Mulberry leaf and konjac flour compound dietary fiber improves digestion and metabolism in elderly mice with high-fish-protein diet by regulating gut microbiota structure and intestinal tissue repair

Abstract

Ensuring sufficient protein intake, efficient digestion, and optimal absorption are crucial for the elderly. This study aims to investigate the potential of a compound dietary fiber, consisting of mulberry leaf and konjac flour (CMK), to enhance the digestion and absorption of a high-fish-protein diet in elderly mice. Results showed that CMK effectively reduced the number of unique peptide segments, generated short-chain fatty acids (SCFA) in feces, improved the content of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), amino acid, and urea nitrogen in serum, activated the contents of pepsin, trypsin, and erepsin, and enhanced the expression of glutamate dehydrogenase (GDH), peptide transporter 1 (PepT1), and aminopeptidase N (APN). Furthermore, CMK demonstrated its ability to decrease the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10), lipopolysaccharide (LPS), and lipopolysaccharide binding protein (LBP), while increase the abundance of beneficial bacteria, such as Lactobacillus and Blautia. In conclusion, CMK proved effective in enhancing the digestion and metabolism of protein in elderly mice through the regulation of gut microbiota structure and intestinal tissue repair.

Graphical abstract

Highlights

• •CMK improve the digestion and metabolism of protein by regulation gut microbiota.
• •CMK improve the digestion and metabolism of protein by intestinal tissue repair.
• •CMK can improve protein digestion and metabolism in elderly mice fed with a high fish protein diet

1. Introduction

As individuals age, the deterioration of intestinal barrier, absorption, and immune functions necessitates ensuring sufficient protein intake to counteract catabolic conditions associated with chronic diseases and age-related decline (Cardon-Thomas, Riviere, Tieges, & Greig, 2017). All countries suggest that the elderly should consume more protein than the recommended amount (1.2 g/kg.d) for healthy individuals, with at least 50% of it being of high-quality, such as animal protein and whey protein. While fish meat is an ideal protein source for the elderly due to its low-fat content, high-protein levels, and essential amino acid composition (Cruz-jentoftj et al., 2019), excessive protein consumption can potentially harm the stomach and intestines, leading to kidney damage and osteoporosis (Widder et al., 2016). Additionally, a high-protein diet's difficulty in digestion and absorption has been linked to an increase in potential pathogenic bacteria and pro-inflammatory microorganisms, resulting in changes to colonic epithelial structure and colitis, detrimental to the intestinal nervous system and motility. Therefore, it is imperative to improve protein digestion and absorption in the elderly while increasing their protein intake for overall health and well-being.

The gastrointestinal tract, particularly the intestinal mucosa, plays a critical role in protein metabolism (Ying et al., 2020), making it a focal point for nutritional intervention aimed at preserving gastrointestinal health and boosting metabolic capacity (Xiao et al., 2023). Studies have shown that digestive enzyme is pivotal in promoting the digestion and absorption of human proteins, and dietary fibers such as xanthan gum, guar gum, and locust bean gum can stimulate the production of these enzymes, aiding in protein digestion and absorption (Chen et al., 2020a, Chen et al., 2020b). Peptidases, small peptide transporters, and amino acid metabolism enzymes all contribute significantly to protein metabolism. Additionally, intestinal microorganisms play important roles in protein metabolism in the human body (Ni et al., 2019). Notably, the gut microbiota in elderly individuals differs significantly from that of their younger counterparts, with a significant increase in the number of Lactobacilli, Bacteroides, and Clostridium in the intestine, contributing to impaired intestinal function and metabolic disorders or diseases.

Research has indicated that protein energy malnutrition in mice affects the local immune system in the intestine, and that supplementation with probiotic fermented milk can increase dendritic cells, macrophages, IgA⁺ cells, and cytokines like TNF-α, IFN-γ, and IL-12, thus restoring the intestinal mucosa's ability to resist Salmonella typhimurium (Ha & Woodward, 1997). Therefore, it is important for the elderly to maintain a balanced diet to control gut microbiota modifications and reduce the rise of intestinal inflammation caused by a high-protein diet (Xiao et al., 2023). Despite the increasing attention to the interaction between intestinal aging and changes in its microenvironment (Zhu et al., 2018), there is a notable lack of comprehensive analysis regarding the impact of high-protein diets on the stomach and intestinal structure in the elderly. The exact mechanism of dietary fiber in regulating protein digestion remains not fully understood. To address this gap, our research aims to improve overall health through diet, focusing on natural, green, and safe food ingredients.

Mulberry leaf and konjac, two abundant sources of dietary fiber commonly grown in China, have been consumed for many years, offering numerous nutritional and physiological benefits. However, their usage has mainly been limited to primary processed food and animal feed, resulting in low added value. Mulberry leaf is rich in polyphenols, flavonoids, polysaccharides, alkaloids, amino acids, and minerals like calcium. It possesses the capability to eliminate inflammatory factors (Guo et al., 2020). Konjac glucomannan (KGM), the main component of konjac flour, is a water-soluble dietary fiber known for its high viscosity and capacity to improve intestinal health and bolster the immune system (Zhang et al., 2021; Zhang et al., 2022). Animal studies have demonstrated that the compound dietary fiber composed of mulberry leaf and konjac flour (CMK) can reduce the pH of the intestine, elevate the content of short-chain fatty acids (SCFA) (Deng, Luo, Zhang, & Zhong, 2020) and enhance beef protein metabolism in mice consuming a high in beef-protein diet (Deng, Zhong, Peng, & Zhu, 2023). In contrast to beef, fish meat is an ideal source of complete protein for the elderly due to its low-fat content, high-protein levels, and essential amino acid composition. The amount and ratio of essential amino acids are most suitable for human needs, and fish muscle fibers are short, with a loose protein tissue structure and high water content, making it easy to digest and absorb (Cruz-jentoftj et al., 2019). Study has found that there is a significant difference in the digestibility of fish and beef in the stomachs of pigs (p ﹤0.05) (Bauchart et al., 2007). Additionally, Wen's in vivo experiments in rats showed that the digestion rate of fish (46.98%) was significantly higher than that of beef (42.75%) during the hydrolysis process of pepsin (p ﹤0.05) (Wen et al., 2015). Our previous study also revealed that CMK can enhance intestinal enzyme activity and regulate the intestinal microbiota of elderly mice consuming a high protein diet rich in beef (Deng et al., 2023). However, whether CMK can improve the digestion and absorption of a high fish-protein diet in aged mice, as well as its effect on gut microbiota structure and intestinal tissue, remains unexplored.

