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ABSTRACT: Studies have reported that theabrownin can moderate the lipid metabolism and intestinal microbiota, thereby affecting the health of humans and model animals, however the research on laying hens is scarce. The present study aimed to investigate the effects of dietary theabrownin supplementation on lipid metabolism, microbial composition and ovarian function in laying hens. A total of 80 laying hens (25 wk of age) were fed with normal diet (CON) and normal diet +100 mg/kg theabrownin (PT group) for 12 wk. The results showed that the addition of theabrownin enhanced villus height of duodenum and decreased crypt depth of jejunum (P < 0.05). At the same time, compared with CON, the concentration of IL-6 and the mRNA expression of IL-1β and IL-6 were decreased significantly in PT group (P < 0.05). Dietary theabrownin reduced the concentration of total cholesterol and glycerol, while decreased lipid droplet optical density in liver (P < 0.05). Compared with CON group, the mRNA expression of PPARγ, HMG-CoAS, ACC were down-regulated and the mRNA expression of CYP8B1 was up-regulated in PT group (P < 0.05). The ACE, Chao1 and Observed_species indexes in cecum microbiota were increased by PT group intervention (P < 0.05). Dietary PT supplementation enhanced the relative abundance of Firmicutes (phylum), Lactobacillus (genus) and the Firmicutes to Bacteroidetes ratio, and reduced the relative abundance of Bacteroidetes (phylum) in cecum (P < 0.05). The organic acids and its derivatives were up-regulated by theabrownin intervention in serum metabolites (P < 0.05). Dietary theabrownin supplementation resulted in higher mRNA expression of Bcl-2 and SIRT1 in ovary and increased the concentration of estradiol in serum (P < 0.05). These discovering indicated that dietary theabrownin supplementation enhanced the intestinal function and influenced serum metabolism by improving intestinal morphology, microbiota community structure and reducing the concentration and expression of inflammatory cytokines in intestine. Dietary theabrownin reduced hepatic lipid deposition and it also decreased the cell apoptosis rate to improve ovarian function and egg weight which were associated with the SIRT1 pathway.

This study was conducted to investigate the effects of betaine on lipid metabolism and m6A RNA methylation in finishing pigs fed a low-energy diet. The results showed that betaine significantly enhanced serum concentration of triglyceride and reduced the relative weight of flare fat (P

ABSTRACT: Yucca schidigera extract (YSE) is a green feed additive that is known to reduce toxic gas emissions and promote intestinal health in animal production. This study investigated the potential of dietary YSE supplementation to mitigate the negative effect of Clostridium perfringens and coccidia infection on productive performance and gut health in laying hens. A total of 48 Lohmann gray laying hens (35 wk of age) were randomly allotted to 1 of 2 groups (n = 24) fed with either a basal diet or a YSE-supplemented diet for 45 d. From d 36 to 45, half of the hens in each group were orally administrated with Clostridium perfringens type A and coccidia. This challenge impaired productive performance and egg quality (P < 0.05), destroyed jejunal morphology and functions (P < 0.05), induced jejunal epithelial cell apoptosis (P < 0.05), and downregulated the antioxidant capacity and Nrf2 pathway expression of jejunal mucosa (P < 0.05) in laying hens. Supplementing YSE in the laying hen diet, to some extents, improved productive performance and egg quality (P < 0.05), and alleviated the effect of challenge on morphology, functions, cell apoptosis, and antioxidant capacity in the jejunum (P < 0.05). Overall, the results suggested that dietary YSE supplementation might mitigate the negative effects of Clostridium perfringens and coccidia infection on gut health, and thereby improve the productive performance and egg quality of laying hens, possibly through enhancing the antioxidant capacity of the jejunum.

