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Applied Biological Chemistry
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Preparation of chitosan oligosaccharides from chitosan of tenebrio molitor and its prebiotic activity

Applied Biological Chemistry volume 67, Article number: 84 (2024) Cite this article

Abstract

This study aimed to establish the optimal production conditions for mealworm chitosan oligosaccharides (MCOS) using the response surface methodology and measure the prebiotic effect of MCOS prepared on cecal microbiota through in vitro anaerobic fermentation. The optimal conditions for MCOS production using chitosanase were 2.5% substrate, 30 mg/g enzyme, and 6 h reaction time. Matrix-assisted laser desorption ionization-time of flight mass spectrometry, Fourier transform infrared spectroscopy, and in vitro assays to confirm that the chemical structure and physicochemical properties of MCOS are similar to those of commercially available chitosan oligosaccharides. The growth of Lactobacillus acidophilus, Lacticaseibacillus casei, and Bifidobacterium bifidum was increased by MOCS and confirmed that the prebiotic effect of MCOS was significant in a concentration-dependent manner. The addition of 1% and 2% MCOS to in vitro anaerobic fermentation resulted in changes in the content of short-chain fatty acids (SCFAs) and an increase in Verrucomicrobiota abundance compared with the control. In the case of Romboutsia, Turicibacter, and Akkermansia, a significant increase was confirmed in the MCOS-containing groups compared to that in the control group. Compared to 2% MCOS, 1% MCOS more significantly affected Lactobacillus levels. MCOS produced by chitosanase under optimal conditions contains oligosaccharides with 2–6 degree of polymerization and exerts a prebiotic effect that affects changes in the SCFA content and microbiota composition in the cecum.

Introduction

Chitin and chitosan are the second most abundant polysaccharides after cellulose; they are mainly extracted from crustaceans such as shells and shrimp [1]. Crustacean shells are the main source of chitin [2]; however, chitin is also present in mollusks, such as squid, and in the cell walls of fungi, yeasts, mushrooms, and insects. As crustacean supplies become unstable due to seasonal factors, insects are emerging as an alternative source of chitin and chitosan. The exoskeletons of insects with a short breeding period and low mineral content comprise 30–45% protein, 25–40% lipids, and 10–15% chitin [3]. Chitin can form up to 40% or more of the exoskeleton depending on the life cycle of insect [4].

Although quantitatively abundant, chitin is not widely used owing to its low solubility, it has excellent biocompatibility and is partially used as a medical material in artificial skin and surgical sutures [5, 6]. Chitosan, produced by the deacetylation of chitin, is used for wastewater treatment and as a heavy metal adsorbent with a strong adsorption capacity [7]. Furthermore, as it has various physiological activities such as anticancer, antibacterial, and cholesterol-lowering activities, it is also used for numerous purposes such as functional foods, functional biomaterials, and pharmaceuticals [8, 9]. Chitosan is a high-molecular-weight polymer that has structural characteristics similar to those of cellulose and is not digested or absorbed in the human gastrointestinal tract. In the gastrointestinal tract, no enzyme can break down the β-l,4-glycosidic linkage, and molecules with a molecular weight of 22 kDa or higher are hardly absorbed [10]. Therefore, to use chitosan efficiently as an active material, it needs to be converted into oligosaccharides.

Chitosan oligosaccharide (COS) is composed of glucosamine, a constituent unit with less than 100 degrees of polymerization (molecular weight 18 kDa). Compared to chitosan, COS has a low molecular weight, low viscosity, and water solubility, enabling various industrial applications. Moreover, it is widely used in functional foods and medicines because it is easily absorbed in vivo [11]. COS with a degree of polymerization (DP) of 2–8 is useful as a prebiotic, and COS at a concentration of 0.1–0.5% contributed to the growth of Bifidobacterium and Lactobacillus strains [12, 13]. Furthermore, since COS with a DP of 9 or higher inhibits the growth of Lacticaseibacillus paracasei and Lactobacillus kefir, it either enhances or inhibits the growth of lactic acid bacteria according to the DP [14].

The production of COS by enzymes, such as chitosanase, has the advantage of minimizing side reactions compared to chemical methods and improving biological activity without side effects due to excellent biocompatibility. Therefore, in this study, the reaction conditions for preparing mealworm COS (MCOS) from mealworm chitosan using chitosanase were optimized using response surface methodology (RSM). The physicochemical properties of MCOS and their effects on LAB growth of lactic acid bacteria were measured. Furthermore, the effect of MCOS on the gut microbiota was analyzed through in vitro anaerobic fermentation.

Results

Deacetylation of mealworm chitosan analysis by 1H-NMR

The degree of deacetylation (DDA) chitosan samples was calculated with the method Lavertu et al. [15]. The structure of deacetylated of the chitosan are presented Fig. 1. The solvent (HOD) proton resonates at 4.81 ppm.

$$\:\:\text{D}\text{D}\text{A}\left(\text{\%}\right)=\:\:\left(\frac{H1D}{H1D+\frac{HAc}{3}}\right)\times\:100\:\:$$

The DDA was calculated using integrals of peak of proton H1 of deacetylated monomer (H1-D: 5.21 ppm) and peak of three protons of acetyl group (H-Ac: 2.1 ppm). Namely, in the vicinity of 2.1 ppm, the resonance peak due to the CD2H residue of CD3COOD overlaps with that due to CH3 residue of N-acetyl. However, as is obvious from the magnification (Fig. 1), CD2H peak can be separated from CH3 peak by drawing symmetric curve for CH3 peak.

The degree of deacetylation commercial chitosan was 91.717% and the degree of deacetylation M-chitosan was 86.923% by using the above equation. Based on these results, it was confirmed that mealworm chitosan was well prepared, and chitosan oligosaccharides were produced with this chitosan.

Fig. 1
figure 1

Deacetylation of mealworm chitosan analysis by 1H-NMR. (A) Peaks indicate acetyl group at 2.1 ppm. The upper line represents the original graph, while the lower line represents the enlarged graph. M-chitosan: the chitosan prepared from mealworm. (B) Deacetylation of chitin to chitosan

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Establishment of optimal conditions for MCOS production by chitosanase using RSM

MCOS production conditions were optimized through a RSM that analyzed the reducing sugar and DE values according to chitosanase reaction conditions (substrate concentration, reaction time, and amount of added enzyme). The equation obtained from the central composite design in Table 1 is as follows:

Y = 242.854-46.8890X + 1.873Y-8.384Z + 2.907 × 2 – 0.0029Y2 + 0.311Z2 – 0.311Z2 – 0.085XY + 0.003YZ + 0.4660XZ (R2 = 0.987).

X: substrate (%), Y: enzyme addition (mg/g of sample), Z: time (h).

