Animal-Origin Prebiotics Based on Chitin: An Alternative for the Future? A Critical Review

The human gut microbiota has been revealed in recent years as a factor that plays a decisive role in the maintenance of human health, as well as in the development of many non-communicable diseases. This microbiota can be modulated by various dietary factors, among which complex carbohydrates have a great influence. Although most complex carbohydrates included in the human diet come from vegetables, there are also options to include complex carbohydrates from non-vegetable sources, such as chitin and its derivatives. Chitin, and its derivatives such as chitosan can be obtained from non-vegetable sources, the best being insects, crustacean exoskeletons and fungi. The present review offers a broad perspective of the current knowledge surrounding the impacts of chitin and its derived polysaccharides on the human gut microbiota and the profound need for more in-depth investigations into this topic. Overall, the effects of whole insects or meal on the gut microbiota have contradictory results, possibly due to their high protein content. Better results are obtained for the case of chitin derivatives, regarding both metabolic effects and effects on the gut microbiota composition.


Introduction
According to United Nations predictions, it is expected that 9.8 billion people will inhabit the planet by around 2050 and 11.2 billion in 2100 [1]. Consequently, it is expected that the demand for food will increase by about 60% in the coming decades [2,3]. Thus, it will be a great challenge to ensure enough and safe food for such a large population. Also, the traditional methods usually employed to obtain food, of both of plant and animal origin, present specific problems, such as high CO 2 emissions or a need for cultivable surfaces, that make difficult their use to meet such an ambitious increase target [4].
Thus, conventional agriculture presents concerns. For example, fresh water (an essential resource) is a very scarce commodity, and progressive desertification of the earth's surface is taking place and will probably be aggravated in the future by global warming and desertification process [5]. According to the Food and Agriculture Organization of the United Nations (FAO) [6], it is expected a progressive decline in global agricultural productivity of about 1% per year.
With respect to animal-origin foods, their global demand is increasing drastically, and it is expected to increase even more in the future [7]. The production of animal-origin food also presents specific challenges, earth [11]. The dominant gut microbial phyla are Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia but the phyla Firmicutes and Bacteroidetes [20] represent about 90% of the total GM. The phylum Firmicutes includes more than 200 different genera, Clostridium being the most relevant as it represents about 95% of the Firmicutes phylum. Other important Firmicutes genera are Lactobacillus, Bacillus, Enterococcus and Ruminococcus [20]. Regarding the phylum Bacteroidetes, the predominant genus is Bacteroides (about 90% of total Bacteroidetes), Prevotella also being a relevant genus [5]. The phylum Actinobacteria is proportionally less abundant and is mainly represented by the genus Bifidobacterium [8]. Proteobacteria includes most food-borne bacteria, such as Salmonella, Klebsiella, Yersinia and Escherichia [21], whereas Fusobacteria is usually related to negative effects as it includes some genera such as Fusobacterium that are related to the development of colorectal cancer [5]. Finally, the phylum Verrucomicrobia includes the genus Akkermansia, a mucin-degrading bacterium believed to contribute to intestinal health and glucose homeostasis, that although is often present in small amounts has important functions and its presence is associated with good gut health [22].
The optimal GM is not yet defined as it presents great variability between individuals [4], but there is a broad consensus that a rich and diverse microbial community leads to a well-balanced and healthy GM composition [23]. Indeed, the human GM is characterized by an inter-individual variability due to various factors relating to the subject's life history, such as infant transitions and antibiotic use, as well as lifestyle, dietary and cultural habits [24].
In recent years, a large amount of scientific literature has been published that shows a close relation between GM composition and functionality and numerous non-transmissible diseases, such as cardiovascular diseases [25], obesity [8], diabetes [26], cancer [27], gastrointestinal diseases [28] and neurological disorders [29]. Although nowadays it is not entirely known if changes in GM composition are a cause or consequence of a given disease, a positive association between the richness and diversity of the GM and human health has been demonstrated [23].
Diet is one of the key modulators of GM composition that directly affects host homeostasis and biological processes but also via metabolites derived from the microbial fermentation of nutrients-particularly short-chain fatty acids (SCFAs) [30]. Among the nutrients than can exert a central role in GM modulation, indigestible carbohydrates are of major interest as functional food ingredients with health benefits [31], as substrates to produce SCFAs.