Dietary fiber (DF) has long been looked as an essential “nutrients” both for animals and humans as it can promote the intestinal tract development and modulate the intestinal epithelium functions and the gut microbiota. This study was conducted to investigate the influences of wheat bran fiber (WBF) on growth performance and intestinal epithelium functions in Xiangcun pigs. Twenty Xiangcun pigs with 60 days of age were divided to two groups and exposed to a basal diet (BD) or BD containing 4.3% wheat bran fiber (WFD). WFD improved the average daily gain (ADG) and feed-to-gain ratio (F:G) (p

The effect of 25-hydroxyvitamin D (25OHD) on the immune response of laying hens is not well elucidated. This study investigated the effects of 25OHD on egg production, egg quality, immune response, and intestinal health of laying hens challenged with Escherichia coli lipopolysaccharide (LPS). One hundred and sixty laying hens at 45 wk of age were randomly divided into 4 dietary treatments with 10 replicates of 4 birds. Hens were fed the corn-soybean based diets contained either 0 or 80 µg/kg 25OHD for 8 wks. At wk of 53 wk, birds of each dietary treatment were injected into the abdomen with 1.5 mg/kg body weight of either LPS or saline a day at 24-h intervals for continuous 7 d. LPS injection significantly decreased (PLPS < 0.05) egg laying rate, feed intake and feed efficiency; while the supplementation of 25OHD increased (PInteraction < 0.05) egg laying rate, feed efficiency and decreased (PInteraction < 0.05) the broken egg rate in layers under LPS injection. LPS challenge decreased (PLPS < 0.05) eggshell strength, eggshell thickness, albumen height and Haugh unit, while dietary 25OHD supplementation increased eggshell strength and eggshell thickness (P25OHD < 0.05). The serum proinflammatory factors [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6)], endotoxin and diamine oxidase (DAO) levels were higher in layers under LPS challenge (PLPS < 0.05); whereas the dietary addition of 25OHD were shown to decrease (P25OHD < 0.05) serum IL-1β and IL-6 concentration irrespective of LPS challenge and led to a higher serum 25OHD level and a reduction in endotoxin concentration in layers under LPS challenge (PInteraction < 0.05). The layers under LPS challenge had higher crypt depth and lower villus height/crypt depth (V/C) ratio in duodenum and jejunum (PLPS < 0.05), while feeding 25OHD were shown to have decreasing effect on crypt depth and increasing effect V/C ratio in layers under LPS challenge (PInteraction < 0.05). Layers under LPS challenge had lower mRNA expression of intestinal barrier associated proteins (claudin-1 and mucin-1) (PLPS < 0.05), while the addition of 25OHD up-regulated claudin-1 and mucin-1 expression (Pinteraction < 0.05). Lower antioxidant enzymes activities, including superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) and higher malondialdehyde (MDA) content in jejunum were found in layers challenged with LPS (P25OHD < 0.05). The effect of 25OHD reversed the effect of LPS on SOD, T-AOC, and MDA content (PInteraction< 0.05). These results suggest that supplementing 80 µg/kg 25OHD in diets may elevate laying performance and egg quality through the improvement of intestinal barrier function, antioxidant capacity, and decreased the proinflammatory cytokines levels in laying hens with Escherichia coli LPS challenge.

ABSTRACT: The underlying mechanism between the gut microbiota and reproductive function is not yet well-known. This study was conducted to investigate the effect of the administration of fecal microbiota transplantation (FMT) from highly laying rate donors on the cecal microbiota, intestinal health and ovarian function in broiler breeders. A total of 60 broiler breeders (53 wk of age) were selected by their laying rate [high (HP, 90.67 ± 0.69%; n = 10) and low (LP, 70.23 ± 0.87%; n = 20)]. The LP breeders were then be transplanted with fecal microbiota from HP hens (FMTHP; n = 10) or the same dosage of PBS (FMTCON; n = 10) for 28 d. The results revealed that FMT from HP donors increased egg-laying rate and serum hormone levels [17β-estradiol (E2), anti-Müller hormone], also decreased proinflammatory cytokine levels (interleukin-6, interleukin-8, tumor necrosis factor-α) of LP breeders (P < 0.05). The FMTHP group breeders had higher villus height, villus height/crypt depth ratio, and upregulated mRNA expression of jejunum barrier-related gene (ZO-2 and mucin-2) and estrogen, follicle-stimulating hormone (FSH) and anti-Müller hormone (AMH) receptor genes (ESR1, ESR2, FSHR, AMHR) (P < 0.05) than FMTCON group. FMT from HP donors led to higher mRNA expression of Bcl2 and sirtuin1 (SIRT1), while it downregulated the proapoptotic genes (Bax, caspase-3, caspase-8, and caspase-9) mRNA expressions in ovary compared with the FMTCON breeders (P < 0.05), and this pattern was also observed in HP donors. Also, HP breeder had higher observed_species and alpha-diversity indexes (Chao1 and ACE) than FMTCON group, while FMTHP can increase observed_species and alpha-diversity indexes (Chao1 and ACE) than FMTCON group (P < 0.05). The bacteria enrichment of Firmicutes (phylum), Bacteroidetes (phylum), Lactobacillus (genus), Enterococcus (genus), and Bacteroides (genus) were increased by FMTHP treatment. The genera Butyricicoccus, Enterococcus, and Lactobacillus were positively correlated with egg-laying rate. Therefore, cecal microbiomes of breeders with high egg-laying performance have more diverse activities, which may be related to the metabolism and health of the host; and FMT from high-yield donors can increase the hormone secretion, intestinal health, and ovarian function to improve egg-laying performance and the SIRT1-related apoptosis and cytokine signaling pathway were involved in this process.