Table 1 Enzyme reaction condition optimization by response surface methodology of mealworm chitosan oligosaccharide
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The optimal reaction conditions predicted by RSM were 2.5% substrate, 30 mg/g of enzyme, and 6 h reaction time; the reducing sugar content was 135.94 mg/g of sample. Optimum reaction conditions by actual measurement were substrate 2.5%, enzyme 30 mg/g of sample, reaction time 6 h, and reducing sugar content was 129.28 mg/g of sample (Table 2).

Table 2 Response surface methodology (RSM) results and actual results for optimal reaction conditions for the production of mealworm chitosan oligosaccharide by chitosanase
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These results were confirmed using a 3D graph (Fig. 1). When the reducing-sugar content was measured as the substrate concentration, enzyme concentration, and reaction time increased in the 3D graph, it tended to decrease as the substrate concentration increased from 2.5 to 7%. Regardless of the amount of enzyme added and the reaction time, the average reducing sugar content was 110.74 mg/g when the substrate concentration was 2.5%, but when the substrate concentration was increased to 5%, the reducing sugar content was 68.86 mg/g (~ 32% decrease). In contrast, when the substrate concentration was increased to 7%, the reducing sugar was 65.6 mg/g. As the amount of enzyme added increased from 10 to 20 mg/g, the reducing sugar content increased slightly. When the amount of enzyme added was increased to 30 mg/g, the reducing sugar content was similar to that of the reaction with 10 mg/g enzyme addition. The amount of enzyme added did not significantly affect the increase in reducing sugar content. Analysis of the effect of reaction time on the reducing sugar content, when the reaction time was 6 h, the highest average reducing sugar content was 81.08 mg/g. As a result of measuring the effect of the reaction time on the reducing sugar content, the highest reducing sugar content was 81.08 mg/g when reacted for 6 h. According to these results, substrate concentration had the greatest effect on the optimal reaction conditions, and the optimal substrate concentration was determined to be 2.5%. The optimal reaction conditions for MCOS production of MCOS are substrate, 2.5%; enzyme, 30 mg/g of sample; and reaction time, 6 h (Fig. 2).

The theoretically optimal reaction conditions according to the response surface analysis method were 2.5% substrate, 30 mg/g enzyme, and 6 h reaction time. The theoretical reducing sugar content was 135.94 mg/g. Even in the actual measurement results, the optimal conditions were substrate 2.5%, enzyme addition amount 30 mg/g of sample, reaction time 6 h, and actual reducing sugar content was 129.28 mg/f of MCOS (Table 2).

Fig. 2
figure 2

Response surface plots for the effect of independent variables on dependent (substrate concentration, enzyme addition and reaction time) for production of chitosan oligosaccharide from mealworm chitosan by chitosanase

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MCOS analysis by MALDI-TOF and FT-IR

The molecular weight was measured by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Figure 3 shows the peaks corresponding to the dimers (375.045), trimers (525.214), tetramers (693.243), pentamers (826.296), and hexamers (987.360).

Fourier-transform infrared (FT-IR) analysis was performed to measure the chemical structures of mealworm chitosan, MCOS, and commercial COS (Fig. 4). Chitosan and MCOS showed similar patterns, whereas commercial COS showed a somewhat weaker peak pattern. Commercial COS showed a weak peak pattern at 3500–2500 cm-1, unlike chitosan or MCOS. There is a difference in the peak at 3200–3500 cm-1, where different vibrations corresponding to OH and amine groups overlap. Chitosan, MCOS, and commercial COS showed peaks at 2849, 2884, and 2885 cm-1, respectively, which appeared due to the absorption of the C-H stretching vibration of methyl or methine. The amide I, amide II, and amide III bands of chitosan, MCOS, and commercial COS showed peaks at 1620/1620/1602, 1553/1555/1505, and 1310/1309/1317 cm-1, respectively. The peak corresponding to the amide band of commercial COS appeared to be somewhat low. This was presumed to show a weakened amide band peak owing to the H-extraction of C-1 and C-2 when preparing COS by chemical decomposition, such as oxidative decomposition. In addition, the reducing sugar content of commercial COS was 117.93 ± 1.70 mg/g and that of MCOS was 14.68 ± 0.24 mg/g (data not shown).

Fig. 3
figure 3

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum of mealworm chitosan oligosaccharide. Peaks indicate [M + H+], dimer (343.18), trimer (504.198), tetramer (665.24), pentamer (826.29), and hexamer (987.36)

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Fig. 4
figure 4

Fourier-transform infrared (FT-IR) spectra of mealworm chitosan, mealworm chitosan oligosaccharide, and commercial chitosan oligosaccharide

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Characteristics of commercial COS and MCOS

To evaluate the properties of commercial COS and MCOS prepared using chitosanase, the solubility, WPC, FBC, ash, and water content were measured (Table 3). There was no significant difference in solubility between commercial COS and MCOS. MCOS showed significantly higher WPC and FBC than commercial COS (p < 0.05 and p < 0.01, respectively). There was no significant difference in the ash and water contents between the commercial OCSs and MCOSs.

Table 3 Physicochemical characterizations of chitosan oligosaccharide
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Effect of MCOS on the growth of lactic acid bacteria

To evaluate the effect of MCOS as a dietary fiber, we measured the cell proliferation activity of L. casei, L. acidophilus and B. bifidum (Fig. 5). As the amount of MCOS increased, the cell masses of the three strains tended to increase. L. casei increased rapidly until 12 h of cultivation and then showed a tendency to increase slowly. L. acidophilus slowly increased after 36 h of culture. B. bifidum showed a slight increase in cell mass after 24 h of culture. The addition of 1.0% MCOS was superior to the addition of 0.5% MCOS in terms of the cell growth of the three lactic acid bacteria. When 1% commercial COS (C-COS 1%) was added, the cell growth of the three strains was similar to that of 1% MCOS. MCOS appears to act as a carbon source for LAB growth of lactic acid bacteria.

Fig. 5
figure 5

Effect of mealworm chitosan oligosaccharide on the growth of L. casei, L. acidophilus and B. bifidum. Data are represented as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. CON by Tukey multiple test. ns: no significant

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Effect of MCOS on SCFA production in vitro anaerobic fermentation

Acetic acid, propionic acid, and butyric acid, the major short-chain fatty acids (SCFAs) produced by intestinal microorganisms, were measured after adding 1% and 2% MCOS via in vitro anaerobic fermentation (Fig. 6). The acetic acid content increased to a higher level than that of the other SCFA. SCFA showed a tendency to gradually increase as fermentation time increased, but when 1% and 2% MCOS were added (MCOS-1 and MCOS-2, respectively), SCFA did not show any significant increase after 24 h. When 1% and 2% MCOS were added, the SCFA content was higher than that in the CON. There was no significant difference in SCFA between 1% MCOS (MCOS-1) and 2% MCOS (MCOS-2). When 1% commercial COS (C-COS 1%) was added, changes in SCFAs similar to those of 1% MCOS were observed. Fermentation for 24 h with 1% MCOS appeared to be the most suitable for SCFA production.

