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2. University of Louisville, Department of Pathology, Louisville, KY, USA.
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Background: Yeast was the first microorganism used as a source of protein and it still is the main source of unicellular protein in human nutrition. Numerous yeast products and yeast derivatives are produced and used in animal feed, pet food, and human food around the world. Although the whole yeast cell and the yeast cell wall have β-1,3-1,6-glucan in their chemical composition, their immunological effects in vivo can be quite different of those of the yeast purified β-1,3-1,6-glucan.
Methods: In order to study these different in vivo effects, we used a traditional package for evaluation of commercial glucans to compare the whole yeast cell, the yeast autolysate, the yeast cell wall, and the yeast purified β-1,3-1,6-glucan produced from the same original yeast raw material. We evaluated the effects of these compounds on phagocytosis, IL-2 secretion, and antibody production.
Results: We found that although the whole yeast cell and the yeast cell wall have β-1,3-1,6-glucan in their chemical composition, their immunological effects in vivo can be quite different of those of the yeast purified β-1,3-1,6-glucan.All activities, ie., phagocytosis, IL-2 production and antibody production were the highest in the group supplemented with purified glucan.
Conclusions: Our results suggest that the yeast derivatives have more limited immunological effects, maybe as a result of local intestinal immune reactions. The purified glucan showed stronger immunological activities.
Keywords: Autolysis, biologically active polysaccharides, glucans, yeast cell wall and yeast derivatives
Historically, mankind and yeast developed a relationship that led to the discovery of fermented beverages. Beer is one of the oldest fermented drinks produced by humans. Some of the earliest evidence comes around10 000 years ago [1]. Beer was initially used as a medicine, due the beneficial properties of herbal extracts (hops), live yeast, and fermented cereal extracts.
Whole yeast cell
Yeast, a unicellular eukaryotes and a typical fungus, fundamentally has the same subcellular structure as higher animal and plant cells (Figure 1). It has been used as food, feed, and medicine. For food and feed applications, it is often used as asource of protein and vitamin B complex, except for vitamin B12. Baker’s yeast obtained a Food and Drug Administration GRAS (Generally Regarded/Recognized as Safe) status [2]. The macromolecular constituents of yeasts include proteins, glycoproteins, polysaccharides, polyphosphates, lipids, and nucleic acids [3]. The nutrients present in whole yeast cells are within the cell, which must be lysed to release them before the absorption by the animal. This lysis can be supported by proteases and glucanases of the microorganisms of the gastrointestinal tract [4]. Therefore, due the limitations in digestibility of these materials, they have mainly been used to foradult animals and ruminants.
Figure 1 : Scanning electron microscopy of whole yeast cell.
Autolyzed yeast
Yeast can be submitted to a process similar to the rotting of fruits. Autolysis is the breakdown of cell constituents by the action of yeast’s endogenous enzymes (lipases, proteases, nucleases, etc.). It occurs naturally when yeast enters the death phase or it can be induced by high temperature, salts, or organic solvents, resulting in a loss of membranous function, degradation of the macromolecules, and the release of degradation products to the extracellular environment [5]. Typical autolyzed yeast has 35-45% of free amino acids, 10-15% of di-, tri-, and tetrapeptides with less than 600 Da, 40-45% of oligopeptides ranging from 2000 to 3000 Da, and 2-5% of polypeptides ranging from 3000 to 100000 Da [6]. Yeast oligopeptides below 10000 Da can reduce body weight and abdominal fat accumulation in obese adult humans [7].
During autolysis, approximately 85-90% of RNA and 25-40% ofDNA are degraded tooligonucleotides, nucleotides, nucleosides, and nitrogenous bases [5]. Nucleotides can improve immune response, liver and intestinal function, composition of microbiota, and animal performance [4]. Autolyzed yeast supplementation can improve performance and modulate the intestinal immune system and microbiota of chickens [8]. Inclusion of yeast extract in the diet of piglets allows a partial replacement for blood plasma [9]. An in vitro study has shown that yeast extract stimulates glucose metabolism and inhibits lipolysis in rat adipocytes [10]. The autolysis process can improve digestibility, palatability, and functionality of yeast material.