Chitin Content in Foods and Chitin Derivatives
Chitin is a polysaccharide composed of N-acetyl-2-amino-2-deoxyglucose (GlcNAc) units linked by β-(1→4) bonds. Chitin is the main fibrous compound in arthropod exoskeletons, mollusk radula, cephalopod endoskeletons, fungal cell walls, and fish and lissamphibian scales [32]. The largest source of chitin globally is suggested to be zooplankton cuticles, with an estimated 379 million tons of Antarctic krill available worldwide [32]. However, fishing these tiny organisms is not commercially viable; and subsequently, considered as shellfish industry waste, such as shrimp, crab and lobster shells with a chitin content of 8-40% are the main source of chitin [30,31]. Fungi provide an alternative source of chitin and, despite having a lower chitin content than crustaceans (10-26% as a chitin-β-(1,3/1,6) glucan complex, GC), are attracting increasing scientific and food industry interest [33,34].
The chitin content can widely vary between different sources, ranging from 16-23% in lobster shells, 25-30% in crab shells and 34-49% in krill shells to 18-38% in cockroach cuticles, 22-64% in butterfly cuticles, 20-44% in silkworm, 8-43% in mushrooms cell walls, 8-27% in mold cell walls and 1-3% in yeast cell walls [33]. Whereas crustacean exoskeletons are not usually employed by the food industry and are considered waste [35], and chitin from fungi is also often extracted from residues [36], chitin from insects can be ingested together with other nutrients because they are usually consumed as whole insects or parts of whole edible insects including ingredients derived from them such as meals/flours [4]. As consumer acceptance in developed countries remains one of the barriers to their use as an entire food [37], the inclusion of insects as flours or other forms of food, for example, cookies, energy bars, hamburgers and sandwich spreads, among others, is promising [38]. Diverse studies have revealed that males are more willing to adopt insects as a protein substitute than females as they are, on average, described as more adventurous eaters [37]. Similarly, younger consumers are less reluctant to eat insects than older consumers [37]. Because of their high chitin content, which represents at least 10% of all dry insects, insects can be a good source of DF in the human diet [4]. In addition to their chitin content, diets containing insects offer other important benefits for both animals and humans. Thus, for the case of animal feed, it was previously reported than insects are a good source of amino acids, fatty acids such as lauric acid, minerals, and most of the B group vitamins [39]. As other important benefit, due to their content in antimicrobial peptides and lauric acid, the inclusion of insects in feed contributed to a decreased need for antibiotics in animal rearing [39]. In the case of humans, insects were reported to be an excellent source of energy, fats, proteins, and minerals [4]. Insect-derived proteins were reported as of a higher biological value than those obtained from plant sources, with an essential amino acid score varying between 46-96%. The fatty acid profile of edible insects was reported as less saturated than those obtained from animal-origin foods, and depending on the species, with lower cholesterol levels, as well as containing plant sterols [40]. With respect to mineral supply, insects were reported as good sources of phosphorous, magnesium, manganese, copper, selenium, zinc, iron, and calcium [4].
Chitin is insoluble in water, but humans have digestive enzymes in their gastrointestinal tract that are capable of degrading chitin to some extent [2]. Chitinolytic enzymes (chitinase and chitobiase) break down the glycosidic bonds between GlcNAc units and degrade chitin into chitosan [2]. Lysozyme is also known to catalyze the deacetylation of 2-acetyl groups and the separation of glycosidic bonds between GlcNAc units from chitin, thus producing chitosan, a partially deacetylated byproduct of chitin degradation. The most beneficial advantage of chitosan is that it can be chemically modified into a wide variety of derivatives, that enhances some characteristics such as water-solubility [41]. Chitosan is a very useful and attractive biopolymer due to its diverse chemical structure. Its structural diversification can be seen by its molecular weight, which ranges from low (≤100 kDa) to high (≥300 kDa), as well as by its degree of deacetylation, which ranges from less than 60% in chitin to more than 60% in chitosan [41]. Hydrolyzed products of chitosan-N-acetyl-D-glucosamine oligomers (chitin oligosaccharide; NACOS) and D-glucosamine oligomers (chitosan oligosaccharide; COS), major degradation products of chitosan/chitin via chemical hydrolysis or enzymatic degradation involving deacetylation and depolymerization processes [3] with molecular weight ≤ 16 kDa [42,43]-are readily soluble in water because of their shorter chain lengths, and this solubility makes them especially useful for food industry purposes.