Fig. 6
figure 6

Effect of mealworm chitosan oligosaccharide (MCOS) on short-chain fatty acid (SCFA) production in vitro anaerobic fermentation. Data are represented as mean ± SD. *p < 0.05, and ***p < 0.001 vs. CON by Tukey multiple test. ns: no significant. CON: basal medium, MCOS1: basal medium with 1% MCOS, MCOS2: basal medium with 2% MCOS, C-COS1: basal medium with 1% commercial COS

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Effect of MCOS on microbiota changes in in vitro anaerobic fermentation

To measure the effect of MCOS on the intestinal microbiota, in vitro anaerobic fermentation was performed by adding MCOS, and C-COS 1% was used. Species richness and diversity according to MCOS addition were investigated using the Chao1, Shannon, and Simpson indices. There was no significant difference in the Chao1 index, which indicates species richness, between the basal medium (CON) and the groups supplemented with 1% and 2% MCOS. C-COS 1% addition (C-COS 1) also showed no significant difference in CHAO1 from other groups. The Shannon and Simpson indices, which are indicators of species diversity, were significantly lower in the 1% and 2% MCOS groups than in the CON group (p < 0.001). As the amount of MCOS increased, the Shannon and Simpson indices decreased in a concentration-dependent manner (Fig. 7). However, the addition of 1% C-COS (C-COS 1) did not show any significant difference in these species diversities with CON.

β-diversity analysis was performed to compare and analyze the similarity between microbial groups using principal coordinate analysis (PCoA) based on weighted UniFrac distance matrices (Fig. 8). The group with 1% MCOS (MCOS-1) showed a clear separation of clusters from the CON group. In addition, the MCOS-2 group showed a tendency for the distribution of clusters to be separated from CON and MCOS-1 groups. However, C-COS1 group showed some cluster overlaps with MCOS-1 and MCOS-2 groups.

Fig. 7
figure 7

Alpha-diversity (CHAO1, Shannon and Simpson index) and β-diversity in vitro anaerobic fermentation by MCOS addition. Data are represented as mean ± SEM. ***p < 0.001 vs. CON by Tukey multiple test. ns: no significant. CON: basal medium, MCOS1: basal medium with 1% MCOS, MCOS2: basal medium with 2% MCOS, C-COS1: basal medium with 1% commercial COS

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Fig. 8
figure 8

Changes in microbiota at the phylum level in vitro anaerobic fermentation by MCOS addition. Data are represented as mean ± SD. *p < 0.05, ***p < 0.001 vs. CON by Tukey multiple test. CON: basal medium, MCOS1: basal medium with 1% MCOS, MCOS2: basal medium with 2% MCOS, C-COS1: basal medium with 1% commercial COS

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Changes in microbiota in vitro anaerobic fermentation according to the addition of MCOS

Changes in the flora were measured after the addition of 1% and 2% MCOS, and 1% C-COS to the basal medium. At the phylum level, Bacillota (formerly Firmicutes) and Pseudomonadota (formerly Proteobacteria) were the main bacterial strains. When 1% MCOS and 1% C-COS were added, the relative abundances of Bacillota were significantly higher than that in the CON group (p < 0.001), whereas the relative abundances of Pseudomonadota were significantly lower (p < 0.001, Fig. 8). The addition of 2% MCOS showed no significant difference in the relative abundances of Bacillota and Pseudomonadota compared to the CON group. The relative abundance of Actinobacteria was significantly lower in the MCOS-2 group than that in the CON group (p < 0.05). MCOS 1% and 2% (MCOS-1 and MCOS-2), and C-COS 1% addition had higher relative abundances of Verrucomicrobiota than CON group (p < 0.001, respectively).

Changes in the microbiota with the addition of MCOS and C-COS were measured at the genus level (Fig. 9). The relative abundance of Escherichia was significantly higher than that in the CON group in a concentration-dependent manner as the amount of MCOS increased. Lactobacillus belong to the Lactobacillaceae family, and changes in their relative abundance following the addition of 1% MCOS and 1% C-COS were similar. The addition of 1% MCOS (MCOS-1) and 1% C-COS (C-COS1) resulted in a significant difference compared with the CON group (p < 0.001 and p < 0.01, respectively), but MCOS-2 showed no significant difference.

The relative abundances of Romboutsia, Turicibacter, and Akkermansia were significantly higher in the MCOS-1, MCOS-2 and C-COS1 groups than in the CON group, but the relative abundance in MCOS-2 was lower than that in MCOS-1 (Fig. 9). With the addition of MCOS and C-COS, the relative abundances of Enterococcus, Bacteroides, Morganella, and Proteus were significantly lower than those in the CON group (p < 0.001). The relative abundance of these strains in both the C-COS1 and MCOS1 groups exhibited similar patterns; however, Morganella showed a tendency to be more prevalent in the C-COS1 group compared to the MCOS1 group.

Fig. 9
figure 9

Changes in microbiota at the genus level in vitro anaerobic fermentation by MCOS addition. Data are represented as mean ± SD. **p < 0.01 and ***p < 0.001 vs. CON by Tukey multiple test. CON: basal medium, MCOS1: basal medium with 1% MCOS, MCOS2: basal medium with 2% MCOS, C-COS1: basal medium with 1% commercial COS

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Discussion

COS prepared from chitosan is a partially hydrolyzed product and is a biopolymer composed of β-(1–4)-linked N-acetyl-D-glucosamine and deacetylated glucosamine units. Furthermore, COS are absorbed by the intestine and induce various biological effects [10, 16]. Deacetylated COS is an indigestible carbohydrate that has a high potential for use as a prebiotic. Therefore, in this study, the conditions for MCOS production by chitosanase were established from mealworm chitosan using RSM.