This study aimed to analyze the effects of CMK on the protein metabolism of elderly mice fed a high-fish-protein diet. Following CMK intervention, we evaluated various parameters related to protein metabolism, including the apparent digestibility of protein in animal feces, serum biochemical indicators, amino acids and peptide sequences in feces, gastric and intestinal well-being, gastrointestinal sections, small intestinal texture, small intestinal mucosal tissue, activities of pepsin, trypsin, and erepsin, and the expression of proteins: glutamate dehydrogenase (GDH), peptide transporter 1 (PepT1), and aminopeptidase N (APN). Additionally, we investigated the impact of CMK on gut microbiota. The findings indicated that CMK can improve the digestion and metabolism of elderly mice with a high-fish-protein diet by regulating gut microbiota structure and intestinal tissue repair. This suggests the potential of CMK as a functional food additive for improving the digestive function of the elderly, further increasing the value of mulberry leaf and konjac agricultural and sideline resources for rural revitalization.

2. Materials and methods

2.1. Materials

The mulberry leaf powder of Jialing No. 20 was supplied by Dazhu County Today Agricultural Products Development Co., Ltd. (Sichuan, China). Its protein content was 19.79%, crude fiber content was 13.63%, crude fat content was 6.71%, moisture content was 3.53%, and total flavonoid content was 3.13%. The konjac flour was provided by Chongqing Kangjiake Food Company (Chongqing, China), and it was found to have a konjac glucomannan (KGM) content of 93%, with an average molecular weight of 9.52 × 10⁵ Da. This KGM is a β-1,4-glycosidic linked glucomannan made up of mannose and glucose in a molar ratio of 1.41:1, and its viscosity in an aqueous solution of 1% (w/w) surpasses 40,000 mPa•s. Jiangzhong Pharmaceutical Co., Ltd. (Jiangxi, China) created a digestive pill as a positive control to promote stomach movement and digestion, containing radix pseudistellariae, yam, malt, orange peel, and other Chinese medicinal herbs. Fresh silver carp was obtained from Chongqing local Supermarket (Chongqing, China).

Elisa kits for Pepsin, Trypsin, and PRSS7 were procured from Shanghai Fusheng Industrial Co., Ltd. (Shanghai, China), while other Elisa kits were acquired from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). LC-MS grade methanol, formic acid, and acetonitrile were acquired from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Antibodies, buffer solutions, and other materials were obtained from Beijing Solebao Technology Co., Ltd. (Beijing, China). Other reagents typically used in laboratories are of analytical grade.

2.2. Preparation of CMK, fish protein powder, and high-fish-protein feed

The preparation of a compound dietary fiber consisting of mulberry leaf and konjac flour followed the same protocol as our preceding experiment, with a 1:1 ratio, and the ingredients were thoroughly mixed (Deng et al., 2023). The KGM content exceeded 90%, while the protein content was below 5%.

The preparation of fish protein powder began by removing the head and tail, followed by cooking and discarding the bones. The fish was then dried and dehydrated in a 50 °C oven, and the residue was crushed and sieved using an 80-mesh sieve. The powder was composed of 1.00% moisture and 90.70% protein.

Two types of feed were procured from Nantong Trophy Feed Technology Co., Ltd. (Nantong, Jiangsu, China). One was normal AIN-93 M feed, the other was a high-protein feed produced according to the formula provided by the author based on the American Nutrition Society's AIN-93 M feed formula. The protein content of the normal AIN-93 M was 13.53%, and the high-fish-protein feed was 50.48%.

2.3. Animal feeding

Fifteen-month-old BALB/c female mice, weighing 28-30 g and certified as special pathogen free (SPF), were procured from the Experimental Animal Center of Chongqing Medical University (Certificate No.: SCXK (Yu) 2018–0003). The mice were housed in the SPF barrier animal laboratory at the School of Pharmacy, Southwest University (Facility license No.: SYXK (Yu) 2014–0002) (Chongqing, China). All procedures involving animals adhered to the ARRIVE guidelines and were approved by the Laboratory Animal Welfare and Ethics Committee of Southwest University (IACUC-20190307-09). The mice underwent adaptive feeding for one week and were then randomly divided into six groups (n = 5). The Normal Control (NC) group was fed the AIN 93 M diet, while the Blank Control (FBC) group received a high-fish-protein diet. The Positive Control (FPC) group was provided with the same high-fish-protein diet along with digestive pills (0.36 g/kg). The High-Dose CMK (FHD) group was given the same high-fish-protein diet with 0.3 g/kg of CMK, while the Middle-Dose CMK (FMD) group received 0.15 g/kg of CMK. The Low-Dose CMK (FLD) group was administered 0.075 g/kg of CMK. All mice had ad lib access to food, and were gavaged at 16:00 every day. The NC and FBC groups were administered the same quantity of normal saline through gavage. The animal feeding laboratory was maintained at a temperature of 23 ± 1 °C, a relative humidity of 53 ± 2%, and a 12 h daily light cycle from 8 am to 8 pm.

2.4. Sample collection

Three days before concluding the experiment, feces were collected at regular intervals, frozen in liquid nitrogen, and stored at −80 °C. After completing the experiment, the mice underwent a 12–14 h fast and were then anesthetized. Blood was drawn from their eyes using a 1.5 mL enzyme-free centrifuge tube. The sample was centrifuged at 3000 r/min for 10 min (Heraeus Fresco 17, Thermo Fisher Scientific, USA) after 1 h of inactivity. The serum was divided into 200 μL aliquots, sealed in centrifuge tubes, and stored at −80 °C. Following cervical dislocation and euthanasia, cecum contents were collected. The heart, liver, spleen, lungs, kidneys, and thymus were removed, washed with 0.9% frozen physiological saline, and any surface water droplets were wiped off with absorbent paper. The weight of each organ was then measured and recorded. The stomach, small intestine, and large intestine were completely collected and preserved with 4% paraformaldehyde. Additionally, the small intestine was washed with cooled normal saline.