Effects of Chitin and Derivatives on Human Health
Additionally to the effects of chitin on the GM, where it has been reported to improve gastrointestinal health due to its prebiotic potential [2], when consumed in foods, chitin and its derivatives are functional DF that can reduce LDL-cholesterol levels in the blood [44,45]. Consumption of chitin has been shown to improve glucose intolerance, increase insulin secretion, relieve dyslipidemia, and protect intestinal integrity and the GM in mice treated with a high saturated fat diet [46]. Chitin, or its derivative, appears to have also antiviral, anticancer and antifungal activity, as well as antimicrobial properties and a bacteriostatic effect on the Gram-negative bacteria Escherichia coli, Vibrio cholerae and Shigella dysenteriae [47].
Chitosan, a naturally occurring bioactive polymer (a copolymer of N-acetyl-D-glucosamine and D-glucosamine) [48] is the most important chitin derivative, and has received increasing attention due to its specific biodegradability by colon bacteria, its well-documented biocompatibility, low toxicity and mutagenic properties [49]. Evidence has shown that chitosan possesses various biological activities, namely antioxidant, antitumor, anti-inflammatory, immunostimulant, wound healing, coadjutant (in aquatic animals), cholesterol-reducing, antibacterial and antifungal properties, and is useful as an active component of the diet for the loss of body fat. Chitosan has also been reported to contribute to decreased blood pressure, control of arthritis, treatment of diabetes mellitus and immunostimulation [21,50].
Previous studies have indicated that chitosan may inhibit the digestion and absorption of visceral fats and interfere with bile acid synthesis and lipid metabolism, demonstrating lipid-lowering effects. This indicates that oral administration of chitosan with Ganoderma polysaccharides improves lipid metabolism disorders [51]. Chitosan has been demonstrated to inhibit pancreatic lipase activity and bind to bile acids, resulting in reduced intestinal fat absorption and increased excretion of fecal fat. At the cellular level, chitosan can suppress adipocyte differentiation, triglyceride accumulation and expression of adipogenic markers, and has also demonstrated an increased ability to remove excess cholesterol and bile acids from tissues and carry them to the liver for excretion [52]. This is important considering that bile acids are potentially toxic [53]. In addition, the conversion of cholesterol to bile acids and their subsequent secretion into the bile is an important pathway for the elimination of cholesterol from the body [52]. Intervention with chitosan has been found to result in the lowest levels of total unsaturated fatty acids in fecal fats. Although the mechanisms involved in lipid metabolism following chitosan intake are not fully understood, enhanced absorption and/or binding function may play an alternative role [52].
Chitosan has also been used to chelate cholesterol in food, inhibiting its absorption in the human intestine [54]. It is important to note that chitosan particles and COS are non-allergenic bioactive nutrients [31,55,56]. It was recently demonstrated that chitosan nanoparticles can protect Caco-2 cells, a model of human enterocytes, from lipopolysaccharide (LPS)-induced cell membrane damage [57]. LPS, also known as lipoglycans and endotoxins, consist in large molecules formed by lipids and a polysaccharide composed of O-antigen, that are found in the outer membrane of Gram-negative bacteria [5]. According to previous reports, reducing the particle size and thus increasing the surface/volume ratio can improve the functional properties of biomolecules [48]. If chitin is degraded into chitosan and COS particles with a low enough molecular weight, they can be absorbed into the bloodstream and transferred to all organs and tissues, where they can have the beneficial effects mentioned above [2].
With respect to chitin derivatives, GC has been indicated as a dietary supplement, with the maximum rate of consumption set at 5 g per day for an average person [58]. Furthermore, GC consumption has been shown to reduce body weight gain in rats, thus pointing to GC as an interesting novel prebiotic for the prevention and treatment of obesity [59].
COS can modulate biological processes to protect the host from inflammation, immunity, obesity, microbial infection and diabetes. In vitro studies suggest that COS may reduce glucose transport in Caco-2 cells and promote glucose uptake by adipocyte cells, whereas in vivo studies show that COS may reduce fasting blood glucose in db/db mice and increase insulin secretion in type-1 diabetic mice [60]. The mechanism of action may be associated with inhibition of enzymes such as intestinal α-glucosidase and pancreatic α-amylase. In addition, it has been reported that COS may slightly affect the GM in cultures of human stool batches [61].
COS with a low molecular weight (<1000 Da) has been reported to significantly inhibit glucose absorption by the intestinal tract by suppressing pancreatic amylase and small-bowel glucosidase activity. In addition, COS increases insulin secretion by promoting the antioxidant capacity of the pancreas and exerts antidiabetic effects in db/db mice and in rats injected with streptozotocin [59]. In addition, COS intake increases serum insulin, which can be produced by beta cell proliferation [59].