Physical, chemical, and enzymatic methods are widely used to produce oligosaccharides from chitosan [17]. This enzymatic method hydrolyzes chitosan under mild reaction conditions and has substrate specificity, allowing the degree of reaction and size of the product to be controlled. However, large-scale production is not economically feasible because the enzymes used to produce COS are expensive. Nevertheless, they are widely used because they have no harmful side effects on the human body and are ecofriendly [18]. MCOS production by chitosanase treatment showed an optimal production efficiency when 2.5% of the substrate and enzyme, corresponding to 3% of the substrate, were added and allowed to react for 6 h (Table 2; Fig. 1). The MCOS produced by chitosanase is composed of oligosaccharides equivalent to DP 2–5 (Fig. 3). COS prepared using enzymes or chemical methods has been reported to have a DP of 2–10 [19, 20]. A comparison of the FT-IR spectra of MCOS and commercial COS (Fig. 4) showed that the FT-IR spectrum of MCOS had a peak pattern similar to that of mealworm chitosan, whereas the commercial COS showed a weak amide band peak (Fig. 4). The amide band appears to be weak because commercial COS are produced by a chemical method rather than by an enzyme treatment [21]. Furthermore, commercial COS and MCOS showed poor solubility; however, WBC count and FBC were significantly higher in MCOS than in commercial COS (Table 3). The difference in the FBC of chitosan increases with the degree of deacetylation or/and molecular weight [22]. These results show that when the particle size is similar, the larger molecular weight causes fat molecules to become embedded in the long chains of chitosan, increasing FBC [23]. Since no separation process was performed when producing chitosan oligosaccharides, the FBC ability can be increased by the chitosan remaining in chitosan oligosaccharides [21]. MCOS have better WBC and FBC counts but similar characteristics to commercial COS.

Low-molecular-weight COS exert prebiotic effects on Lactobacillus spp. and Bifidobacterium sp. [24,25,26]. Deacetylated COS appears to have a prebiotic effect as it is not digested by intestinal enzymes [27]. COS, which has a DP of 3–6 and is 90% deacetylated, also increases the level of lactic acid bacteria in the mouse cecum and lowers the level of harmful intestinal microorganisms [26]. Prebiotics are fermented by intestinal microorganisms to produce organic acids and SCFA, and the amount or ratio of SCFA produced is affected by the type of monosaccharide that makes up the prebiotics, type of bond, degree of polymerization, and solubility [28]. SCFAs are used as nutrients by mucosal cells, and human or rat colonic mucosal cells receive 5–10% of their energy from SCFAs. Furthermore, SCFA promote proliferation of the colonic mucosa [29] and plays an important role in the prevention and treatment of intestinal diseases [30]. Addition of MCOS 1% showed a significant increase in SCFA during in vitro anaerobic fermentation, and the acetic acid content was the highest among the SCFA (Fig. 6). Acetic acid is the main SCFA in the intestines, produced by lactic acid-producing bacteria using COS as a carbon source, and is recognized as a very important factor in the regulation of the intestinal environment. It has been reported that the production of SCFAs and the resulting decrease in pH inhibit the growth of harmful intestinal bacteria, promote immune function, and increase the absorption of minerals [31]. As a result of changes in SCFA and microbiota due to COS in vitro fermentation by human feces, an increase in the relative abundance of Bacillota contributed to the increase in SCFA [32]. Additionally, the relative abundances of Bacillota and Verrucomicrobiota were related to an increase in butyric acid content. In contrast, an increase in the butyric acid levels resulted in a decrease in the relative abundance of Bacteroides. The increase in acetic acid resulted in a decrease in the relative abundance of Bacteroides and Enterococcus at the genus level. These changes were also observed in vitro anaerobic fermentation with MCOS, with similar SCFA content and changes in the microbiota (Fig. 8). Addition of 1% MCOS showed similar effects to addition of 1% C-COS.

As MCOS is also composed of oligosaccharides corresponding to DP 2–6, anaerobic fermentation was performed in vitro to determine its effect on the intestinal microbiota (Figs. 8 and 9). The addition of MCOS significantly increased the relative abundances of Romboutsia, Turicibacter, and Akkermansia compared to the CON group. The relative abundance of these strains further increased when 1% MCOS was added. Romboutsia, which increases with the addition of MCOS, is frequently observed in the human intestine and is presumed to play an important role in maintaining host health [33, 34]. The relative abundance of Turicibacter increases [35, 36], some studies have reported conflicting results [37, 38]. The relative abundance of Turicibacter, which is closely associated with fat intake, decreased with the addition of MCOS. Akkermansia is a representative beneficial intestinal bacterium like Lactobacillus and is involved in the proliferation of intestinal cells, suppression of inflammation, and metabolic diseases [39, 40]. The addition of 1% MCOS significantly increased the relative abundance of Lactobacillus. Although research on changes in the intestinal microbiota caused by COS is limited, Lactobacillus increased in the cecum and feces of broilers and weaned pigs administered COS. Additionally, an increase in Bifidobacterium sp., Lactobacillus sp., and Prevotella and a decrease in harmful bacteria have been reported [41, 42]. The relative abundances of Enterococcus and Bacteroides, which are commensal bacteria, decreased with the addition of MCOS. Enterococcus are commensal bacteria found in the intestines of various mammals [43]. Bacteroides play various roles as intestinal microorganisms, including commensal, mutualist, and beneficial. Some Bacteroides strains act as beneficial or harmful microorganisms, depending on the host’s intestinal environment. Low levels of Bacteroides have been observed in the intestines of inflammatory bowel disease patients [44]. The relative abundances of Morganella observed in cancer tissues [45] and Proteus, a known susceptible pathogen [46], were reduced by the addition of MCOS. MCOS increased the relative abundance of beneficial bacteria, such as Lactobacillus, Romboutsia, Turicibacter, and Akkermansia and lowered the relative abundance of commensal bacteria, such as Enterococcus and Bacteroides and harmful bacteria, such as Morganella and Proteus. The effect of improving the intestinal microbiota by the addition of 1% and 2% MCOS was somewhat different, and a better prebiotic effect was expected with 1% MCOS than with 2% MCOS. Given that 1% MCOS exhibited comparable effectiveness in improving gut microbiota as 1% C-COS, it may serve as a practical substitute for commercial COS. These results are presumed to be related to the physiological functions of COS, such as enhanced immunity and suppressed fat accumulation. Mealworm MCOS produced by chitosanase contain oligosaccharides with a degree of polymerization of 2–6, and MCOS containing these oligosaccharides showed a prebiotic effect similar to commercial COS. Further studies will be the changes in microbiota caused by chitosan oligosaccharide derived from mealworm using human feces.

Materials and methods

Materials

Chitosan was prepared according to a previously reported method [47]. Chitosanase (Bacillus sp., 35,000 U/g) was purchased from Amicogen Co., Ltd. (Jinju, Korea), a company specializing in food and bio enzymes, and was used in this study. Commercial COS (C-COS) prepared from crustacea was purchased from TCI Chemicals (Tokyo, Japan) and d-glucosamine was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Deacetylation of mealworm chitosan analysis by 1H-NMR (Nuclear Magnet Resonance spectrometer)

For the check of the acetyl group of mealworm chitosan to make chitosan oligosaccharides, it was analyzed using 1H-NMR. To dissolve reagent grade chitosan (Sigma-Aldrich 417963., St. Louis, MO, USA) and mealworm chitosan, 0.06 g of chitosan was added to 40 ml of 1% HCL and sonicated in a water bath at 60 °C for 2 h. The sample supernatant after centrifuge, and the concentration of chitosan in the sample solution was 0.2%. Deutrium oxide (system peak: 4.81) was used as a solvent for NMR analysis, and 100 mL of Deutrium oxide was added to 1.0 mL of the sample and analyzed (AVANCE 600., 600 Hz, Bruker Co.).