Colitis is a common and complex intestinal inflammatory disease in which lactate, a metabolite of anaerobic glycolysis, plays a crucial role. Our study aimed to investigate the alleviated effect of lactate in colitis, and to provide a nutritional measure to alleviate colitis injury. The variations in colonic lactate in piglets with DSS-induced colitis were investigated in Experiment 1 (Exp.1). Thirty weaned pigs were allotted into three groups and sampled at different stages of DSS-induced colitis (days 0, 5, and 7). The colonic level of lactate and interleukin 10 (IL-10) was significantly decreased on day 5 when compared to day 0. Colonic lactate, IL-10, and G protein receptor 81 (GPR81) levels were significantly increased on day 7 when compared to day 5. Sixty weaned piglets were assigned to control (basal diet), DSS (basal diet with DSS gavage), or lactate (2% lactate supplementation diet with DSS gavage) groups to investigate the effects of lactate on DSS-induced colitis in Experiment 2 (Exp.2). Lactate reduced the disease activity index (DAI), DSS-induced impairment of colonic structure in response to the critical inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) when compared with the DSS group. Furthermore, GPR-81 levels, colonic M2 macrophages, and IL-10 levels, the colonic antioxidant capacity, colonic butyrate levels were increased, and eventually improved growth performance post-colitis. The results of this study show that lactate was decreased at the peak of colitis, accumulated in subsidized colitis. Furthermore, dietary lactate supplementation helped to alleviate DSS-induced colitis injury.

Resveratrol (RV) is associated with protection against oxidative stress to improve health, however the effect of RV in layers under oxidative stress (OS) is limited. The objective of this experiment was to investigate the negative effect of OS and protective effects of RV against OS in laying hens. 40 Lohmann layers (25-wk-old; BW = 1.44±0.10 kg) were allocated to four treatments in a 2 × 2 factorial arrangement with either RV (0 or 600 mg/kg) or intraperitoneal injection of tert-butyl hydroperoxide (tBHP) (0 or 800 μmol/kg BW) for 31 days. The results shown that the hens challenged with tBHP presented lower egg-laying rate, feed intake, feed efficiency and higher defective egg rate (P(tBHP)

The difference in microbiota was examined for breeders with different egg-laying rates, and the impact of dietary Enterococcus faecium (EF) was also determined in the present study. A total of 256 Arbor Acres broiler breeders (48-wk-old) were used in a 2 × 2 factorial design, which encompassed 2 egg-laying rate levels [average (average egg laying: AP, 80.45 ± 0.91%) and low (lower egg laying: LP, 70.61 ± 1.16%)] and 2 different dietary groups [control (no additive), 6 × 108 cfu/kg EF]. The results showed that the AP breeders presented a lower egg weight, feed conversion ratio, abdominal fat rate, and serum leptin level (P(laying) ≤ 0.05) as well as a higher egg-laying rate (P(laying)