2.5. Organ index calculation

The organ index of each mouse can be calculated by dividing the organ weight by the body weight according to the given formula. $\text{Organ Index}\left(\%\right)=\frac{\text{Total organ weight}\left(\mathrm{g}\right)}{\text{Body weight}\left(\mathrm{g}\right)}\times 100\%.$

2.6. Determination of the pH and water content of cecal contents and feces

The cecal contents and feces of mice in the same group were accurately measured at 0.2 g each. Subsequently, 10 times deionized water was added to both, mixed evenly, and the pH value was measured with a pH meter after standing still for 30 min. Additionally, 0.2 g was precisely weighed into a constant-weight aluminum box, dried to a constant weight at 105 °C, and then the moisture content was measured.

2.7. Determination of in vivo apparent digestibility

The kit method was used to calculate the total protein in the feed and feces, and the apparent digestibility was calculated according to the following formula (Yang et al., 2020).

$$\mathit{Apparent\; digestibility}\left(\%\right)=\frac{\text{Total protein in feed}\left(\%\right)-\text{Total protein in feces}\left(\%\right)}{\text{Total protein in feed}\left(\%\right)}$$

2.8. Determination of serum biochemical indicators and gastrointestinal enzyme activity

According to the instructions of the reagent kit, the main serum indexes to be measured include serum urea nitrogen, glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total amino acid, albumin, total protein, and the serum inflammatory factors of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10), lipopolysaccharide (LPS), and lipopolysaccharide binding protein (LBP). The enzyme activity of gastric and small intestinal contents, such as pepsin, trypsin, and erepsin (PRSS7), was measured.

2.9. Determination of SCFA in cecum contents

The Agilent 7890 series gas chromatography (Agilent Technologies, Inc., California, USA) was used for SCFA determination, following the prior test method (Deng et al., 2023). A DB-FFAP column (30 m × 0.32 mm, 0.25-μm film thickness, Agilent, USA) was utilized.

2.10. Metabolomics identification of the fecal supernatant

Metabolomics identification of the fecal supernatant followed a previous method used by Wei et al. (2019) with slight modifications. A specific amount of mouse feces was weighed into an Eppendorf tube and mixed with 600 μL of extraction solution (the ratio of acetonitrile: methanol: water was 2:2:1, −20 °C). After vortexing for 30 s, steel balls were added, and the sample was ground at 45 Hz for 4 min, then sonicated in an ice water bath for 5 min. This process was repeated thrice. Finally, the sample was soaked for 1 h at 20 °C. Subsequently, the sample was centrifuged at 4 °C and 12,000 r/min for 15 min (Thermo Fisher Scientific, USA). 100 μL of the supernatant was taken and placed into an LC sample injection bottle. Using an Agilent 1290 Infinity II series UPLC BEH Amide liquid chromatographic column (Agilent Technologies Inc., USA), the target compound could be separated. An Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source was employed to perform mass spectrometry analysis through the multi-reaction monitoring (MRM) mode (Agilent Technologies Inc., USA). All data collection and quantitative analysis of the target compound were carried out through Agilent MassHunter Workstation Software (B.08.00).

2.11. Identification of peptide sequences in feces

The determination of peptide sequences in feces followed a previous method with slight modifications (Macvicar et al., 2019). The lysate and fecal sample were blended together, then stirred and blended three times for 400 s each, and finally broken down further while on ice for 30 min. The supernatant was obtained after high-speed centrifugation for 15 min at 4 °C and 15,000 r/min (Thermo Fisher Scientific, USA). 500 μg of protein from each sample was placed into a new EP tube and freeze-dried. Subsequently, 1 mL of pre-cooled methanol was added to the tubes and they were shaken at 4 °C for 4 h. The supernatant was harvested after a high-speed centrifugation at 15,000 r/min and 4 °C (Thermo Fisher Scientific, USA). Desalination was carried out using a C₁₈ desalination column, and the samples were analyzed by LC-MS/MS with an online nano-spray ion source. LC-MS/MS is a Q-Active HF-X mass spectrometer (Thermo Fisher Scientific, USA) connected in series with an EASY-nano-LC 1200 (Thermo Fisher Scientific, USA). Finally, 3 μL of sample was analyzed by tandem mass spectrometry with PEAKS Studio X.

2.12. Gut microbiota analysis

The Power Soil DNA Isolation Kit (MO BIO Laboratories) was used to extract total bacterial DNA from samples, following the manufacturer's instructions. The quality and quantity of the DNA were evaluated by measuring the ratio of 260 nm/280 nm and 260 nm/230 nm. The V₃-V₄ region of the bacterial 16S rRNA gene was then amplified using a specific primer pair that included adapter and barcode sequences, following the method of Zhan et al. (Zhan et al., 2019). The PCR products were quantified using Quant-iT™ dsDNA HS Reagent and pooled. The sample was then purified and subjected to high-throughput sequencing analysis of the bacterial rRNA genes with the Illumina Hiseq 2500 platform (2 × 250 paired ends) at Biomarker Technologies Corporation (Beijing, China).

2.13. Texture determination of small intestines

The samples, 10 cm in length and cooled to room temperature with the fat removed, were attached to an A/MTG fixture (Stable micro system, England) at a spacing of 5 cm, straightened and tightened naturally. The test mode adopted was a two-time stretching procedure. The first stretch was 6 mm and held for 10 s to measure the elastic index, and the second stretch was 30 mm until breaking to measure the fracture index. The speed before measurement was 1 mm/s, and 10 mm/s after measurement, with the measurement speed being 1 mm/s.