Preparation of mealworm chitosan oligosaccharide (MCOS)

MCOS was prepared by treatment with chitosanase using chitosan prepared using a previously described method [47] as a substrate. RSM was used to optimize the chitosanase reaction and to produce MCOS. The substrate concentration (S), reaction time (t), and amount of enzyme added to the substrate (E/S) were selected as factor variables, and response surface analysis was performed using the central composite design method. To optimize the chitosanase reaction conditions, the substrate concentration (2.5, 5.0, and 7.5%), reaction time (6, 8, and 10 h), and enzyme addition amount (10, 20, and 30 mg/g) were used as independent variables were used as independent variables to treat chitosanase with 50 mM acetate buffer (pH 5.5; Sigma-Aldrich) and shaking at 160 rpm at 50 °C (Table 1). After the chitosanase reaction, the reducing sugar content and dextrose equivalents (DE) were measured as dependent variables. The experimental results were analyzed using the RSM of Minitab software (version 16, Minitab Inc., State College, PA, USA). Data were analyzed by multiple regression analysis by applying a quadratic reaction model equation, and the quadratic reaction equation used was as follows.

Y = A + aX + bY + cZ + dX2 + eY2 + fZ2 + hYZ + iXZ.

X is (substrate concentration), Y (enzyme addition amount), and Z is (reaction time).

Reducing sugar analysis and DE calculation

The reducing sugar content of chitosan oligosaccharides, a chitosanase product, was measured using the dinitrosalicylic acid (DNS) method [48]. After mixing 3 mL of the DNS solution with 1 mL of the sample solution, the mixture was heated in boiling water for 5 min, and then cooled at room temperature. The absorbance was measured at 550 nm using a microplate reader (Biotek Synergy Mx, Biotek Instruments, Winooski, VT, USA), and the content was calculated using a calibration curve with glucose as a standard. The dextrose equivalent (DE) was calculated by quantifying sugar and reducing sugars [49].

Dextrose equivalents (%) = reducing sugar content (%) / solid content (%) × 100.

Matrix-assisted laser desorption/ionization- mass spectrometry (MALDI-TOF MS) and fourier-transform infrared (FT-IR) analysis

Matrix-assisted laser desorption/ionization- mass spectrometry (MALDI-TOF MS) was performed to confirm the presence of MCOS oligomers. Before MALDI-TOF MS analysis, MCOS were desalted using a microanalyzer (Asahi Chemicals Co., Tokyo, Japan) equipped with a Neosepta cartridge (AC-110-10, ASOM Co., Tokyo, Japan). The molecular weight of MCOS obtained from the enzymatic hydrolysis of mealworm chitosan was measured using MALDI-TOF MS (Voyager-DETM STR Biospectrometry Workstation Inc., NCIRF, Applied Biosystems Inc.). The MCOS molecular weight was calculated based on the molecular weight of glucosamine. The structure of MCOS was determined by fourier-transform infrared (FT-IR) analysis, and the FT-IR spectrum was measured at a wavelength of 4000 –700 cm-1 by the UATR technique, using PerkinElmer® Spectrum™100 (Waltham, MA, USA).

Physiochemical characteristic evaluation of MCOS

To evaluate the physiochemical properties of MCOS and commercial COS, their solubility, water binding capacity (WBC), fat binding capacity (FBC), ash content, and water content were measured. Solubility was measured using 1% acetic acid [50]. WBC and FBC counts were measured according to the method described by Wang and Kinsella [51]. The ash content was measured by weighing after incineration at 600 °C. Moisture content was determined by measuring the weight before and after drying at 105 °C.

Growth activity of MCOS on lactic acid bacteria

Lactobacillus acidophilus KCTC3140, Lactocaseibacillus casei KCTC3110, and Bifidobacterium bifidum KCTC3357, obtained from the Korean Collection for Type Cultures (KCTC, Daejeon, Korea), were used to measure the effects of MCOS on cell growth. The strains were cultured in a modified peptone yeast extract fructose (PYF) medium. The modified PYF medium was 10 g of yeast extract, peptone 5 g, tryptone 5 g, L-cysteine hydrochloride monohydrate 0.5 g, and salt solution 40 mL per liter, and the pH was adjusted to 7.5. The composition of the salt solution was 0.2 g of CaCl2·2H2O, 0.5 g of MgSO4·7H2O, 1 g of K2HPO4, 1 g of KH2PO4, 10 g of NaHCO3, and 2 g of NaCl per liter. MCOS were added at 0.5 and 1%, respectively, and cultured at 37°C for 48 h, and absorbance was measured at 600 nm to evaluate cell mass.

In vitro anaerobic fermentation

In vitro anaerobic fermentation was performed as previously described [52]. The basic medium used was 2 g of peptone, 2 g of yeast extract, 0.1 g of NaCl, 0.04 g of K2HPO4, 0.04 g of KH2PO4, 0.01 g of MgSO4(H2O)7, 0.01 g of CaCl2(H2O)6, 2 g of NaHCO3, 2 mL of Tween 80, 0.02 g of hemin, 10 mL of Vitamin K1, 0.5 g of L-cysteine hydrochloride monohydrate and 0.5 g of bile salts per liter. MCOS was added to basal medium at final concentrations of 1% and 2% (w/v). The pH of the medium was adjusted to 6.8 with a 0.1 M NaOH solution, resazurin (1 mg/L) was added, and anaerobic conditions were maintained by replacing the medium with oxygen-free nitrogen gas. Fresh cecal contents were homogenized for 5 min using BOSCH ultracompact dissolved in PBS (100mM, pH 7.0), inoculated in an amount equivalent to 4% of the medium volume, and incubated at 37°C for 48 h. To minimize differences in cecal microbiota between mice, cecum from three mice was mixed, and the experiment was repeated three times.

Analysis of short-chain fatty acids

Samples were collected from the culture medium at 24 h intervals using a sterile syringe, centrifuged (8000 × g) for 15 min at room temperature, and the filtrated supernatant was used for analysis. Gas chromatography (GC; YL-6100 GC system, Young Lin Co., Anayang, Korea) was equipped with a capillary column (DB-FFAP 123–3253, 50 m × 0.32 mm × 0.50 µM), flame ionization detector, and an autosampler. The injector and detector port temperatures were 200 and 240 °C, respectively. The carrier gas was N2 at a flow rate of 1.4 mL/min.

