**1. Introduction**

The phenomenon of subclinically elevated levels of endotoxin in the bloodstream has recently been termed "metabolic endotoxemia" [1,2]. This elevation is not evident in the clinical setting, but is currently being studied as a significant potential etiology of chronic diseases such as atherosclerosis, type II diabetes mellitus, Parkinson's disease, pancreatitis, amyotrophic lateral sclerosis, Alzheimer's disease, and cancer metastasis that arise in the context of chronic low-severity inflammation [1–3]. Endotoxins are known to consist of amphiphilic lipopolysaccharide (LPS) macromolecules located on the surface of Gram-negative bacteria. Released from bacteria in vivo or administered in an isolated form, endotoxins exert both physiological and powerful pathophysiological e ffects in higher organisms and, thus, represent important virulence factors of Gram-negative bacteria [4]. Circulating LPS is bioactive in vivo and correlates with measures of innate and adaptive immune activation [5]. LPS is the primary target for interaction with components of the host immune system, which can be activated by LPS to produce synthesis of tumor necrosis factor-alpha (TNFα), interleukin (IL)-6, IL-8, and free radicals, such as reactive oxygen species (ROS) [6]. The overexpression of these pro-inflammatory mediators may result in fever, severe damage of tissues, coagulopathy, endothelial dysfunction, vascular instability, apoptosis, multiorgan failure, and septic shock. The gastrointestinal tract is involved in the initial response to the systemic inflammatory reaction [7]. Impaired intestinal barrier function and/or increased epithelial permeability may promote the translocation of bacteria and endotoxin from the gu<sup>t</sup> into the body, increasing the susceptibility to infections [8].

The gu<sup>t</sup> microbiota seems able to promote systemic low-grade inflammation, insulin resistance, and enhanced cardiovascular risk through a mechanism that involves the increased exposure to bacterial products coming from the gut, particularly to the LPS [9,10]. Rich sources of LPS live in the upper respiratory and gastrointestinal tracts, especially in the mouth and colon. LPS may also be found in foodstu ffs [11].

Emerging human clinical studies have demonstrated that diet and nutritional supplements have demonstrated the ability to provide clinically important reductions in circulating endotoxins and improve related e ffects, such as inflammation and other negative health markers [12]. Food fibers are in close contact with the large intestine's immune system, which composes a significant part of the common immune system. The deficiency of food fibers in the diet promotes the appearance of many gastrointestinal and metabolic diseases [13].

Seaweed polysaccharides, such as carrageenans (CRGs), are one source of soluble dietary fibers [14]. CRGs are sulfated linear galactans, whose basic structural units are disaccharide-carrabiose, consisting of alternating β-1,3-linked and α-1,4-linked galactose residues. Variation in this basic structure results from the 3,6-anhydrogalactose content, location, and number of sulfate groups [15]. The three most industrially exploited types, namely κ-, ι-, and λ-CRG, are distinguished by the presence of one, two, and three ester-sulfate groups per repeating disaccharide unit, respectively. Native carrageenans always represent complex hybrid structures or are generally a mixture of galactans composed of di fferent carrabiose types. The hybrid nature of CRGs at the molecular level is responsible for changes in the biological and physico-chemical properties of CRGs compared with those of their homopolymeric ideal types [16].

Although CRGs have been used as safe food ingredients for a long time, they are also used as a classical model for inducing inflammation. Inflammation induced by carrageenan was originally described by Winter [17]. The CRG-induced edema, as an inflammation model, is usually used to assess the contribution of natural products to resist the biochemical changes associated with acute inflammation. When CRG is injected, acute inflammation with edema appears, along with a production of free radicals as well as a release of the inflammatory mediators [18]. At the same time, intensive studies have shown that CRGs can be regarded not only as foodstu ff ingredients, due to a wide spectrum of biological and physiological activities, such as antiviral [19], anticoagulant [20], antitumor [21], and immunomodulatory activities [22]. At present, CRG has been included in the United States Pharmacopeia 35-National Formulary 30S1 (USP35-NF30S1), the British Pharmacopoeia 2012 (BP2012), and the European Pharmacopoeia 7.0 (EP7.0), implying that CRG may have a promising future as a pharmaceutical excipient. According to the JECFA, only degraded carrageenans were associated to adverse e ffects and should not be used as food additives. It known that degraded low-molecular-weight carrageenan exhibits toxicological properties at high doses, stimulates ulceration of the fecal mucosa, histopathological changes, epithelial thinning, slight erosion, cellular infiltration, and other negative changes in animal organisms [23]. Native high-molecular-weight carrageenan is safe and non-toxic product. The safety of CRGs is supported by a large number of animal oral safety studies in which no

adverse e ffects were reported at high doses (up to 5% in the diet) [24]. The wide range of potential pharmacological applications of di fferent types of CRGs is the main reason for the increased interest in these polysaccharides. The e ffect of sulfated polysaccharides on metabolic endotoxemia has only been studied in animals. It has been shown that the administration of sulfated polysaccharides to high fat-diet-fed mice increased the amount of short-chain fatty acids in the intestinal tract, decreased the blood LPS or LPS binding protein concentration, and attenuated weight gain [25,26]. The influence of CRG on LPS and on the antibacterial host defense systems, such as the chemotactic responses to heat-killed bacteria or LPS in the pleural cavity and interleukin-1 production by pleural macrophages, was studied by Tatede et al. [27]. Their results demonstrate the potential activity of iota-CRG to enhance antibacterial host defense systems in mice. The administration of κ-CRG has been reported to increase the phagocytic activity of carp, *Cyprinus carpio*, and its resistance against *Edwardsiella tarda* and *Aeromonas hydrophila* administered via intraperitoneal injection [28]. The administration of iota-CRG has been reported to increase the innate immune response of the orange-spotted grouper, *Epinephelus coioides*, and its resistance against *Vibrio alginolyticus* administered via injection [29]. It has been shown that white shrimp, *Litopenaeus vannamei*, that received lambda-CRG exhibited higher immune ability as well as resistance against Gram-negative bacterium *V. alginolyticus* [30].

In a previous study, we showed that the administration of CRGs significantly prolonged the survival of mice against LPS challenge [31]. The degree of protection depended on the structure of the CRGs, their dose, route, and time of administration. We previously demonstrated a potential application of CRGs as protectors against S and R forms of *Proteus mirabilis* LPS. We concluded that CRGs might be considered as anti-endotoxin agents, neutralizing LPS and abolishing their proapoptotic activity [32]. The results obtained in this study by three serological methods indicate the role of *P. mirabilis* lipid A in the binding to di fferent CRGs. The lipid A component is responsible for much LPS toxicity. At the same time, it is known that in the macromolecular mechanism of endotoxin induction many of the observed biological e ffects of LPS can be related to their physical state, mainly to the three-dimensional supramolecular structure of the LPS assembly [33]. We previously demonstrated that LPS interact with polysaccharide chitosan, which is accompanied by the essential modification of some immunological properties of LPS [34,35]. Later we discovered a transformation of the supramolecular structure of LPS after binding to chitosan [36]. It was necessary to establish whether a change in the macromolecular structure of LPS occurs in the presence of CRG.

In the present study, we examined the e ffect of CRGs on LPS-induced endotoxemia in mice as well as the influence of the biologically active food supplement "Carrageenan-FE" on the immune system and hemostasis parameters of patients with food borne infections caused by Gram-negative bacteria. To explain the mechanism of the protective e ffect of CRGs on LPS, the e ffect of these polysaccharides on the supramolecular structure of LPS was studied.