Yeast cell wall (MOS)
After autolysis, yeast cream can be centrifuged in order to re-move the yeast extract and isolate the yeast cell wall (Figure 2). The mannan oligosaccharides, part of the yeast cell wall, belong to a group of beneficial compounds for intestinal health called prebiotics. They have stimulatory effects for some parameters of local immune function, the property of blocking the adhesion of pathogenic bacteria to the epithelial cells of the intestine mucosa, and direct stimulating effects for beneficial microbial populations that are able to use them as a substratefor growing. Together, these competitive exclusion factors can be favorable to beneficial microbial populations and explain their typical effects on increasing butyrate concentrations and on decreasing concentrations of putrefaction compounds in the gut of dogs [11].
Figure 2 : Transmission electron microscopy (8.400x) of yeast cell wall.
The use of prebiotics might be beneficial to puppies, dogs under stress, and geriatric dogs for conditions that may predispose for an unbalance in the immune system or microbial dysbiosis in the gut. MOS tend to enhance microbial populations and modulate immune function [12].
Purified yeast β-glucan
Finally, the yeast cell wall can be submitted to further purification process in order to remove the mannan-protein outer darker layer to form a purified β-glucan (Figure 3). Various extraction methods for β-glucan exist, generating various products with distinct, but not always identical, immunomodulatory effects [13-15]. β-Glucans, the main part of the purified yeast cell wall,are macromolecules that present β conformation linkages in different degrees of polymerization, where the β-1,3 glucose linear chain core has abundant lateral branches initiated with β-1,6 linkages. The term glucan, in amore general meaning, represents a group of biologically active polysaccharides that can alsooriginate from other sources, including the cell wall of filamentous fungus, seaweed, oat, and barley [16]. Their cell’s functions include protection, energy reserve, and osmotic stability [17]. They are recognized as actives GRAS in a process submitted to the FDA (Government revenue number [GRN]: 000239), without long-term chronic toxicity, according to EFSA’s Additives Safety [18].
Figure 3 : Transmission electron microscopy (8.400x) of yeast β-glucan.
In its purified form the β-1,3;1,6-glucan can be recognized and captured in the gut by cooperation of specialized cells (M cells and macrophages), with subsequent unrolling of a complex biodynamics process, generating an important systemic effect in addition of the microbial immunomodulatory effect. The biological effect of β-glucans depends on the route of application, as well as on other characteristics such as origin, solubility, size of the molecules, conformation and purity [16]. However, direct relation between structure and function has never been fully established [19].
In the present study, we fed mice with four yeast actives for 28 days: whole yeast cell, yeast autolysate, yeast cell wall and purified yeast β-glucan; all were produced from the same original yeast raw material. We measured the serum IL-2 production as an indicator of innate immune response, the macrophages, neutrophils, and monocytes phagocytosis index as indicators of cellular immune response and antibody production against ovalbumin as an indicator of adaptive immune response. This is the first report of this nature for comparison of immunological effects of yeast and yeast derivatives.
Experimental animals and welfare statement
The use of animals was approved by the University of Louisville IACUC committee (#12029). All mice (40 total) used were 6-8–weeks old, both sexes,and of the BALB/c strain from Jackson Laboratory (Bar Harbor, ME, USA). All animals used in our study were grown in conventional conditions with monitored light, temperature, and air. All animal work was done according to the University of Louisville IACUC protocol. Animals were sacrificed by CO2 asphyxiation.
Experimental design
After acclimation, animals were fed with the same standard rodent diet 50001 (Purina). The test actives were disperse in phosphate buffered saline (PBS) and given daily by gavage; control group received only PBS, Group 1 received 762μg of whole yeast cell with 18.38% of β-glucan, Group 2 received 902μg of yeast autolysate with 15.52% of β-glucan, Group 3 received 492μg of yeast cell wall with 28.47% of β-glucan, and Group 4 received 200μg of purified yeast β-glucan with 70.01% of β-glucan. Supplementations were designed to beiso-glucan, based on the differences for the concentrations of glucan in the actives.All actives used in this study were produced from the same original raw material of Saccharomyces cerevisiae by the Department of Research and Development of Biorigin Company (São Paulo, Brazil). After feeding for 14 days, blood was drawn by puncture of the jugular vein.
Phagocytosis
The technique employing phagocytosis of synthetic polymeric microspheres was described previously [20]. Briefly: peripheral blood cells or isolated peritoneal cells were incubated in vitrowith 0.05ml of 2-hydroxyethyl methacrylate particles (HEMA; 5×108/ml). The test tubes were incubated at 37°C for 60min, with intermittent shaking. Smears were stained with Wright stain. The cells with three or more HEMA particles were considered positive, and cell types were distinguished based on their morphology. All experiments were performed in triplicate. At least 300 cells in 60 high-power fields were examined in each experiment.