ABSTRACT: The purpose of this experiment is to explore the effects of dietary supplementation of benzoic acid, Enterococcus faecium, and essential oil complex (BEC) on coccidia and Clostridium perfringens challenge in laying hens. A total of 80 Lohmann gray laying hens (35 wk old) were allocated to 4 treatments in a 2 × 2 factorial arrangement with the main effects of Clostridium perfringens type A (CP) and coccidia challenge (with or without challenge) and 2 BEC levels (0 and 1,000 mg/kg). The total experimental period was 6 wk. The results showed that: the challenge group significantly decreased the laying rate and average daily feed intake (ADFI) of laying hens (PChallenge < 0.01). The BEC + challenge group significantly increased the laying rate and decreased the feed conversion ratio (FCR) of laying hens (PBEC < 0.05). The challenge significantly decreased the thickness, strength, and relative weight of eggshell (PChallenge < 0.05). The BCE + challenge group significantly increased the relative weight and strength of the eggshell (P BEC < 0.05). The challenge significantly increased the crypt depth of the duodenum, jejunum and ileum, and decreased the villus-to-crypt ratio (V/C) (PChallenge < 0.01). The BEC + challenge group decreased the crypt depth of the duodenum and jejunum, and increased the V/C of the duodenum (PBEC < 0.01). The pathological scores of duodenum and jejunum of the challenge group were significantly higher than other groups (PChallenge < 0.01), while the BEC + challenge group had lower pathological scores of jejunum (PBEC < 0.01). The challenge significantly decreased the mRNA expression of Occludin, Mucin-2, Zonula occluden-1 (ZO-1) (P challenge < 0.05); whereas the BEC group significantly increased the expression of Occludin, Mucin-2, and Claudin-1 mRNA (P BEC < 0.05). The challenge significantly increased the level of interleukin 1β (IL-1β) in the jejunum (PChallenge < 0.05). Taken together, adding BEC to the diet can improved production performance and egg quality of layers, by protecting intestinal health against Clostridium perfringens type A (CP) and coccidia challenge.

This study was conducted to determine the effects of 25-hydroxycholecalciferol (25-OH-D3) on performance, egg quality, tibia quality, and serum hormones concentration in laying hens reared under high stocking density. A total of 800 45-week-old Lohmann laying hens were randomly allotted into a 2 × 2 factorial design with 2 levels of dietary 25-OH-D3 levels (0 and 69 μg/kg) and 2 rates of stocking densities [506 (low density) and 338 (high density) cm2/hen]. Laying hens were monitored for 16 wk. High stocking density decreased laying rate, egg weight, and feed intake compared with low stocking density (P < 0.01) during 1 to 8 wk and 1 to 16 wk. Overall, high stocking density increased eggshell lightness value and decreased shell redness and yellowness value, strength, thickness, and relative weight compared with low stocking density (P < 0.05). Dietary supplementation with 25-OH-D3 reduced the value of the eggshell lightness and increased its yellowness and eggshells weight (P ≤ 0.05). The increase in eggshell thickness was more pronounced when 25-OH-D3 was supplemented to layers under high stocking density (interaction, P < 0.05). Layers under high stocking density had lower ash content and calcium content in the tibia than layers under low stocking density (P = 0.04); dietary 25-OH-D3 increased tibia strength compared with no addition (P = 0.05). Layers under high stocking density had higher serum concentrations of 25-OH-D3, corticosterone (CORT), lipopolysaccharide (LPS), and osteocalcin (OC; P < 0.05), lower content of parathyroid hormone (PTH) compared with layers under low stocking density (P < 0.01). Dietary 25-OH-D3 increased serum concentration of 25-OH-D3, carbonic anhydrase (CA), and calcitonin (CT) (P < 0.01) and reduced corticosterone, lipopolysaccharide and osteocalcin concentration (P ≤ 0.05). The increase effect in PTH was more pronounced when 25-OH-D3 was supplemented to layers under high stocking density (interaction, P = 0.05). Overall, the results gathered in this study indicate that high stocking density result in reducing production performance, shell color and quality, and tibia health, whereas dietary 25-OH-D3 was able to maintain tibia health and to mitigate the negative impact of high stocking density on productive performance.