Effect of Chitin and Its Derivatives on Gut Microbiota
Traditionally, both chitin and its derivatives are well known as inhibitors of the bacterial growth of pathogenic microorganisms such as Salmonella Typhimurium, enteropathogenic E. coli and V. cholerae, among others [23,61]. More recently, it has been reported that chitin ingestion, additional to inhibiting pathogen growth, does not show the same effect on some potentially beneficial bacteria, such as Bifidobacterium and Lactobacillus [4]. In two separate recent studies, chitin ingestion promoted the growth of beneficial bacteria in the GM. Stull et al. [62] reported that the presence of cricket chitin increased the multiplication of Bifidobacterium animalis by 5.7 times in the human adult GM. These effects were confirmed in a recent work that noted that the inhibition pathway of chitin against Lactobacillus rhamnosus (stimulation) and E. coli (inhibition), respectively, was different, the inhibitory activity against Gram-negative bacteria being higher than against Gram-positive [2].
In recent years, different research works have investigated the effect of whole insects, chitin and its derivatives on the GM in vitro, and in animal models and humans ( Table 1). Some of these studies were performed in fish species [63,64].
Obviously, the results obtained in fish models are less applicable to humans because fish GM are very different to those of humans and terrestrial animals. Thus, it was reported that the predominant phylum in fish GM is Proteobacteria, followed by Fusobacteria and Firmicutes, whereas Bacteroidetes and Actinobacteria are often less numerous [48]. However, in the rainbow trout (Oncorhynchus mykiss) the most common phylum is Tenericutes, that is very scarce or non-existent in the human GM [56,64].
With respect to the effects of administering whole insects and insect flour on the GM, the results can be observed in Table 1. It shows works carried out in humans [62], on in vitro trials simulating the human gastrointestinal tract and using human feces [65], in laying hens or broilers [7,[66][67][68], in fish species such as rainbow trout (O. mykiss) [63,64], zebrafish (Danio rerio) [69] and Siberian sturgeon (Acipenser baerii) [70], and in mice [71]. As can be seen in Table 1, the effects of insect administration or supplementation do not have a consistent effect on the GM. In most cases, there were a relevant number of beneficial effects, but in any case, it was found some harmful effects in GM composition were also found. These contradictory effects can be explained by the fact that both the insects and insect meal employed have a higher protein content that of chitin. A diet with a high protein content is well known to be dysbiotic, as usually occurs in the Western diet [10].
In vitro systems replicate the human GM but are less dynamic than the real human gastrointestinal environment. An in vitro trial simulating the human tract, investigating the administration of Tenebrio molitor flour, found an increase in Bacteroidaceae and Prevotellaceae, but not of harmful bacteria such as Clostridium histolyticum, Desulfovibrionales and Desulfuromonales. Bacteroides assists the host in degrading polysaccharides and contains genes codifying glucosidase enzymes [41], whereas Prevotella has the potential to participate in the metabolism and utilization of plant polysaccharides. Contrariwise, the relative abundance of Desulfovibrionales and Desulfuromonales is related to harmful effects and may contribute to the development of colorectal cancer [70], and C. histolyticum is potentially pathogenic in several species, including humans [65]. The beneficial effects were also reinforced in a study with an observed increase of SCFAs, very important for good colonic epithelial maintenance [65].