DNA extraction and 16 S rRNA gene sequencing

The frozen samples were thawed and centrifuged at 10,000 rpm for 10 min. DNA was extracted from the centrifuged pellet using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany), and the concentration was standardized to 5 ng/µL using the Qubit dsDNA HS assay kit (Invitrogen, Carlsbad, CA, USA). DNA was subjected to two-step PCR using the 341 F and 806R primer sets to amplify V3-4 of the variable region of the 16 S rRNA gene [53, 54]. Illumina MiSeq (Illumina) library production was performed according to the manufacturer’s protocol, and sequencing was performed using CH Lab (Seoul, Korea).

Statistical analysis

All experiments were repeated thrice, and the significance of the experimental values was analyzed using the SPSS software package (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA). One-way ANOVA was performed at p < 0.05, and significance was verified using Tukey’s multiple range test.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

COS:

Chitosan oligosaccharide

DE:

Dextrose equivalentes

DNS:

Dinitrosalicylic acid

DP:

Degree of polymerization

FBC:

Fat binding capacity

FT-IR:

Fourier-transform infrared

GC:

Gas chromatography

MALDI-TOF MS:

Matrix-assisted laser desorption/ionization-mass spectrometry

MCOS:

Mealworm chitosan oligosaccharides

PCoA:

Principal coordinate analysis

PYF:

Peptone yeast extract fructose

RSM:

Response surface methodology

WBC:

Water binding capacity

References

  1. Islam N, Hoque M, Taharat SF (2023) Recent advances in extraction of chitin and chitosan. World J Microbiol Biotechnol 39:28. https://doi.org/10.1007/s11274-022-03468-1

    Article  CAS  Google Scholar 

  2. Hahn T, Tafi E, Paul A, Salvia R, Falabella P, Zibek S (2020) Current state of chitin purification and chitosan production from insects. J Chem Technol Biotechno 95:2775–2795. https://doi.org/10.1002/jctb.6533

    Article  CAS  Google Scholar 

  3. Khayrova A, Lopatin S, Varlamov V (2021) Obtaining chitin, chitosan and their melanin complexes from insects. Int J Biol Macromol 167:1319–1328. https://doi.org/10.1016/j.ijbiomac.2020.11.086

    Article  CAS  PubMed  Google Scholar 

  4. Mohan K, Ganesan AR, Muralisankar T, Jayakumar R, Sathishkumar P, Uthayakumar V, Chandirasekar R, Revathi N (2020) Recent insights into the extraction, characterization, and bioactivities of chitin and chitosan from insects. Trends Food Sci Technol 105:17–42. https://doi.org/10.1016/j.tifs.2020.08.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang ST, Tsai CC, Shih MC, Tsai ML Flavor-related applications of chitin and chitosan in foods: Effect of structure and properties on the efficacy. In Chitosan for Biomaterials Iii: structure-property relationships, Jayakumar R, Prabaharan M (eds); Springer International Publishing: Cham, Switzerland, 287:169–202. https://doi.org/10.1007/12_2021_90

  6. Agarwal T, Chiesa I, Costantini M, Lopamarda A, Tirelli MC, Borra OP, Varshapally SVS, Kumar YAV, Koteswara Reddy G, De Maria C, Zhang LG, Maiti TK (2023) Chitosan and its derivatives in 3D/4D (bio) printing for tissue engineering and drug delivery applications. Int J Biol Macromol 246:125669. https://doi.org/10.1016/j.ijbiomac.2023.125669

    Article  CAS  PubMed  Google Scholar 

  7. Pal P, Pal A, Nakashima K, Yadav BK (2021) Applications of chitosan in environmental remediation: a review. Chemosphere 266:128934. https://doi.org/10.1016/j.chemosphere.2020.128934

    Article  CAS  PubMed  Google Scholar 

  8. Thambiliyagodage C, Jayanetti M, Mendis A, Ekanayake G, Liyanaarachchi H, Vigneswaran S (2023) Recent advances in Chitosan-based applications-A review. Materials 16:2073. https://doi.org/10.3390/ma16052073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Petroni S, Tagliaro I, Antonini C, D’Arienzo M, Orsini SF, Mano JF, Brancato V, Borges J, Cipolla L (2023) Chitosan-based biomaterials: insights into chemistry, properties, devices, and their biomedical applications. Mar Drugs 21:147. https://doi.org/10.3390/md21030147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chae SY, Jang MK, Nah JW (2005) Influence of molecular weight on oral absorption of water soluble chitosans. J Control Release 102:383–394. https://doi.org/10.1016/j.jconrel.2004.10.012

    Article  CAS  PubMed  Google Scholar 

  11. Muanprasat C, Chatsudthipong V (2017) Chitosan oligosaccharide: Biological activities and potential therapeutic applications. Pharmacol Ther 170:80–97. https://doi.org/10.1016/j.pharmthera.2016.10.013

    Article  CAS  PubMed  Google Scholar 

  12. Roberfroid M, Gibson GR, Hoyles L, McCartney AL, Rastall R, Rowland I, Wolvers D, Watzl B, Szajewska H, Stahl B, Guarner F, Respondek F, Whelan K, Coxam V, Davicco MJ, Léotoing L, Wittrant Y, Delzenne NM, Cani PD, Neyrinck AM, Meheust A (2010) Prebiotic effects: metabolic and health benefits. Br J Nutr 104:S1–S63. https://doi.org/10.1017/s0007114510003363

    Article  CAS  PubMed  Google Scholar 

  13. Mohan K, Rajan DK, Ganesan AR, Divya D, Johansen J, Zhang SB (2023) Chitin, chitosan and chitooligosaccharides as potential growth promoters and immunostimulants in aquaculture: a comprehensive review. Int J Biol Macromol 251:126285. https://doi.org/10.1016/j.ijbiomac.2023.126285

    Article  CAS  PubMed  Google Scholar 

  14. Lee HW, Park YS, Jung JS, Shin WS (2002) Chitosan oligosaccharides, dp 2–8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp. Anaerobe 8:319–324. https://doi.org/10.1016/s1075-9964(03)00030-1

    Article  PubMed  Google Scholar 

  15. Lavertu M, Xia Z, Serreqi AN, Berrada M, Rodrigues A, Wang D, Buschmann MD, Gupta A (2003) A validated 1H NMR method for the determination of the degree of deacetylation of Chitosan. J Pharm Biomed Anal 6:1149–1158. https://doi.org/10.1016/s0731-7085(03)00155-9