2.14. Pathological observation of stomach, small intestine, and large intestine

The fixed tissue is dehydrated and embedded using an automated dehydrator. Subsequently, the tissue slices undergo the following steps: dewaxing in water, staining with hematoxylin for 10–20 min, rinsing in tap water for 1–3 min, differentiation in hydrochloric acid alcohol for 5–10 s, another rinse in tap water for 1–3 min, immersion in warm water at 50 °C or a weakly alkaline solution until a blue colour develops, followed by another tap water rinse for 1–3 min. The slices are then treated with 85% alcohol for 3–5 min, stained with eosin for 3–5 min, washed with water for 3–5 s, dehydrated with gradient alcohol, cleared with xylene, and finally sealed with neutral resin. The image of the slices (200 times magnification) was collected for analysis with a BA400 Digital Three Eye Camera Microscopic Camera System (McAudi Industrial Group Co., Ltd., China).

2.15. Data analysis

All values were assessed three times concurrently. The data obtained from the experiment were expressed as “⁻x ± s”, calculated, and plotted using Excel 2010. Statistix 9.0 was employed to check for significant differences via a one-way ANOVA, and p < 0.05 indicated statistical significance.

3. Results and discussion

3.1. Beneficial effects of CMK on protein metabolism in elderly mice consuming a high-fish-protein diet

In the initial week, there was no marked difference in food intake among the groups (Fig. 1A). As the feeding period extended, the food intake of the Blank Control (FBC) group was notably less compared to the other groups. Although the weight of each mouse decreased over the experimental period, the amount of weight change was not significantly different (p > 0.05) (Fig. 1B). This phenomenon can be attributed to the satiating effect of a high in protein diet, leading to reduced and stable weight maintenance (Leidy et al., 2015). Additionally, the KGM in the diet can stimulate intestinal movement, contributing to a certain degree of weight loss (Zhang et al., 2022). Furthermore, considering the physiological decline in elderly mice, the relative changes of organ indices were not significant (p > 0.05) (Fig. 1C), suggesting that the diet had no toxic or adverse effects on the animals.

Fig. 1.

Effects of CMK on protein metabolism in elderly mice consuming a high-fish-protein diet. Analysis of Food intake (A), Weight changes (B), Organ index (C), Moisture content of the cecal and facal (D), pH of the cecal and facal (E), SCFA content (F), and the Apparent digestibility of high-fish-protein (G). No significant differences are indicated by the same uppercase and lowercase letters (p > 0.05), while different uppercase and lowercase letters signify meaningful distinctions (p < 0.05).

In comparison to the FBC group, the moisture content in the cecal and fecal samples of FHD, FMD, and FLD groups increased, exceeding the levels observed in the NC and FPC groups (p < 0.05) (Fig. 1D). Relative to the NC group, the FBC group showed a significant increase in pH levels in both cecal and fecal samples (p < 0.05), while CMK intervention caused a significant decrease (p < 0.05) (Fig. 1E).

Elderly mice in the high-fish-protein diet groups displayed significantly lower SCFA content in their cecal contents than the NC group (p < 0.05) (Fig. 1F). In the FHD group, the concentrations of SCFAs were significantly greater than those in the FBC and FPC groups (p < 0.05). Furthermore, the levels of acetic acid, isobutyric acid, and isovaleric acid were higher than in the NC group. KGM, rich in soluble dietary fiber, exhibits a remarkable capacity to absorb up to 80–100 times its own volume of water, contributing to significant water-retention (Zhang et al., 2022). The presence of 1-deoxynojirimycin (DNJ), food fiber, crude fat, minerals, and undigested sugars from mulberry leaf powder in the cecum, enhances the water content of cecal contents (Chen et al., 2020a, Chen et al., 2020b). CMK fermentation in the cecum generates SCFAs, reducing the pH of cecal contents and feces (Deng et al., 2023). SCFAs play a crucial role in maintaining the tight junctions of colon cells, which is vital for intestinal health. Additionally, SCFAs reduce the production of proinflammatory factors, stimulate the proliferation and differentiation of epithelial cells, accelerate the repair of damaged epithelium, enhance intestinal epithelial barrier function, and lower pH (Dalile, Van, Vervliet, & Verbeke, 2019).

The evaluation of dietary protein bioavailability typically involves the assessment of digestibility, representing the percentage of protein absorbed in the digestive tract compared to protein intake. This measurement and reflects the efficiency of food breakdown by digestive enzymes and the absorption of amino acids and peptides (Bax et al., 2013). In our study, all four groups (FPC, FHD, FMD, and FLD) exhibited significantly higher protein digestibility than the FBC group, surpassing a digestibility rate of 96.78% (p < 0.05) (Fig. 1G). This suggested that both CMK and digestive tablets improved protein digestibility, particularly evident in the FHD group, where the apparent digestibility of protein reached 97.05%. Zhang et al.'s study demonstrated that KGM has the potential to prevent loperamide-induced constipation in mice through regulating various microorganisms in the gastrointestinal tract (Zhang et al., 2022). Moreover, Zhong et al.'s research illustrated that CMK effectively aids consumers in digesting high-protein and high-fat dishes, such as Chongqing hotpot (Zhong & Zhong, 2018). Our previous study also showed that CMK could modulate the intestinal microbiota and strengthen the immune system (Deng et al., 2023). We speculate that the improved digestibility associated with CMK may be linked to the promotion of intestinal probiotics.