Clinical studies investigating prebiotic effects in humans have some issues with respect to ethical constraints, as well as limited sampling possibilities from the colon and limited measurements of in situ SCFA production [4]. In an in vivo clinical trial in humans [62], the intake of 25 g/day of Gryllodes sigillatus cricket powder provided contradictory results: an increase in probiotic species such as Bifidobacterium animalis and a decrease in other beneficial species such as Lactobacillus reuteri, as well as SCFAs. Both Bifidobacterium and Lactobacillus are well-known probiotic bacteria with a long history of safe use [5]. Increased GM diversity and richness, and increased proportions of Oscillospira, while proportions of Fusobacterium decreased Increase in SCFA production, and lower triglyceride content in serum and cholesterol content in serum and egg yolks [7] In vivo using 20 healthy adults Gryllodes sigillatus cricket powder 14 days of eating prepared study breakfast meals that included cricket powder (25 g/day) or control Bifidobacterium animalis increased by a log fold-change of 5.7 on the cricket diet compared to the control diet. Probiotic species Lactobacillus reuteri and two other lactic acid-producing bacteria were decreased by 3-to 4-fold relative to the control after 2 weeks of cricket powder consumption Acetate in the stool was reduced by an estimated 2.31 µM/g during the cricket diet. Similarly, cricket consumption was also associated with reduced propionate content [61] In vivo trial in rainbow trout (Oncorhynchus mykiss) H. illucens insect meal 10%, 20% and 30% partial substitution of fish meal with insect meal for 12 weeks Actinobacteria and Proteobacteria phyla were increased after inclusion of insect meal in the diet. An increase in richness, diversity and lactic acidand butyrate-producing bacteria was also observed Not provided [64] In vitro fermentation system using fresh fecal samples from 5 healthy donors In vivo using 42 specific-pathogen-free mice 3-week-old stable fly larvae Normal control group (n = 7); Escherichia coli control group (n = 7); ciprofloxacin (0.13 mg/kg; n = 7), 2 mg/kg metabolites of stable fly (n = 7), 4 mg/kg metabolites of stable fly (n = 7) and 8 mg/kg metabolites of stable fly (n = 7) groups. All groups but controls were injected intraperitoneally with E. coli (0.3 mL of 2.50 × 10 11 CFU/mL) once a day for 3 days, respectively, to induce diarrhea Cecal microbial sequencing showed a significant difference in the prevalence of Firmicutes, Clostridium, Bacteroidetes and Alistipes in the metabolites of stable fly-treated groups, suggesting that this treatment could be used to manage diarrhea in mice Metabolites derived from stable fly are rich in amino acids that may affect intestinal health by regulating intestinal immunity, antioxidant capacity and microbial population in mice with diarrhea [71] CFU: Colony formit units; GM: Gut microbiota; SCFA: Short Chain Fatty acids.
Diptera metabolites [68] showed beneficial effects regarding the management of E. coli-induced diarrhea in mice by modulating the immune system, antioxidants and GM composition. The intestinal microbiota imbalance was reversed, as shown by the increase in Firmicutes to Bacteroidetes ratio and Clostridium levels caused by E. coli-induced diarrhea.
With respect to trials carried out in mice, hens and chickens, the results showed benefits in metabolic parameters, such as an increase in the activity of glycolytic enzymes [69], a decrease in the triglyceride and cholesterol content in serum [7] and eggs [7,67], and an increase in the activity of αand β-glucosidases and α-galactosidase [69], stimulating immunity and antioxidant capacity [71], but also harmful effects such as a decrease in villi mucin production [66] or laying frequency, feed intake and egg weight [67]. Production of mucin is important for correct maintenance of cecal epithelial integrity [66]. The changes in GM varied from an increase in richness [7,63,64] and diversity [7,64,66], or a decrease in diversity when used at higher concentrations [66], to increases in beneficial bacteria such as Bacteroides and Prevotella [65], Bifidobacterium [62,68], Lactobacillus [66] and Ruminococcus [66,71]. A reduction of Clostridium spp. was also found [71]. Ruminococcus, Bacteroides, Bifidobacterium, Roseburia and Faecalibacterium are considered primary degraders of complex polysaccharides, and their presence in abundance in the GM ensures good utilization and fermentation of DF [70].
However, harmful effects were also found, such as a decrease in L. reuteri [62], increase in Helicobacter [66], a cancer-related genus [68], decrease in Bacteroides and Prevotella [69], increase in the pathogen Clostridium perfringens [69] and increase in the phylum Proteobacteria [64], that contains pathogenic genera and species [21]. Thus, whole insects or their meal cannot be entirely considered as GM-enhancing foods. In any case, it should be considered that whole insects or insects meals have less dysbiotic effects on GM than other sources of animal protein [4]. Additionally, with respect to other chitin sources, such as crustaceans, insects are not overexploited than some crustaceans, specially shrimp, that are strongly exploited in all fishing areas, particularly in the Atlantic Ocean, where is considered fully exploited and in the Indian Ocean, where it were seems some signs of overexploitation [72].
The results obtained in fish species, although the changes at phylum levels are more difficult to extrapolate to humans due to the wide differences between the GM of fish and humans, showed beneficial effects in terms of greater GM diversity [63,64] or richness [64]. With respect to beneficial groups, an increase was observed in the butyrate-producing Clostridium coccoides-Eubacterium rectale cluster [71] and other lactic acid and butyrate producers [64]. Less consensus was obtained for other phyla such as Proteobacteria, that increased [64] or decreased [63] depending on the work, or Enterobacteriaceae, that also increased in Siberian sturgeon [70], whereas it decreased in zebrafish [69] supplemented in both cases with Hermetia illucens.
