organisms, widely presented in cell walls of fungi and plant cells [10][11][12]. Three types of β-glucans are present in the cell walls of higher plants: (i) β(1,3)-glucan, (ii) cellulose, and (iii) xyloglucan [11]. In yeast, three different β-glucans types are present and they Mannoproteins are glycoproteins with attached mannan residues, constitute between 20 and 50% of the total proteins of the cell wall, while the greatest contribution to this proportion lies in β(1-6)-glucans (65-90 % of total β-glucans fraction) of the total weight [13]. As previously mentioned, β-glucans has an important healthy effect on vertebrates especially on mammalian organisms at improving their innate and acquired immunity, and overall organic response to environmental changes and adverse effects triggered [14][15][16][17]. The molecular structure and composition of β-glucans as well as their derived physicochemical characteristics are determinant on physiological and immune functions of vertebrates. In yeast and fungi, β glucans are mainly present β (1-3)-glucans backbone bearing branches composed by β (1-6)-linked side groups, which are very important to modulate in vertebrates both immune responses, innate and adaptive [18,19]. Other features determining their biological activity are molecular mass, solubility and type of aggregation, their spatial folding and the resulting tertiary structure as well as the relative charge they carry [8,18,20].
Selenium is an essential micronutrient and well antioxidant naturally found in soil, water and in some foods. Selenium compounds, although in trace quantities, are indispensable for proper physiological functioning of vertebrate organisms.
The beneficial effects of selenium can be linked mainly with the selenoproteins and their relevant role in the organisms such as, endocrine, muscular, cardiovascular, nervous, reproductive, antioxidant and immune functions [19,20]. Clinical evidence indicates that consumption of Se-supplemented diet can increase phagocytosis and the activity of natural killer cells, in sheep and humans respectively [21][22][23][24][25][26]. In vertebrates, the mentioned biological effects of this microelement are mostly attributed to the insertion of selenium in a group of proteins, named selenoproteins (SeP), where the selenocysteine as take as a truly 21st amino acid residue. To the present, 25 genes coding for SeP have been identified.
In some cases, the SeP are enzymes with catalytic functions, responsible for biological reactions of the reduction-oxidation type, antioxidant defense, DNA repair systems, epigenetic processes, and thyroid hormone metabolism [26]. Those are the cases of three thioredoxin reductases (TRs), glutathione peroxidases (GPx), methionine sulfoxide reductase (MsrB1), and 3 thyroid hormone deiodases (DIs) [27].
Lactotransferrin also known as Lactoferrin (LF), is a 78 kDa iron-binging glycoprotein which belongs to the Transferrin Protein family Talalactoferrin and LTF [28]. These proteins are able to transport proteins which can bind two Fe 3+ ions in association with the binding of an anion, usually bicarbonate. LF is commonly found in colostrum and milk of vertebrates but also, although in smaller quantities, in tears, nasal secretions, saliva and intestinal fluids [29][30][31]. LF has antibacterial and antiviral actions retarding their proliferation and even killing them due its iron-binding capacity that results in microbial membrane destabilization [30].This protein is also an important modulator of inflammatory processes and immune response, probably by cytokine and chemokine production as well as interaction with oxidative/antioxidative processes, regulating the production of intracellular levels Reactive

Probiotic Preparation
The normal diet consists in NUTRICUBOS-LabChows

Blood Biochemistry
Blood samples were collected through the ophthalmic plexus

Immunological Status
The immunological status of animals was followed up 0, 15 th and 30 th days of the probiotic assay by using 5 animals. The levels of lymphocytes, neutrophils and monocytes, were determined as well as the production of some cytokines were determined. Leucocytes numbers were established by using hemocytometer [38]; remaining parameters were evaluated by commercial kits.

Isolation of blood monocytes
Blood monocytes were extracted from two mL of peripheral blood from the wing vein of five selected animals from each experimental groups using standard procedure [39]. The total number of mononuclear-containing cells was counted by a standard hemocytometer and cell viability was determined.

Isolation of Bone Marrow derived macrophages
The five animals selected for monocytes isolation, were used to collect bone marrow cells according the standard described methodology [40,41].