Biochemical indicators in animal serum serve as markers for the overall metabolic state of the body. Research suggests that the urea nitrogen level decreases when amino acids consumption aligns with the body's absorption capacity. Conversely, an excess of amino acids surpassing the digestive capacity elevates urea nitrogen content (Wang, Qiao, Liu, & Ma, 2006). A high-fish-protein diet resulted in a marked rise in serum urea nitrogen content (p < 0.05) and a significant decrease in GPT activity (p < 0.05) compared to other groups (Fig. 2). This indicates that amino acid levels in the FBC group exceeded the body's processing ability, resulting in elevated serum urea nitrogen. Consequently, some amino acids were excreted with urine, reducing the effective protein utilization rate, consistent with the findings of Wang et al. (Wang et al., 2006). CMK intervention resulted in reduced serum urea nitrogen content (p < 0.05) (Fig. 2A), along with a substantial rise in total amino acid content (Fig. 2C), GPT and GOT activities (Fig. 2B), and serum total protein content (Fig. 2D) (p < 0.05). CMK was observed to enhance protein digestibility and amino acid absorption. The use of digestive tablets had no significant effect on urea nitrogen (Fig. 2A) and serum total protein (Fig. 2D) (p > 0.05); however, GPT and GOT activity (Fig. 2B) and total amino acid content (Fig. 2C) increased (p < 0.05). It appears that both CMK and digestive tablets improved protein digestibility, consistent with the results of apparent digestibility in Fig. 1G. Moreover, digestive tablets promoted urinary nitrogen excretion while CMK augmented amino acid absorption in the body. Studies have demonstrated that CMK can modulate enzyme activity associated with intestinal protein metabolism, alleviating harm from a beef-protein-rich diet to the intestines of elderly mice and promoting intestinal health (Deng et al., 2020). This study suggests that CMK is beneficial for protein synthesis and metabolism, leading to enhanced utilization of a high-fish-protein diet.

Fig. 2.

Serum biochemical analysis. Urea nitrogen (A), Transaminase activity (B), Total amino acids (C), and Total serum protein (D). Different uppercase and lowercase letters signify a significant difference (p < 0.05).

The digestibility of CMK was further evaluated using metabolomics and peptide omics targeting amino acid in mouse fecal supernatant. Amino acids, the building blocks of proteins, are essential for protein metabolism. The study showed that the FBC group had significantly higher fecal amino acid content than the other groups, except for glycine, 1-methyl-L-histidine, and L-citrulline (p < 0.05) (Fig. 3A). This higher content is attributed to the slower absorption of polar amino acids like glycine and histidine compared to non-polar amino acids (Li, Zhang, & Dai, 2006). The concentration of L-citrulline in the fecal metabolites of each group was consistent at 105 nmol/g, with no notable differences (p > 0.05). Citrulline is a reliable marker for assessing the performance and integrity of small intestinal epithelial cells, which is a key factor in determining intestinal barrier dysfunction (Jones et al., 2015). The similarity in citrulline absorption among groups implies normal gastrointestinal function in elderly mice.

Fig. 3.

Effects of CMK on the digestibility of elderly mice fed a high-fish-protein diet. Effect of CMK on amino acid metabolism (A), PCA of metabolites (B), the number of peptide segments in the feces (C), a Wayne plot (D), and the localization of feces in actin (E) and myosin (F), the sequences with a bold gray background are confidently identified. The blue bars below the sequences demonstrate the exact sequence matching of each reliable qualitative peptide segment, while the gray bars indicate the de novo peptide segment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The FBC group exhibited the highest total fecal amino acid content (3078.98 ± 0.01 nmol/g), with the NC group at 2049.05 ± 0.54 nmol/g. Other groups (FPC, FHD, FMD, and FLD) showed significantly lower amino acid content in comparison to the FBC group (p < 0.05). Digestive tablets and CMK led to a reduction in amino acid concentration in fecal metabolites (p < 0.05), indicating augmented amino acid absorption. PCA analysis (Fig. 3B) showed that after intervention with digestive tablets and CMK, the fecal metabolites of elderly mice in each group were similar to those of the NC group, suggesting a positive effect on amino acid absorption.

Digestibility and digestion product sequences of proteins are associated with their composition and the size of peptide fragments produced during digestion (Kim et al., 2004). To gain insight into digestive product sequences, Nano-HPLC-MS/MS was employed, with a particular focus on the range between 500 and 4000 Da with high accuracy (> 20). A total of 194 peptide segments were identified and categorized based on their molecular weight: 500–1000 Da, 1000–1500 Da, 1500–2000 Da, 2000–2500 Da, 2500–3000 Da, 3000–3500 Da, and 3500–4000 Da. After this, Wayne intersection analysis was conducted on the peptide sequences. It can be seen that 93% of peptide segments had a molecular weight below 3000 Da, with no difference between the FBC and NC groups (Fig. 3C). However, CMK intervention decreased the number of peptide segments in feces (p < 0.05), most falling between 1000 and 1500 Da, indicating an increase in peptide segments absorbed by the body. The NC, FBC, FPC, FHD, FMD, and FLD groups had 14, 25, 15, 14, 15, and 17 unique peptide segments in their feces, respectively (Fig. 3D). Evidently, a diet rich in fish protein increased the number of peptide segments in feces, while CMK decreased the amount of distinct peptide segments. This indicates that CMK contributes to the digestion and absorption of proteins in a high-fish-protein diet.

In the exploration using PEAKS DB software (ver. 201907) for Oreochromis niloticus, actin and myosin emerged as the peptides of highest abundance. The amino acid sequences displayed high conservation, particularly at the ends, and the identified peptide segments exhibited high reliability (Fig. 3E, F). Notably, the C-terminal was mainly composed of polar amino acids, including lysine and threonine, while the N-terminal consisted mainly of non-polar amino acids like methionine and alanine, accompanied by a presence of a few polar amino acids like cysteine and aspartic acid. This observation suggests that the C-terminal domain had fewer degraded peptide segments than the N-terminal domain. Such a pattern implies that these two proteins, actin and myosin, are less prone to breakdown in the body, contributing to their higher abundance.

3.2. CMK safeguards the gastrointestinal tissue of elderly mice fed a high-fish-protein diet

The gastrointestinal system plays a crucial role in human health, contributing to digestion, absorption, metabolism, and serving as a vital physical, chemical, immune, and microbial barrier (Wang, Chen, Zhang, Lu, & Chen, 2020). Preserving the health of the gut involves maintaining its barrier function and overall homeostasis (Arbizu, Chew, Mertens-Talcott, & Noratto, 2020). Indicators such as firmness, elasticity, breaking force and breaking distance in the small intestine are linked to its propulsive function. The study showed that the FBC group exhibited significantly lower values for these indicators compared to the NC group and CMK intervention groups (p < 0.05) (Table 1). Notably, the FLD group, in comparison to the FBC group, experienced an increase in firmness, elasticity, breaking force, and breaking distance by 8.89%, 16.57%, 37.83%, and 35.56%, respectively. This suggests that the addition of CMK may improve the texture of the small intestine in elderly mice on a high-protein diet, thereby enhancing its propulsive function.