As can be seen in Table 2, the effects of chitin derivatives on both GM and metabolic effects are more favorable than those of whole insects or insect meal. Interestingly, an in vitro trial reported that the beneficial effects on GM of chitosan-carrying foods are limited to foods that possess a low protein content [23]. This could be a reasonable explanation for the better effects of chitin derivatives, due to the high protein content of both insects and insect-derived meals [4], and the dysbiotic effect of protein-rich diets [82][83][84].

Significant Changes in Metabolites and Metabolic Effects Reference
In vitro determination of bacteria grown in tryptone soy broth comparing E. coli vs.

Lactobacillus rhamnosus
Chitin was tested at concentrations of 5 and 1 g/L, whereas COS was tested at concentrations of 5, 1, and 0.5 g/L COS powder ≤ 1.5 kDa and degree of deacetylation ≥ 90%. Chitin was prepared at a concentration in the range 1-5 g/L. COS was prepared in the range 0.5-5 g/L COS reduced the growth of E. coli, whereas chitin totally inhibited E. coli growth. COS stimulated the growth of L. rhamnosus, whereas chitin inhibited its growth Not provided [2] In vitro determination of minimal inhibitory concentrations against E. coli and Staphylococcus aureus in broth, milk and apple juice Minimal inhibitory concentrations were tested in the range 0-0.6% w/v. 0.5% w/v chitosan and COS in milk and apple juice Chitosan with average molecular weights of 628, 591 and 107 kDa and a degree of deacetylation in the range 80-85%. COS with molecular weight of < 5 and < 3 kDa and a degree of deacetylation in the range 80-85% COS showed higher antibacterial activity than chitosan against E. coli, whereas chitosan showed higher antibacterial activity than COS in S. aureus. The use of chitosan in foods will be limited to foods that possess a low protein content Not provided [23] In vivo trial using 24 C57BL/6J mice  [48] In vivo trial using 144 piglets (Duroc × landrace × Yorkshire) 100, 200 or 400 mg/kg in feed for 28 days Chitosan nanoparticles with a particle size of about 50 nm, average molecular weight of 220 kDa and degree of deacetylation of 95% Increase in GM diversity and Bacteroidetes, Prevotellaceae and Ruminococcus while Firmicutes and Clostridiaceae family decreased Improvement in growth performance. Improvement in immunoglobulin IgA, IgG, C3 and C4. Decrease in plasma cortisol, PEG2, IL-6 and IL-1ß [49] In vitro trial investigating effects on growth of 100 Bifidobacterium and in vivo trial in 24 Groningen rats In vitro trial at 0.5% GC w/v concentration. Rat diet was supplemented with 10% GC (w/w) GC was obtained from A. niger by a tyndallization procedure Increase of Bifidobacterium adolescentis and B. longum. Decrease in Firmicutes to Bacteroidetes ratio and improved colonization efficiency of B. breve Decrease in body weight gain with respect to controls [56] In vivo trial using 20 C57BL/6J mice 1 mg/mL chitin oligosaccharide (NACOS) in drinking water (about 200 mg/kg/day) for 5 months in a high-fat diet NACOS with a polymerization degree 2-6 Increase of Lactobacillus, Bifidobacterium, Akkermansia and Bacteroides whereas Desulfovibrio and Firmicutes to Bacteroidetes ratio decreased Decrease in mRNA of cytokines, including TNF-α, IL-6, MCP-1 and LPS in serum. Improved bacterial motility, oxidative stress, energy metabolism and inflammation process

Significant Changes in Metabolites and Metabolic Effects Reference
In vivo using 130 subjects free of diabetes mellitus Participants were randomly assigned to receive chitin-glucan (GC) (4.5 g/day; n = 33), GC (1.5 g/day; n = 32), GC (1.5 g/day) plus olive oil extract (135 mg/day; n = 30) or matching placebo (n = 35) for 6 weeks GC derived from Aspergillus niger mycelium Not provided Administration of 4.5 g/day GC for 6 weeks significantly reduced oxidized low-density lipoprotein. At the end of the study, GC was associated with lower LDL-C levels, although this difference was statistically significant only for the GC 1.5 g/day group [73] In vitro trial using trypticase phyton yeast inoculated with different Bifidobacterium strains 0.025%, 0.1% and 0.5% low-molar-mass chitosan, chitosan succinate; chitosan glutamate and 0.1% and 0.5% COS in anerobic trypticase phyton yeast medium Chitosan molecular weight 75 kDa; degree of deacetylation 83%, prepared by enzyme hydrolysis to obtain different fractions Both chitosan and all derivatives inhibited Bifidobacterium growth Not provided [74] In vitro fermentation using fresh feces of C57BL/6J mice 1 g/L of COS in drinking water, about 200 mg/kg/day for 5 months COS with deacetylation degree over 95% and average molecular weight < 1 kDa

Increase of Bacteroidetes and Verrucomicrobia phyla whereas Proteobacteria and Firmicutes phyla decreased
Increase in colonic H 2 , acetate and butyrate [75] In vivo using 24 C57BL/6J mice GC (10% w/w). Food intake was recorded, taking into account spillage, twice a week for 4 weeks GC was derived from the cell walls of the mycelium of A. niger GC supplementation increased the quantities of Bacteroides-Prevotella spp., whereas the Clostridium coccoides-Eubacterium rectale cluster group and Roseburia spp. were completely restored after GC treatment. Bifidobacteria in the high-fat GC-fed mice were higher than in the high fat-fed mice or control mice GC decreased body weight gain by about 28% as compared to high-fat diet. This effect was accompanied by lower fat mass development.