    Article  Google Scholar 

  16. Fernandes JC, Eaton P, Nascimento H, Belo L, Rocha S, Vitorino R, Amado F, Gomes J, Santos-Silva A, Pintado ME, Malcata FX (2008) Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure. Biomacromolecules 9:3346–3352. https://doi.org/10.1021/bm800622f

    Article  CAS  PubMed  Google Scholar 

  17. Naveed M, Phil L, Sohail M, Hasnat M, Baig MMFA, Ihsan AU, Shumzaid M, Kakar MU, Mehmood Khan T, Akabar MD, Hussain MI, Zhou QG (2019) Chitosan oligosaccharide (COS): an overview. Int J Biol Macromol 129:827–843. https://doi.org/10.1016/j.ijbiomac.2019.01.192

    Article  CAS  PubMed  Google Scholar 

  18. Jeon YJ, Shahidi F, Kim SK (2000) Preparation of chitin and chitosan oligomers amd their applications in physiological functional foods. Food Rev Int 16:159–176. https://doi.org/10.1081/fri-100100286

    Article  CAS  Google Scholar 

  19. Jeon YJ, Kim SK (2000) Continuous production of chitooligosaccharides using a dual reactor system. Process Biochem 35:623–632. https://doi.org/10.1016/s0032-9592(99)00118-1

    Article  CAS  Google Scholar 

  20. Kaur S, Dhillon GS (2015) Recent trends in biological extraction of chitin from marine shell wastes: a review. Crit Rev Biotechnol 35:44–61. https://doi.org/10.3109/07388551.2013.798256

    Article  CAS  PubMed  Google Scholar 

  21. Chen YX, Li JN, Li QQ, Shen YY, Ge ZC, Zhang WW, Chen SG (2016) Enhanced water-solubility, antibacterial activity and biocompatibility upon introducing sulfobetaine and quaternary ammonium to Chitosan. Carbohydr Polym 143:246–253. https://doi.org/10.1016/j.carbpol.2016.01.073

    Article  CAS  PubMed  Google Scholar 

  22. Xia WS, Liu P, Zhang JL, Chen J (2011) Biological activities of chitosan and chitooligosaccharides. Food Hydrocoll 25:170–179. https://doi.org/10.1016/j.foodhyd.2010.03.003

    Article  CAS  Google Scholar 

  23. Zhang CX, Tang XD, Cheng JA (2008) The utilization and industrialization of insect resources in China. Entomol Res 38:S38–S47. https://doi.org/10.1111/j.1748-5967.2008.00173.x

    Article  Google Scholar 

  24. Matute AIR, Cardelle-Cobas A, García-Bermejo AB, Montilla A, Olano A, Corzo N (2013) Synthesis, characterization and functional properties of galactosylated derivatives of chitosan through amide formation. Food Hydrocoll 33:245–255. https://doi.org/10.1016/j.foodhyd.2013.03.016

    Article  CAS  Google Scholar 

  25. Macchi G (1996) A new approach to the treatment of obesity: Chitosan’s effects on body weight reduction and plasma cholesterol’s levels. Acta Tox Th 17:303–322. https://doi.org/10.4236/jbbs.2018.81001​

    CAS  Google Scholar 

  26. Lee HW, Park YS, Jung JS, Shin WS (2002) Chitosan oligosaccharides, dp 2–8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp. Anaerobe 8:319–324. https://doi.org/10.1016/s1075-9964(03)00030-1

    Article  PubMed  Google Scholar 

  27. Pan XD, Chen FQ, Wu TX, Tang HG, Zhao ZY (2009) Prebiotic oligosaccharides change the concentrations of short-chain fatty acids and the microbial population of mouse bowel. J Zhejiang Univ Sci B 10:258–263. https://doi.org/10.1631/jzus.b0820261

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kumar V, Sinha AK, Makkar HPS, de Boeck G, Becker K (2012) Dietary roles of non-starch polysachharides in human nutrition: a review. Crit Rev Food Sci Nutr 52:899–935. https://doi.org/10.1080/10408398.2010.512671

    Article  CAS  PubMed  Google Scholar 

  29. Ou LB (2004) Study on immunoregulatory function of dietary fiber. Food Ind Nutr 9:26–30. https://koreascience.kr/article/JAKO200414714202568.page

    Google Scholar 

  30. Wong JMW, de Souza R, Kendall CWC, Emam A, Jenkins DJA (2006) Colonic health: fermentation and short chain fatty acids. J Clin Gastroenterol 40:235–243. https://doi.org/10.1097/00004836-200603000-00015

    Article  CAS  PubMed  Google Scholar 

  31. Hijova E, Chmelarova A (2007) Short chain fatty acids and colonic health. Bratisl Med J 108:354–358. https://www.researchgate.net/profile/Emilia-Hijova/publication/5647158_Short_chain_fatty_acids_and_clonic_health/links/549290280cf2302e1d072e37/Short-chain-fatty-acids-and-colonic-health.pdf

    CAS  Google Scholar 

  32. Liu W, Li XQ, Zhao ZL, Pi XG, Meng YY, Fei DB, Liu DQ, Wang X (2020) Effect of chitooligosaccharides on human gut microbiota and antiglycation. Carbohydr Polym 242:116413. https://doi.org/10.1016/j.carbpol.2020.116413

    Article  CAS  PubMed  Google Scholar 

  33. Milani C, Ticinesi A, Gerritsen J, Nouvenne A, Lugli GA, Mancabelli L, Turroni F, Duranti S, Mangifesta M, Viappiani A, Ferrario C, Maggio M, Lauretani F, De Vos W, van Sinderen D, Meschi T, Ventura M (2016) Gut microbiota composition and Clostridium difficile infection in hospitalized elderly individuals: a metagenomic study. Sci Rep 6:25945. https://doi.org/10.1038/srep25945

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ricaboni D, Mailhe M, Khelaifia S, Raoult D, Million M (2016) Romboutsia Timonensis, a new species isolated from human gut. New Microbes New Infect 12:6–7. https://doi.org/10.1016/j.nmni.2016.04.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Peters BA, Dominianni C, Shapiro JA, Church TR, Wu J, Miller G, Yuen E, Freiman H, Lustbader I, Salik J, Friedlander C, Hayes RB, Ahn J (2016) The gut microbiota in conventional and serrated precursors of colorectal cancer. Microbiome 4:69. https://doi.org/10.1186/s40168-016-0218-6

    Article  PubMed  PubMed Central  Google Scholar 

  36. Geng JW, Fan H, Tang XD, Zhai HQ, Zhang ZG (2013) Diversified pattern of the human colorectal cancer microbiome. Gut Pathog 5:2. https://doi.org/10.1186/1757-4749-5-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Akutko K, Matusiewicz K (2017) Campylobacter concisus as the etiologic agent of gastrointestinal diseases. Adv Clin Exp Med 26:149–154. https://doi.org/10.17219/acem/61428

