Skip to main content
NCBI home page
Bioscience of Microbiota, Food and Health logoLink to Bioscience of Microbiota, Food and Health
. 2024 Dec 17;44(2):171–181. doi: 10.12938/bmfh.2024-078

The effects of functional biscuits on intestinal mucosal microbiota composition, brain function, and antioxidant activity

Junxi SHEN 1, Leyao FANG 1, Zhoujin TAN 1, Nenqun XIAO 2,*, Maijiao PENG 2,*
PMCID: PMC11957763  PMID: 40171390

Abstract

Protecting brain health is one of the current focal points of public concern. Medicinal foods that promote brain health, such as Gastrodia elata Bl, black sesame seeds (Sesamum indicum L.), walnuts (Juglans regia L.), jujube (Ziziphus jujuba Mill.), Poria cocos, and Coix seeds, possess antioxidant and neuroprotective properties, as well as modulating effects on the intestinal microbiota. This study evaluated the effects of functional biscuits formulated with these medicinal foods on the intestinal mucosal microbiota, brain function, and antioxidant activity in mice. Forty male SPF-grade C57BL/6J mice were randomly divided into a blank control group (NG), low-dose functional biscuit group (GLG), medium-dose functional biscuit group (GMG), and high-dose functional biscuit group (GHG). After 42 days of continuous feeding with the functional biscuits, changes in the richness, diversity, and community structure of the intestinal mucosal microbiota were observed. Compared with the NG group, norepinephrine (NE) levels in the hippocampus significantly increased in the GLG, GMG, and GHG groups, while gamma-aminobutyric acid (GABA) levels showed no significant difference. In the GMG and GHG groups, malondialdehyde (MDA) levels in the liver significantly decreased, and acetylcholine transferase (ChAT) levels in the hippocampus significantly increased. Additionally, multiple bacterial genera were found to be correlated with the NE, ChAT, and MDA levels. These findings indicate that functional biscuits have effects on modulating the intestinal mucosal microbiota composition, enhancing brain function, and exhibiting antioxidant activity, making them a beneficial functional food for brain health.

Keywords: intestinal mucosal microbiota, functional food, functional biscuit, medicinal homologous foods, intestinal microecology

INTRODUCTION

In recent years, due to modernization and changes in the demand of people for a healthy lifestyle, coupled with the expansion of applications in food science and technology research, there has been a significant increase in consumer interest and demand for health foods [1]. Consumer attitudes have gradually shifted towards value-added foods. These foods are not only more nutritious but also healthier than traditional standard foods [2]. This concept of food is precisely termed functional food. Functional foods contain ingredients that provide various health benefits in addition to basic nutritional components. Increasing clinical evidence suggests that regular consumption of functional foods as part of a diversified diet can help control blood sugar, activate antioxidant enzymes, regulate intestinal microbiota, and enhance immune system activity, cognitive responses, and overall health status [3]. Therefore, it is necessary to actively develop new types of functional foods to meet the diverse needs of consumers.

Biscuits have become one of the widely favored baked goods globally due to their flavor, crisp texture, easy storage, and extended shelf life [4]. Regular biscuits are characterized by higher levels of sugars, fats, and starch, providing high energy to the body. However, they tend to have low dietary fiber, antioxidant, and other nutritional components [5]. As a result, long-term consumption of regular biscuits is not recommended for children or the elderly [6, 7]. The development of new cookies with high dietary fiber, abundant vitamins, and bioactive ingredients would enhance the nutritional and functional properties of biscuits, positively impacting the health status of consumers [8].