Consumption of GC showed potential beneficial effects with respect to the development of obesity and associated metabolic disorders such as diabetes and hepatic steatosis [76] In vivo trial using 40   Chitosan, the most simple chitin derivative, showed metabolic effects in pigs, such as a reduction in feed intake and body weight [79], and metagenomic evidence of promoting important metabolic pathways and vitamin synthesis [81], and improved serum lipidic profile, such as a decrease in triglycerides, cholesterol and aspartame aminotransferase [80], a marker of liver health [82]. Regarding its direct effect on the GM, chitosan addition was related to a decrease in Firmicutes [79,81], and increase in the counts of Bacteroidetes [81]. A higher ratio of Firmicutes to Bacteroidetes has previously been associated by some authors with a higher risk of obesity in humans [10,85]. Additionally, high proportions of Firmicutes have been associated with increased susceptibility to inflammation, infection, oxidative stress and insulin resistance [86,87]. Contrariwise, Bacteroidetes species such as Bacteroides and Prevotella possess strong peptidase activity and are associated with isovalerate and isobutyrate production [88]. At genus level, chitosan was found to increase Prevotella [81], decrease Lactobacillus [79,81] and increase Bifidobacterium [74,79], as well as increasing Ruminococcus, Oscillibacter, Bifidobacterium, Prevotella, Alloprevotella and Paraprevotella [80]. All the variations at genus level, with the exception of the decrease in Lactobacillus, are considered beneficial to health because they include SCFA producers, such as Prevotellaceae and Oscillibacter, that produce anti-inflammatory metabolites, which subsequently regulate proinflammatory immune cells [89][90][91].
With respect to GC, its administration in non-diabetic subjects reduced the serum content of oxidized low-density lipoproteins, a major risk in cardiovascular diseases, the leading mortality cause in Western countries [92]. GC administration also decreased the body weight of rats [56] and mice, accompanied by a decrease in fat mass [69].
Regarding the effect on GM composition, GC decreased the Firmicutes to Bacteroidetes ratio [56], and increased the amounts of lactic acid bacteria and SCFA-producing genera such as Bifidobacterium [56], Bacteroides-Prevotella spp., C. coccoides-E. rectale and Roseburia spp. All of these are genera that can grow in the presence of complex carbohydrates, and some of them such as Roseburia carry carbohydrate degradation genes, being producers of SCFAs [93]. C. coccoides produces metabolites, like SCFAs, secondary bile acids and indolepropionic acid, that play a probiotic role primarily through energizing intestinal epithelial cells, strengthening the intestinal barrier and interacting with the immune system [94].
NACOS has shown several pharmacological effects, including antimicrobial activity and protection against pathogen-induced infections [56]. The abundance of four beneficial bacteria, Bifidobacterium, Lactobacillus, Akkermansia and Bacteroides, were reduced in mice after a high-fat diet, which was significantly restored after supplementation with NACOS [56]. NACOS also clearly reduced the abundance in mice fed a high-fat diet of Desulfovibrio [56], a bacterial genus which is closely related to this type of diet [95] and is responsible for inflammation due to its lipid A structures of LPS [96]. Desulfovibrio can use hydrogen or organic compounds such as lactate and formate to reduce sulfate to generate hydrogen sulfide (H 2 S), which is toxic in nature and can have pathological consequences for the host [95].