    Article  PubMed  Google Scholar 

  38. Warren RL, Freeman DJ, Pleasance S, Watson P, Moore RA, Cochrane K, Allen-Vercoe E, Holt RA (2013) Co-occurrence of anaerobic bacteria in colorectal carcinomas. Microbiome 1:16. https://doi.org/10.1186/2049-2618-1-16

    Article  PubMed  PubMed Central  Google Scholar 

  39. Everard A, Belzer C, Geurts L, Ouwerkerk JP, Druart C, Bindels LB, Guiot Y, Derrien M, Muccioli GG, Delzenne NM, de Vos WM, Cani PD (2013) Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 110:9066–9071. https://doi.org/10.1073/pnas.1219451110

    Article  PubMed  PubMed Central  Google Scholar 

  40. Alam A, Leoni G, Quiros M, Wu HX, Desai C, Nishio H, Jones RM, Nusrat A, Neish AS (2016) The microenvironment of injured murine gut elicits a local pro-restitutive microbiota. Nat Microbiol 1:15021. https://doi.org/10.1038/nmicrobiol.2015.21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kong XF, Zhou XL, Lian GQ, Blachier F, Liu G, Tan BE, Nyachoti CM, Yin YL (2014) Dietary supplementation with chitooligosaccharides alters gut microbiota and modifies intestinal luminal metabolites in weaned Huanjiang mini-piglets. Livest Sci 160:97–101. https://doi.org/10.1016/j.livsci.2013.11.023

    Article  Google Scholar 

  42. Guan ZW, Feng Q (2022) Chitosan and chitooligosaccharide: the promising non-plant-derived prebiotics with multiple biological activities. Int J Mol Sci 23:6761. https://doi.org/10.3390/ijms23126761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dubin K, Pamer EG (2017) Enterococci and their interactions with the intestinal microbiome. Microbiol Spectr 5:1–7. https://doi.org/10.1128/microbiolspec.bad-0014-2016

    Article  CAS  Google Scholar 

  44. Rajilic-Stojanovic M, Biagi E, Heilig HGHJ, Kajander K, Kekkonen RA, Tims S, de Vos WM (2011) Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology 141:1792–1801. https://doi.org/10.1053/j.gastro.2011.07.043

    Article  CAS  PubMed  Google Scholar 

  45. Chen WG, Li FL, Ling ZX, Tong XJ, Xiang C (2012) Human intestinal lumen and mucosa-Associated microbiota in patients with colorectal cancer. PLoS ONE 7:e39743. https://doi.org/10.1371/journal.pone.0039743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Armbruster CE, Mobley HLT (2012) Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis. Nat Rev Microbiol 10:743–754. https://doi.org/10.1038/nrmicro2890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kim H, Kim H, Ahn Y, Hong KB, Kim IW, Choi RY, Suh HJ, Han SH (2023) The Preparation and physiochemical characterization of chitin using Alcalase. Molecules 28:3254. https://doi.org/10.3390/molecules28073254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. https://doi.org/10.1021/ac60147a030

    Article  CAS  Google Scholar 

  49. Delheye G, Moreels E (1988) Dextrose equivalent measurements on commercial syrups. Starch-Starke 40:430–432. https://doi.org/10.1002/star.19880401107

    Article  CAS  Google Scholar 

  50. Tammineni N, Rasco B, Powers J, Nindo C, Ünlü G (2014) Bovine and fish gelatin coatings incorporating tannins: effect on physical properties and oxidative stability of salmon fillets. J Food Chem Nutr 2:93–102. https://esciencepress.net/journals/index.php/JFCN/article/view/741

    Google Scholar 

  51. Wang JC, Kinsella JE (1976) Functional properties of novel proteins - alfalfa leaf protein. J Food Sci 41:286–292. https://doi.org/10.1111/j.1365-2621.1976.tb00602.x

    Article  CAS  Google Scholar 

  52. Mou J, Li Q, Shi W, Qi X, Song W, Yang J (2020) Chain conformation, physicochemical properties of fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus and its in vitro fermentation by human gut microbiota. Carbohyd Polym 228:115359. https://doi.org/10.1016/j.carbpol.2019.115359

    Article  CAS  Google Scholar 

  53. Singh V, Muthuramalingam K, Kim YM, Park S, Kim SH, Lee J, Hyun C, Unno T, Cho M (2021) Synbiotic supplementation with prebiotic Schizophyllum commune derived β-(1,3/1,6)-glucan and probiotic concoction benefits gut microbiota and its associated metabolic activities. Appl Biol Chem 64:7. https://doi.org/10.1186/s13765-020-00572-4

    Article  CAS  Google Scholar 

  54. Jeon D, Singh V, Unno T (2021) In vitro investigation of food effects on human gut microbiota. J Appl Biol Chem 64:75–81. https://doi.org/10.3839/jabc.2021.01

    Article  Google Scholar 

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Acknowledgements

Thank you to the Rural Development Administration for supporting us in carrying out the research.

Funding

This research was supported by the Rural Development Administration (RS-2021-RD009646).

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  1. Hyemi Kim and Ga Yeong Cheon contributed equally to this work.

Authors and Affiliations

  1. Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, Seoul, 02841, Republic of Korea

    Hyemi Kim & Hyung Joo Suh

  2. Department of Food Science and Nutrition, Jeju National University, Jeju, 63243, Republic of Korea

    Ga Yeong Cheon & Ki-Bae Hong

  3. Biomedical Research Center, Research Affairs, Korea University Anam Hospital, Seoul, 02841, Republic of Korea

    Jae Hee Kim & Sung Hee Han

  4. Department of agricultural biology, Wanju, 55365, Republic of Korea

    Ra-Yeong Choi & In-Woo Kim

  5. Transdisciplinary Major in Learning Health Systems, Department of Healthcare Sciences, Graduate School, Korea University, Seoul, 02841, Republic of Korea

    Hyung Joo Suh

  6. Institute of Human Behavior & Genetics, Korea University, Seoul, 02841, Republic of Korea

    Sung Hee Han

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Contributions

Conceptualization: SHH; data curation: HK and GYC; formal analysis: HK and GYC; methodology: JHK, R-YC and I-WK; software: K-BH; validation: R-YC and I-WK and HJS; writing—original draft: HK and GYC and SHH; writing—review and editing: K-BH and SHH All authors have read and agreed to the published version of the manuscript.

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Kim, H., Cheon, G.Y., Kim, J.H. et al. Preparation of chitosan oligosaccharides from chitosan of tenebrio molitor and its prebiotic activity. Appl Biol Chem 67, 84 (2024). https://doi.org/10.1186/s13765-024-00937-z

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