Table 1.

Results of intestinal texture.

	NC	FBC	FPC	FHD	FMD	FLD
Firmness (g)	30.1 ± 1.6a	27.0 ± 3.3b	27.0 ± 0.8b	29.9 ± 0.1a	29.5 ± 0.8a	29.4 ± 5.1a
Elasticity (%)	79.6 ± 0.1a	67.6 ± 13.9c	75.0 ± 3.8b	79.5 ± 0.1a	79.2 ± 2.7a	78.8 ± 15.3a
Breaking force (g)	39.2 ± 5.0a	30.4 ± 2.1c	36.0 ± 9.6b	40.3 ± 0.3a	40.6 ± 1.0a	41.9 ± 3.9a
Breaking distance (mm)	8.9 ± 0.7c	9.0 ± 1.6c	10.9 ± 2.5b	12.3 ± 0.2a	12.0 ± 0.1a	12.2 ± 4.0a

Note: Variations between two pairs in the same column are shown by different superscripts (p < 0.05).

The surface area of the small intestine is a direct indicator of its absorption function, and the morphological characteristics of villi and crypts reflect the maturation and absorption function of epithelial cells. A higher villus length and shallower crypt depth (V/C) value indicate a healthier mucosal state (Zhang, Wang, Chen, Wang, & Cao, 2015). The FBC group exhibited significantly lower height and width of villi in the duodenum (Fig. 4A), jejunum (Fig. 4B), and ileum (Fig. 4C). However, CMK administration resulted in increased villi height and width, shallower crypt depth, thicker muscular layer, and a significant higher V/C value (p < 0.05). Observation of whole gut sections, including the stomach (Fig. 5A, B), small intestine (Fig. 5C), and large intestine (Fig. 5D), showed that a high-protein diet induced typical pathological changes in mice, including increased inflammatory cell infiltration and epithelial shedding. However, CMK exhibited positive effects on intestinal health, highlighting its protective and regulatory impact on the intestines of elderly mice fed a high-protein diet. Numerous studies support the positive impact of dietary fiber on gastrointestinal function. Zhang's research, for instance, concluded that the consumption of KGM as a functional dietary fiber could ameliorate constipation-related symptoms by influencing various microorganism populations in the gastrointestinal tract (Zhang et al., 2021). Shen et al. discovered that KGM is capable of protecting against chronic alcoholic intestinal damage by reducing lipid peroxides in the gastric mucosa, augmenting antioxidant capacity, improving the intestinal microecology, and reinforcing the barrier effect of the gastric mucosa and intestinal tissue (Shen et al., 2018). In the context of a high-fish-protein diet, which poses a risk of harm to the small intestine of elderly mice, CMK demonstrates potential in safeguarding intestinal structure and enhancing nutrient absorption capacity.

Fig. 4.

Positive impacts of CMK on the gastrointestinal tissue of elderly mice fed a high-fish-protein diet. Comparison analysis of Duodenal tissue (A), Jejunum tissue (B), and Ileum tissue (C).

Fig. 5.

Effects of CMK on reprogramming the gastrointestinal environment of elderly mice fed a high-fish-protein diet. A. Panorama of gastric histomorphology (20×). B. Gastric histomorphology (200×). C. Duodenal histomorphology (200×) (a), Jejunum histomorphology (200×) (b), Ileum histomorphology (200×) (c). D. Cecum histomorphology (200×) (a), Colonic histomorphology (200×) (b), Rectum histomorphology (200×) (c).

Aging in the small intestine is characterized by various changes, such as weakened mucosal barrier, increased intestinal permeability, and decreased operational ability. These changes impact intestinal morphology, histology, nutrient absorption, and the overall microecology of the intestine, inducing host microecology imbalance and leading to a sustained state of intestinal immune aging (Ni et al., 2019). In this context, persistent inflammation can activate conditioned pathogenic bacteria, disrupt the balance of gut microbiota, affect the microecological environment of the gastrointestinal tract, induce mucosal damage, and lead to persistent intestinal injury and systemic inflammatory response (Grosicki, Fielding, & Lustgarten, 2018). The results of serum cytokine determination (Table 2) showed that the high-protein diet caused a significant rise in the concentrations of the inflammatory cytokines TNF-α and IL-1β (p < 0.05). However, the intervention with CMK considerably diminished these concentrations (p < 0.05). Additionally, the levels of IL-10 notably increased (p < 0.05), while the concentrations of LBP and LPS decreased significantly (p < 0.05). These findings indicate that the use of CMK has the potential to reduce the levels of intestinal immune antigens, thus reducing the body's inflammatory response. This observation is consistent with the results obtained from the examination of small intestine sections (Fig. 5), reinforcing the potential anti-inflammatory effects of CMK in the context of a high-fish-protein diet for elderly mice.

Table 2.

Results of serum immune factors.

	TNF-α (pg/mL)	IL-1β (pg/mL)	IL-10 (pg/mL)	LBP (μmol/L)	LPS (EU/L)
NC	119.11 ± 8.97d	57.65 ± 3.78c	228.39 ± 6.03c	30.42 ± 1.01c	13.56 ± 2.49ab
FBC	267.66 ± 2.11a	88.92 ± 7.72a	353.05 ± 5.04b	68.44 ± 5.12a	16.47 ± 3.65a
FPC	228.69 ± 1.49b	72.05 ± 9.92b	232.71 ± 5.28c	48.16 ± 12.12b	13.00 ± 0.46b
FHD	205.36 ± 9.22c	68.29 ± 3.94b	409.73 ± 8.05a	44.57 ± 2.32b	12.98 ± 0.86b
FMD	201.65 ± 3.14c	68.97 ± 3.84b	400.71 ± 8.20a	43.53 ± 4.55b	13.13 ± 0.97b
FLD	200.23 ± 4.08c	69.42 ± 4.42b	402.22 ± 8.49a	43.54 ± 7.96b	13.61 ± 0.76ab

Note: Variations between two pairs in the same column are shown by different superscripts (p < 0.05).