ABSTRACT: The objective of this experiment was to investigate the influence of dietary tributyrin on reproduction performance and ovary function of broiler breeders with different egg laying rate. Two hundred fifty-six AA broiler breeders (48-wk-old) were allocated to 4 treatment in a 2 × 2 factorial arrangement with the main effects of tributyrin supplementation (0 and 1,000 mg/kg tributyrin [TRI]) and 2 egg laying rate levels (average [AR, 81.01 ± 0.79%] and low [LR, 70.98 ± 0.95%]). The results shown that the LR breeders presented higher egg weight, but lower egg laying rate, qualified egg rate and feed efficiency than the AR breeders (P(laying) < 0.05). Also, the superoxidase dismutase (SOD) activity in magnum was lower while malondialdehyde (MDA) was higher in ovary and magnum of LR breeders than that in the AR breeders (P(laying) < 0.05). Dietary supplementation with tributyrin significantly enhanced egg weight (P(TRI) < 0.05), increased albumen height as well as Haugh unit (HU) in AR breeders (P(interaction) < 0.05), and also had higher total antioxidant capacity (T-AOC) and lower MDA in ovary (P(TRI) < 0.05). The cell apoptosis rate and proapoptosis related gene expression (caspase 8, 9 and Bax) in the ovary of LR breeders was higher, while anti-apoptosis related gene (Bcl-2) expression were lower in LR breeders when compared with the AR breeders (P(laying) < 0.05). Dietary supplementation with tributyrin decreased the cell apoptosis rate and downregulated caspase 9 expression in LR breeders (P(Interaction) < 0.05), up-regulated the Bcl-2 expression in both 2 breeders (P(TRI) < 0.05). These findings suggest that the breeders with lower egg laying rate also characterized by deteriorate ovary function indicated by lower antioxidant capacity and higher cell apoptosis rate. Dietary supplementation with tributyrin increased egg albumen quality, decreased ovarian proapoptosis related gene expression to improve reproductive tract function; and the positive effect on egg albumen quality is more pronounced in average reproductive breeders.

ABSTRACT: Essential oils (EO) are concentrated hydrophobic liquids containing volatile aromatic compounds obtained from plants, which have properties as withdrawn antibiotic growth promoters. The objective of this study was to explore the effects of EO on growth performance, digestibility, immunity and intestinal health in broilers. A total of 500 1-day-old Arbor Acre broilers were randomly put into five groups with 10 replicate cages containing 10 birds each. Birds in the 5 groups were fed a basal diet (CON), and basal diet with 50, 100, 200 or 400 mg/kg EO (EO0.5, EO1, EO2 and EO4) for 42 d respectively. Birds were euthanized at 21d and 42 d, blood and tissue samples were collected. In the study, the digestibility of DM, GE and EE in groups with EO supplementation were significantly increased compared with CON group (P < 0.05). However, only EO2 and EO4 significantly increased the digestibility of CP compared with CON group (P < 0.05). In contrast to CON group, EO0.5 and EO1 in jejunum at 21 d, and EO1 in jejunum at 42 d markedly increased the activity of sucrase (P < 0.05). In addition, the level of SOD of EO2 and EO4 in serum at 21 d was significantly increased compared with CON group (P < 0.05). What's more, the concentration of intestinal mucosa SIgA in jejunum and ileum at 21 d of groups with EO supplementation was significantly increased compared with CON group (P < 0.05). Moreover, V/C in jejunum at 21 d of groups with EO supplementation, CD in jejunum at 42 d was also significantly increased to compare with CON group (P < 0.05). Furthermore, the expression levels of critical genes associated with nutrient transportation (i.e., GLUT2, SGLT1, SLC38A, SLC79A and SLC27A4) and barrier function (TJP1) were quadratically and linearly up-regulated in jejunum and ileum with EO supplementation (P < 0.05). These results suggest that EO has a positive impact on growth, immunity and intestinal health in broilers, and 200 mg/kg of EO was recommended in broiler diet.