Interestingly, it seems that treatment with NACOS significantly reduces mRNA levels of cytokines related to inflammation processes, including TNF-α, IL-6 and MCP-1, and also significantly decreases plasma concentrations of LPS in mice fed a high-fat diet [56]. NACOS also dramatically increased the abundance of Bifidobacterium, Lactobacillus, Akkermansia and Bacteroides in mice fed a high-fat diet. Evidence suggests that increased abundance of Bifidobacterium, Lactobacillus, Akkermansia muciniphila and Bacteroides has positive effects on intestinal integrity, glucose tolerance and attenuated obesity [97][98][99][100][101].
With respect to COS, some authors have hypothesized that its administration could provide better results than that of chitin or chitosan because of a more efficient digestion of chitin by AMCase in the gastrointestinal tract [2]. According to Mateos-Aparicio et al. [60], COS with many acetylated residues are more efficient in promoting the growth of beneficial Lactobacillus than deacetylated COS. These effects depend on the molecular weight and the degree of acetylation of COS [60]. The metabolic effect of COS showed beneficial effects on body weight maintenance and reduction, and glucose and insulin management, as well as decreasing inflammation-related markers [46,102]. When combined with resistant starch, COS administration decreased protein-fermentation markers such as H 2 S 2 , ammonia, phenols and indole as well as increasing the excretion of bile acids in feces, the thickness of the mucosal layer and SCFA production [78].
With respect to its effects on GM composition, COS did not have a significant effect on GM richness and diversity [46,75,77], but did stimulate the growth of L. rhamnosus, whereas chitin inhibited its growth [2], suggesting the above-mentioned more efficient digestion of COS than chitin in the gastrointestinal tract. It was also reported to have a favorable effect on the Firmicutes to Bacteroidetes ratio [46,75,77], decrease the phylum Proteobacteria [75], and reduce Lachnospiraceae NK4A136 group, Alistipes, Helicobacter, Ruminococcus and Odoribacter, while increasing Lachnospiraceae UCG 001 and Akkermansia [46]. These findings suggest benefits related to COS intake, as several studies indicate that the high abundance of Alistipes and decrease in the population of Akkermansia are closely related to the development of diabetes [95]. These results indicate the potential beneficial effects of COS on host health by reforming the structure of the GM.
Other chitosan derivatives such as low molecular weight chitosan [49,81] and chitosan-silver nanoparticles also showed a beneficial effect, both with respect to metabolic parameters such as goblet cell density and villi height [48], and to improving the metabolism of terpenoids and polyketides, digestive systems, cell growth and death, glycan biosynthesis and metabolism as well as the metabolism of cofactors and vitamins [81]. Effects on the GM were similar to those obtained from other chitin derivatives and consisted of benefits in the Firmicutes to Bacteroidetes ratio [49,81], a decrease in Proteobacteria [49], and increase in the genus Prevotella [81]. The only discordant element with respect to the beneficial effects was a decrease in Lactobacillus [49].

Conclusions
The decrease in terrestrial agriculture and disposable water is likely to increase the consumption of non-vegetable DF by humans in the near future, because its adequate consumption is essential to maintain human health. Chitin, as one of the most abundant biopolymers in nature, is an interesting candidate to partially substitute or complement cellulose-based DF, the main sources of chitin in nature being insects, crustaceans and fungi.
In view of the results obtained, the use of whole insects or meal, although showing beneficial effects on the modulation of GM composition in most cases, also showed some harmful effects on both GM composition and other metabolic parameters. This divergence could be explained by the high protein content of insects and meal that could contrast with the beneficial effects of chitin on GM composition and human health. Thus, as recent works have indicated, chitin derivatives such as chitosan only exert their potential prebiotic activity if carried by foods that possess a low protein content. However, chitin derivatives show better results in the modulation of GM, enhancing the growth of beneficial bacteria and inhibiting the growth of some potentially pathogenic bacteria. Additionally, chitin derivatives also show positive effects in terms of anti-inflammatory capacity, stimulation of the immune response, prevention of diabetes, and prevention and management of obesity. Thus, their potential as a source of DF should be investigated in more depth, to enhance the valorization of these products as human foods. Funding: The authors thank the European Regional Development Funds (FEDER), grant ED431C 2018/05, and Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CyTED), grant PCI2018-093245 for covering the cost of publication.

Conflicts of Interest:
The authors declare no conflict of interest.