3.3. Effects of CMK on the enzyme activity of stomach and intestines and the expression of metabolism protein of elderly mice fed a high-fish-protein diet

Enzyme activity in the stomach and intestines plays a crucial role in protein metabolism. After pepsin's initial action on food proteins, subsequent proteases like trypsin, chymotrypsin, elastase, and peptidases take over to further break down the digestive products and undigested proteins, ultimately forming amino acids (Ito et al., 2019). Digestive enzyme activity serves as a significant physiological indicator of the body's digestive capability, and alterations in dietary nutrients can affect the secretion and activity of intestinal digestive enzymes, as they form the substrate for the enzymes after ingestion (Brzek et al., 2013). Research has revealed that a high-protein diet can activate digestive enzyme activity (Zhu et al., 2018). In elderly mice fed a high protein diet, intestinal enzyme activity becomes a critical factor in protein metabolism, influencing protein efficiency and deposition rates, contributing to the restoration of the hepatopancreas and intestinal tissue structure (Wu, Ji, Yu, Sun, & Zhou, 2020). In the current study, a high-fish-protein diet significantly increased the activities of trypsin and erepsin (p < 0.05) (Fig. 6A). The addition of CMK further increased enzyme activity (p < 0.05) in a dose-dependent manner, with the FLD group showing an upsurge in pepsin activity from 0.91 U/g to 0.97 U/g, trypsin activity from 0.35 U/g to 0.51 U/g, and erepsin activity from 2.30 U/g to 3.71 U/g, compared to the FBC group. This enhancement is beneficial for protein digestion and absorption. Conversely, the use of digestive tablets significantly raised pepsin activity (p < 0.05), but significantly reduced erepsin activity. These findings align with the observed facilitation of urinary nitrogen excretion (Fig. 2A) and enhanced protein digestibility (Fig. 1G) by digestive tablets. CMK, on the other hand, is associated with improved digestion and absorption, potentially strengthening the internal structure of elderly mice and assisting in decreasing the amount of protein in food.

Fig. 6.

Effects of CMK on enzyme activity of the stomach and intestines in elderly mice fed a high-fish-protein diet. Determination of gastrointestinal enzyme activity (A), the protein expression of GDH (C), PepT1 (D), and APN (E), with significantly different mean values indicated by different letters in the bar (p ˂ 0.05).

Moreover, the study investigated the expression of protein metabolism markers in the liver, such as APN, PepT1 and GDH, which play a significant role in facilitating protein digestion and absorption. The addition of CMK to a high-fish-protein diet increased the expression of these protein metabolism markers in the liver of elderly mice. The FMD group displayed significantly higher expression levels compared to the FBC group, indicating a potential molecular basis for the enhancement of protein metabolism in elderly mice on a diet rich in fish-protein with the supplementation of CMK (Fig. 6B-E). This finding aligns with our previous research demonstrating CMK's ability to up-regulate the expression of GDH, PepT1, and APN proteins in the context of a high-beef-protein diet (Deng et al., 2023).

3.4. Effects of CMK on reprogramming gut microbiota in elderly mice fed a high-fish-protein diet

OTU clustering analysis of mouse cecal contents yielded 1,351,807 pairs of reads, which were then spliced and filtered to generate 1,206,076 clean tags. Each sample had a minimum of 45,321 and an average of 67,004 clean tags. Taxonomic annotation of OTUs revealed 12 phyla, 22 classes, 41 orders, 78 families, 154 genera, and 167 species of bacteria (Fig. 7A). Cluster analysis results (Fig. 7B) indicated 481 OTUs, with each sample yielding between 386 and 439 OTUs. 317 OTUs were shared among the six sample groups, representing 68% of the total OTUs. The unique OTUs were 1, 0, 1, 8, 7, 1 (Fig. 7C), indicating that CMK supplementation to the high-fish-protein diet impacted the gut microbiota of elderly mice.

Fig. 7.

Effects of CMK on reprogramming gut microbiota of elderly mice consuming a diet with high-fish-protein. The classifications of gut microbiota at different levels (A), OTU numbers (B), and Venn diagram (C).

The Ace index (Fig. 8A) and Chao1 index (Fig. 8B) revealed that the FHD group had the most abundant intestinal species, significantly greater than the NC group (p < 0.05). This indicates a dose-dependent effect of CMK in increasing intestinal species abundance in elderly mice on a high-fish-protein diet. Additionally, compared to the FBC group, the Shannon index (Fig. 8C) of other groups was significantly lower (p < 0.05), and the Simpson index (Fig. 8D) was significantly higher, with no considerable disparities between the other groups (p > 0.05). This suggests that the FBC group had the lowest species diversity, indicating that the gut microbiota of elderly mice can be altered by a high-fish-protein diet, and CMK intervention could increase the species diversity of the gut microbiota in elderly mice.

Fig. 8.

Assessment of alpha and beta diversity. Ace index (A) and Chao1 index (B) are used to measure species abundance. Shannon index (C) and Simpson index (D) evaluate species diversity. When species abundance is equal, a more even distribution of species in a community increases its diversity. A high Shannon index and low Simpson index demonstrate greater species diversity in the sample. PCoA (E) and PLS-DA (F) reveal differences in species diversity among samples. Those closer in distance on the coordinate map exhibit greater similarity. NMDS (G) is similar to PCoA, aiming to identify inter- and intra-group differences through the distribution of samples. The closer the samples are located together on the coordinate chart, the greater the similarity between them. A Stress value <0.2 indicates that the NMDS analysis is reliable. PermANOVA (H) is a statistical method used to analyze the similarity between multi-dimensional data groups. Following PCoA analysis, PermANOVA assesses whether there is a significant difference in Beta diversity between samples in different groups. The higher the R² value, the greater the explanatory power of the group for the difference, and the larger the difference. A P-value <0.05 indicates a higher level of credibility for the test.