Phagocytosis Assay
Peripheral blood monocytes and bone marrow derived macrophages from animals exposed to normal and probiotic supplemented feed during 30 days were subjected to the phagocytosis assay analysis. The assay was carried out by using

Data Analysis
Data were analyzed using one-way ANOVA followed by Dunnett's or Fisher's protected least significant difference multiple comparison testing in SPSS13.0 (SPSS, Chicago, IL, USA).
When necessary, data were transformed for normalization and to reduce heterogeneity of variance p-values <0.05 were statistically considered significant.

Results/Observations
The Saccharomyces cerevisiae strain LX36 showed higher β-glucan content was selected from our culture collection to and probiotic supplement 2 (T2). The animals were monitored by determining blood, hepatic and renal biochemical markers related with specific organic functions, oxidative status and immune system. functions. We observe that mice in both, control and test groups are healthy and in a good physiological condition (Table 1).   Table   2). When compared the production of proinflammatory (IFNγ, IL2, IL12) with the production of antiinflammatory (IL 10, IL4) cytokines the increments observed at 15 th and 30 th days reflex a conserved balance between the production of both types of cytokines.

Discussion
Living organisms are in permanent interaction with internal and environmental conditions and the ability to adapt the organic processes to new reality is crucial for survival. Those processes in animals frequently resulted in oxidative stress, metabolic dysfunction, loss of essential nutriments, decreased availability to maintain homeostasis of essential nutrients, energetic balance, retain electrolytes, and overall physiological dysregulation leading to poor health condition [43]. diseases [18,57,58]. Selenium was considered toxic element since its discovery in 1857 until 1957 when its beneficial effects started to be recognized and studied for the first time [26]. The beneficial effects of Se are attributed at its in incorporation, instead of S, in the cysteine residue of selenoproteins [59,60].
The way of action of selenium incorporated in form of selenoproteins (SeP) over the different types of immune response is not completely elucidated and is matter of many researches, more exhaustive studies could be conducted but there are sufficient evidences for its practical use in human and livestock healthcare.
In our experiments Se-supplemented diet increases phagocytosis of monocytes and macrophage, improving antioxidative and immunological responses [26,[61][62][63]. As in the case of β-glucans, the adequate selenium intake stimulates the innate immune systems.
This process includes the activation of macrophages through the keep stable the homeostasis in tissues where it's present. These effects were fund also when combinational supplements T1 and T2 were tested. It has been reported LF stimulates the production of proinflammatory cytokines like TNF-α, Il-1β, IL-6 and IL-18 and enhancing phagocytosis [30,34,71].
We obtained that production of proinflammatory (IFNγ, IL2, IL12) with the production of antiinflammatory (IL 10, IL4) cytokines the increments observed at 15 th and 30 th days reflex a conserved balance between the production of both types of cytokines as a result combinational effect. LF also interacts with membrane glycosaminoglycan, blocking the viral entry to host cells and/or inferring subsequent viral transmission and probably enhancing the activity of β-glucans [30,34,72,73]. It has been reported that ingestion of probiotic supplements brings the described beneficial effects on vertebrate organisms, but synergic combinational effect of β-glucans has not been evaluated [17,25,32,61]. We obtained the similar results with both supplements, the fact that same positive and beneficial was reached using 30% less amount of yeast β-glucans, organic selenium and lactoferrin indicate synergic effect of all three components. The reported studies demonstrate that they share some common mechanisms of action but at the same time there are some particularities allowing overall synergic activity over the different physiological functions of the vertebrates [8,17,50,57,61].

Concluding Remarks
These results support the idea of systemic positive effect of dietary supplementation with probiotic in humans and animal.
In animal production during handling operations that implies the manipulation of animals, change of feeding, transfers to another location, transport to a new region, etc., and changes in animal life cycle (birth, breastfeeding or breeding, weaning, calving, etc.).
These drastic changes in living conditions and stages of the life cycle can have a dramatic impact on the productivity of the livestock by weight loss, slow weight gain, organ physiological malfunction and diminish of immunological defenses. We seek to minimize its consequences with dietary supplements as preventive treatment with h use of probiotic with several key compounds with the ability to overall activate multiple physiological mechanisms with overall beneficial effect to the organic vital functions and to the animal health.