Innate immune response–serum production of interleukin 2 (IL-2)
Purified spleen cells (2x106/ml in RPMI 1640 medium with 5% FCS) from mice injected with tested samples were added into wells of a 24-well tissue culture plate. After addition of 1mg of Concanavalin A (ConA; positive control), cells were incubated for 48hrs in a humidified incubator (37oC, 5% CO2). At the endpoint of incubation, supernatants were collected, filtered through 0.45mm filters and tested for the presence of IL-2 using a Quantikine mouse IL-2 kit (R&D Systems, Minneapolis, MN). The optical density was determined by using a STL ELISA reader (Tecan U.S., Research Triangle Park, NC) at 492nm, and the amount of IL-2 was calculated from the standard curves included in the kit.
Adaptive immune response – antibody formation
The formation of antibodies was evaluated using ovalbumin as an antigen and ELISA assay [14]. Animals were subcutaneously injected twice (two weeks apart) with albumin (100μg per mice) and the serum was collected 7 days after the last injection. The Freund adjuvant was used together with the antigen in a positive control group. Total amount of specific Ig was measured using appropriate anti-mouse Ig-AP antibodies. The optical density was determined using a STL ELISA reader (Tecan U.S.) at 492nm.
Electron microscopy
Electron microscopy was performed by Electronic Microscopy Center (Botucatu Biosciences Institute, Botucatu, Brazil) following previously described specific methodology for yeast [21].
Statistical Analysis
All parameters were validated for normality (Shapiro–Wilk test) and homogeneity of variances (Levene test). Comparisons between treatments were made using one-way ANOVA and Tukey post hoc test or Kruskal–Wallis test when appropriate (SIGMA PLOT 11.0, Systat Software, San Jose, CA, USA). The minimum significance level was set at P≤0.05. The tables contain the following data: mean, median, standard deviation, standard error, size of the sample, total, minimum and maximum values of the sample and P-value. Different letters indicate statistically significant differences between groups. Data figures were expressed as the mean±standard error of the mean.
Most of natural immunomodulators affect nonspecific immunity. Therefore, we started our study by measuring the effects on phagocytosis by peripheral blood cells and peritoneal macrophages. We used a well-established model of synthetic microspheres based on 2-hydroxymethacrylate, which offers a low nonspecific adhesion to the membrane (and fewer false positives) [20]. This technique has been routinely used in the evaluation of various glucans, advantageously allowing better comparison with published data [21]. Our results show that all groups significantly stimulated phagocytic activity of peritoneal macrophages, with the Group 4 (glucan) showing the strongest activity (Table 1).
Table 1 : Effect of glucans on phagocytosis.
Our experiments were focused on the use of tested compounds as an adjuvant in an experimental model of ovalbumin immunized mice. Animals were injected twice (two weeks days apart) with 0.1mg of ovalbumin and the serum was collected 7 days after last injection. Experimental groups were getting daily doses of tested material. Freund adjuvant was used as a positive control. The results (Table 2) showed that all samples increased the antibody production, but only glucanshowed stimulation closer to positive control with Freund adjuvant. Next, we focused our attention on effects of tested materials on production of IL-2. Under normal conditions, the level of IL-2 in mouse serum is very low, therefore all samples caused significant stimulation of IL-2 synthesis and secretion. Like in case of phagocytosis, Group 4 showed the highest effects (Table 3) and the level of IL-2 secretion was close to the positive control (ConA) and significantly higher than that of the remaining samples.
Table 2 : Effect of glucans on antibodies.
Table 3 : Effect of glucan on IL-2 production.
Modern lifestyle (air pollution, dietary changes, use of antibiotics, etc.) appears to decrease a desirable exposureto microbial antigens, opportunistic pathogens and nonpathogens. This can lead to an inappropriate stimulus of macrophages and dendritic cells and an increase the severity and prevalence of chronic diseases, like allergies and autoimmune diseases in pets and humankind, due an incorrect modulation of the immune system [22]. Recently, evidence has indicated that obesity and type 2 diabetes are characterized by chronic inflammatory processes. In adipose tissues, especially visceral tissues, this type of inflammation contributes to the onset of metabolic disturbances and increased insulin resistance [23].