QIIME software was used to evaluate the variation in species diversity between samples through beta diversity analysis. PCoA (Fig. 8E) and PLS-DA (Fig. 8F) were used to analyze the gut microbiota of elderly mice, revealing a significant impact of the high-fish-protein diet on microbial communities. The NC group was mainly located in the first quadrant of the PCoA and the second quadrant of PLS-DA, while the other groups were mainly situated in the fourth quadrant of both graphs. Furthermore, the Stress = 0.1538 < 0.2 (Fig. 8G), Permanov analysis of p = 0.009, and R² = 0.367 (Fig. 8H) demonstrated the validity of the changes in the gut microbiota.

Uutilizing Unweighted Unifrac analysis, the association between species and abundance of each group at the genus level was explored. Results (Fig. 9A, B) highlighted that the high-fish-protein diet changed the species composition of the gut microbiota in elderly mice, while CMK intervention caused Lactobacillus to become the most prevalent bacteria in their gut microbiota.

Fig. 9.

Effects of CMK on reprogramming gut microbiota of the intestines in elderly mice fed a diet of high-fish-protein. UPGMA tree column (A), Heat map of distance between samples (B), Taxonomic composition distribution at phylum level (C), The proportion of Firmicutes to Bacteroides in every group (D), Taxonomic composition of the top 20 at genera level of relative abundance (E), Correlation analysis of the top 20 genera of gut microbiota and the 25 amino acid metabolites in NC, FBC, and FHD groups (F). Red, blue, and white colors represent correlations of 1, −1, and 0, respectively, with an asterisk indicating correlations with p < 0.05. The vertical axis represents differential bacteria, and the horizontal axis represents metabolites. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

All samples showed Firmicutes are the most abundant phylum, with the combined relative abundance of Firmicutes, Bacteroidetes, and Proteobacteria exceeding 80% (Fig. 9C). A dose-dependent increase in Firmicutes and a corresponding decrease in Bacteroidetes were observed when CMK was supplemented, resulting in a Firmicutes to Bacteroidetes ratio (F/B) of >2.0, which was higher than the ratios of the FBC and NC groups (Fig. 9D). Elderly individuals typically exhibit a higher abundance of Bacteroidetes than Firmicutes in their gut microbiota (Ni et al., 2019). This implies that supplementation with CMK can improve the gut microbiota composition of elderly mice consuming a high-fish-protein diet.

At the genera level, the NC group was dominated by Uncultured_bacterium_f_Desulfovibrionaceae and Bacteroides (Fig. 9E). The high-fish-protein diet caused a gradual increase in the abundance of Lactobacillus. The utilization of CMK yielded an increase in the abundance of Uncultured_bacterium_f_Desulfovibrionaceae, Lactobacillus, and Blautia. The Blautia is a SCFA-producing bacterium (Zang et al., 2018). Lactobacillus, a probiotic found in the human gut, has been demonstrated to improve gastrointestinal functioning, digestion, and nutrition, as well as reducing aging immunity (Bukhari et al., 2020). Notably, the elderly usually have fewer Lactobacilli in their bodies (Ni et al., 2019). Furthermore, this microorganism has been observed to protect the intestinal lining, as evidenced by the results of intestinal examinations (Fig. 5). Qin et al.'s research has demonstrated that Lactobacillus can impede the infiltration of inflammatory cells in the colon by controlling inflammatory cytokines in mice (Qin et al., 2023). The rise in TNF-αand IL-10 (Table 2) as a result of CMK may be associated with an augmented concentration of Lactobacilli in the body. Additionally, consuming CMK can regulate the type and amount of gut microbiota in elderly mice fed a high-protein diet, thereby decreasing the abundance of harmful microbiota (Deng et al., 2023), protecting gut barriers, promoting intestinal health, and improving protein digestibility. This likely contributes to the promotion of protein digestion and metabolism.

To further explore the connection between microbial communities and high-fish-protein metabolism, a heat map was constructed to compare the top 20 genera of microorganisms in the NC, FBC, and FHD groups with 25 amino acids in their metabolites (Fig. 9F). Results showed that the genera negatively correlated with amino acid content include Ruminococcaceae, Muribaculaceae, Desulfovibrionaceae, Lactobacillus, and Ruminococcus_1, Rikenellaceae_RC9_ gut_ group. Fig. 9E reveals an increase in Ruminococcaceae, Desulfovibrionaceae, and Lactobacillus genera after the intervention of CMK, suggesting a decrease in amino acid content in feces and an increase in the amount of amino acids absorbed by the body. Thus, it can be concluded that the intervention of CMK regulates the types and abundance of gut microbiota related to amino acid metabolism in elderly mice fed with a high-fish-protein diet, thus promoting protein digestion.

4. Conclusion

This study aimed to evaluate the effectiveness of combining mulberry leaf powder and konjac flour in improving digestive function in elderly mice. Through an in vivo system, we assessed its impact on intestinal metabolite SCFA, focusing on protein metabolism stability, intestinal microbiota, and the inflammatory response induced by a high-protein diet. The results revealed a positive influence of CMK intervention, including enhanced integrity of intestinal barrier cells, increased levels of anti-inflammatory factors, and a notable improvement in the quality of the caecum. Additionally, inflammatory factors decreased, and the SCFA content increased, thus affecting protein metabolism positively. These results elucidate the effectiveness and underlying mechanism of these components in promoting human health, providing valuable insights and practical strategies for utilizing natural ingredients to promote health outcomes.

CRediT authorship contribution statement

Liling Deng: Writing – review & editing, Writing – original draft, Data curation. Geng Zhong: Project administration, Conceptualization. Qiong Wang: Software, Data curation. Zhaojing Zhu: Validation, Investigation. Yongbo Peng: Writing – review & editing, Investigation.

Declaration of competing interest

The authors declare no conflict of interest.

Data availability

Data will be made available on request.