To explored the protective effect of manno-oligosaccharides (MOS) on intestinal epithelium exposure to Enterotoxigenic Escherichia coli (ETEC), thirty-two pigs were assigned into four treatments and fed with basal or MOS-containing (0.3% MOS) diet. Pigs were challenged by ETEC or culture medium. Results show that MOS not only reduced diarrhea incidence and fecal E. coli abundance but decreased serum D-lactate and DAO concentrations upon ETEC-challenge (P

ABSTRACT: The high stocking density is a major stress factor that adversely affects the health and performance of poultry. Therefore, the object of this study was conducted to explore whether dietary 25-hydroxyvitamin D (25-OH-D3) could improve gut health of laying hens reared under high stocking density. A 2 × 2 factorial design was used in this 16-week study, in which 800 45-week-old Lohmann laying hens were allocated into two levels of dietary 25-OH-D3 levels (0 and 69 µg/kg) and two rates of stocking densities [506 (low density, LD) and 338 (high density, HD) cm2/hen]. Compared with the layers with LD, the layers with HD had lower crypt depth in duodenum (P(Density) < 0.05), lower short chain fatty acid (propionic and butyric acid) contents in cecum (P(Density) < 0.05), and lower mRNA expression of intestinal barrier associated protein (claudin-1, mucin-1 and mucin-2). Exposed layer to HD also led to lower intestinal antioxidative capacity [superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and higher malondialdehyde (MDA) content] in small intestine (P(Density) < 0.05), lower bacterial abundance of Bacteroidetes (phylum), Spirochaetes (phylum) and Bacteroides (genus; P(Density) < 0.05), higher bacterial enrichment of Lactobacillaceae (genus) and Firmicutes/Bacteroidetes ratio (P(Density) < 0.05) in cecum. Dietary 25-OH-D3 increased the villus height in duodenum and jejunum (P(25-OH-D3) < 0.05), decreased Chao 1 and ACE indexes in cecum (P(25-OH-D3) < 0.05), and it also up-regulated the mRNA expression of claudin-1, mucin-1 and mucin-2 (P(25-OH-D3) < 0.05). Layers treated with 25-OH-D3 led to an enhanced antioxidative enzyme activity of CAT (P(25-OH-D3) < 0.05). Additionally, the effect of 25-OH-D3 reversed the effect of HD on T-AOC and MDA content (P(Interaction) < 0.05). In HD layers, 25-OH-D3 administration decreased the enrichment of Bacteroidetes (phylum), increased Firmicutes (phylum), and Firmicutes/Bacteroidetes ratio (P(Interaction) < 0.05). These results suggest that supplementing 25-OH-D3 in diets may elevate gut health through the improvement of intestinal barrier function, antioxidant capacity and cecal microbiota composition in laying hens with high stocking density.

This study was conducted to explore whether dietary pectic oligosaccharide (POS) supplementation could improve gut health of broiler breeders with different egg-laying rates. A 2 × 2 factorial design was used in this study. Two hundred fifty-six Arbor Acres broiler breeders (48 wk of age), including 128 average egg-laying rate and 128 low egg-laying rate (LELR) birds, were randomly fed with the diets supplemented with or without 200 mg kg−1 of POS (n = 8). The trial lasted for 8 wk. Compared with average egg-laying rate broiler breeders, LELR broiler breeders had lower laying rate and qualified egg rate (P

In this study, we investigated the effects of alginate oligosaccharide (AOS) on the intestinal microbiota and intestinal barrier integrity in weaned pigs. Twenty-four weaned pigs were equally assigned to either a control group (fed the basal diet) or an AOS group (fed the basal diet supplemented with 100 mg/kg AOS). AOS upregulated caecal and colonic tight junction protein expression levels but downregulated caecal pro-inflammatory cytokine expression levels. AOS increased the caecal and colonic abundance of phospho-adenosine 5′-monophosphate-activated protein kinase α (p-AMPKα) and decreased the abundance of caecal intra-nuclear nuclear factor-κB (NF-κB) p65. Furthermore, AOS increased the caecal prevalence of several bacteria belonging to Bacteroidetes and Firmicutes as well as the caecal and colonic concentrations of short-chain fatty acids (SCFAs). Thus, AOS alters the intestinal microbiota composition and SCFA concentrations in weaned pigs, thereby influencing the intestinal inflammatory responses and barrier integrity by regulating NF-κB and AMPK pathways.

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the β-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the β-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14–52 (disulfide bond between β-sheets B2 and B3) and Xyn2C59–149 (disulfide bond between β-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the β-sheet core is due to the stable structure of the entire protein.