In this context, purified yeast β-glucan could be a soughtafter option to recover the appropriate stimulus of macrophages and dendritic cells. With over 30,000 published studies, it is the most studied immunomodulator. Beneficial effects of β-glucans on animal and human health have been widely described in the literature and include immunomodulatory, prebiotic, adsorptive to zearalenone, anti-infective, anti-inflammatory, antitumor, antidiabetic, antimutagenic, anti-allergic, anti-enteritis, regenerative, antithrombogenic, anticoagulant, antioxidant, lipid-lowering, and radioprotective [15-17, 24-28]. They have also been widely used in animal feed to improve the growth performance of farm animals [29-30]. Finally, they have been used in petfood to reduce clinical signs of canine osteoarthritis and atopy, to prevent alveolar bone loss in feline periodontal disease, to modulate glucose levels in dogs, and in obesity control and maintenance after weight loss via their effects on metabolism glucose, fat, and in appetite control [23,31-33]. The most important action resulting in adequate stimulation is probably the way glucan interact with their receptors. The main glucan receptors are complement receptor 3 (CR3, CD11b/CD18) and Dectin-1. The first receptor belongs to the b2-integrin family and is found mostly on macrophages, leukocytes, and NK cells. Glucan bind to the lectin site of this receptor and the overlapping I-domain of CD11b. The stimulation of cells relies on simultaneous binding of glucan and iC3b-opsonized material [34]. On the other hand, Dectin-1 is a type II transmembrane protein present on neutrophils, macrophages and dendritic cells. Upon binding of glucan, an immunoreceptor tyrosine-based activating motif is phosphorylated [35].
However, even with extensive literature on glucan andits biological effects, some limitations exist. Not all studies are in agreement, with the most common reason being that the glucans used in these experiments differed widely in purity and biological activity. In addition, crude extracts of glucan have been used in commercial products for pets and humans arguing the same functionality of purified glucans.
In this study, we compared the immunological effects of the yeast-derived materials, from whole yeasts to purified glucan, with crescent content of glucans. All were produced from the same yeast raw material. The dosages of the tested material were adjusted in order to give the same level of glucans. The intention was to suggest that the whole immunomodulatory effect of yeast glucans can be related with the process of purification, making purified glucans more available for recognition by its specific receptors.
All polysaccharide-based natural immunomodulators act primarily on innate immunity and particularly on its cellular branch. Therefore, the first reaction tested was phagocytosis, where we evaluated the effects on the phagocytic activity of peritoneal macrophages and peripheral blood neutrophils and monocytes. We found that the stimulation was consistently significant only in the case with fully isolated glucan and the other;the less purified fraction showed activity only in some cases. In addition to direct effects on activities of immunocytes, it is assumed that the immunomodulators bind to the specific receptors with subsequent signaling resulting in cell activation and secretion of cytokines and other biologically active molecules. Immunostimulators in general and glucans in particular stimulate secretion of various cytokines [36]. Evaluating the effects on IL-2 production, our study confirmed the strong effects of isolated glucan [37]. These results are potentially important, as glucan is known to affect the balance of Th1/Th2 cytokines [38]. After overcoming the theory that glucans are only nonspecific stimulators, the next common dogma assumed that they affect cellular immunity only. Lately, attention is beginning to focus on possible effects on humoral immunity, and glucans are now considered to have important potential as a part of vaccines [38,39]. Our results showed that even crude extracts supported antibody response.
This studyadds more informationto the three previous studies, testing over 40 different glucans [14,22,39]. Our results suggest that the yeast derivatives have more limited immunological effects, maybe as a result of local intestinal immune reactions. The purified glucan showed stronger immunological activities. Since the digestive tract of animals cannot degrade β-glucan, after ingestion, one portion of glucans has a specific prebiotic effect to the intestinal microbiota (mainly to the genus Akkermansia) and the other portion is captured by specific immune cells in the small intestine to generate a systemic response. The latter can explain the increased response when compared with the other yeast derivatives.
The authors declare that they have no competing interests.
| Authors' contributions | CAFO | VV |
| Research concept and design | √ | √ |
| Collection and/or assembly of data | √ | √ |
| Data analysis and interpretation | √ | √ |
| Writing the article | √ | √ |
| Critical revision of the article | √ | √ |
| Final approval of article | √ | √ |
| Statistical analysis | √ | √ |
The authors would like to thank Biorigin for their β-glucan donation and financial support. The funders had no role in the study design, data collection and analysis, or decision to publish. The authors have no competing interests to declare.
EIC: Markus H. Frank, Harvard Medical School, USA.
Received: 14-April-2020 Final Revised: 04-July-2020
Accepted: 07-July-2020 Published: 14-July-2020
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