Based on traditional Chinese health maintenance theories, we have developed a novel type of biscuit (hereinafter referred to as “functional biscuits”) with Gastrodia elata Bl, black sesame seeds (Sesamum indicum L.), walnuts (Juglans regia L.), jujube (Ziziphus jujuba Mill.), Poria cocos, and Coix seeds as its primary functional ingredients. The tuber of G. elata Bl is a valuable and widely used traditional Chinese herbal medicine extensively used in both clinical practice and daily health maintenance [9]. Its active components primarily include phenolic compounds, polysaccharides, glycosides, organic acids, and sterols, exhibiting various effects such as anti-anxiety, anti-inflammatory, antioxidant, neuroprotective, memory enhancement, and anti-aging properties [10, 11]. Both walnuts and black sesame seeds, which belong to the nut and seed category, are rich sources of unsaturated fatty acids, dietary fiber, and minerals [12]. They play a crucial role in anti-inflammatory and antioxidant modulation, regulation of disrupted gut microbiota, and neurodegenerative disease management. Additionally, they contribute rich aromatic fragrances and a robust taste, enhancing the flavor of biscuits [13, 14]. Jujube is a highly nutritious and functionally significant fruit commonly used in clinical traditional Chinese medicine. Jujube contains a variety of bioactive substances, including polysaccharides, polyphenols, amino acids, and dietary fiber, which significantly enhance the neuroprotective activity of the organism and reduce the toxicity of other herbal medicines [15, 16]. P. cocos and Coix seeds are traditional Chinese medicinal herbs frequently employed in clinical practice to treat conditions such as edema, insomnia, and rheumatism. They are also essential health foods, both rich in polysaccharides and triterpenoids, imparting pharmacological effects such as antioxidation, anti-inflammation, immune modulation, neuroprotection, and regulation of intestinal microbiota [17, 18].

The human intestinal microbiota consists of numerous bacteria that play a crucial role in influencing various physiological functions. Furthermore, it significantly regulates aspects such as the host immune response, intestinal endocrine function, neural signal transmission, food digestion, and metabolism [19]. Communication between the intestinal microbiota, neuroendocrine system, and brain is primarily established through humoral pathways, immune pathways, and neural pathways [20]. The intestinal microbiota communicates with the central nervous system through microbial metabolites that influence neurotransmitters such as norepinephrine (NE) and gamma-aminobutyric acid (GABA), participating in altering their synthesis and degradation [21]. Therefore, modulating the intestinal microbiota represents an ideal target for influencing the health status of the gut-brain axis.

The aim of this study was to observe, through animal experiments, the effects of a novel biscuit with the aforementioned medicinal food as its primary functional components on the general condition, antioxidant-related indices, brain neurotransmitters, and intestinal mucosal microbiota of mice. This research aimed to further investigate the functional roles of a functional biscuit and provide a reference for the development and production of functional foods beneficial to brain health.

MATERIALS AND METHODS

Animals

Considering the influence of gender on the gut microbiota [22], this experiment utilized mice of a single gender. Forty male SPF-grade C57BL/6J mice, aged 6–8 weeks, were obtained from Hunan Slaccas Jingda Laboratory Animal Co., Ltd. (Hunan, China) (animal license number SCXK [Hunan] 2019-0004). All experiments and procedures involving animals were conducted in accordance with the approved protocols of the Institutional Animal Care and Use Committee of Hunan University of Chinese Medicine.

Feeds

During the experimental procedures, Co-60 irradiated feed for the growth and reproduction of experimental mice was provided by the Experimental Animal Center of Hunan University of Chinese Medicine [23].

Production of functional biscuits

The ingredients for making the biscuits were as follows: low-gluten wheat flour (Binzhou Zhongyu Food Co., Ltd., Binzhou, China), edible corn starch (Weihaomei Wuhan Food Co., Ltd., Wuhan, China), edible baking soda (Angel Yeast Co., Ltd., Yichang, China), maltitol (Shandong Futian Pharmaceutical Co., Ltd., Dezhou, China), G. elata Bl (Yunnan, production batch: 210701), Coix seeds (Guizhou, production batch: 21102301), P. cocos (Yunnan, production batch: 2205008), black sesame seeds (Anhui, production batch: 211104), walnuts (Xinjiang, production batch: 211101), jujube (Xinjiang, production batch: P20220102), and Yibao purified water. G. elata Bl, P. cocos, Coix seeds, walnuts, and black sesame seeds were ground in a pulverizer, and the G. elata Bl powder, P. cocos powder, and Coix seed powder were passed through an 80-mesh sieve for later use. Jujubes were pitted, weighed, and blended with water in a juice extractor to make a jujube puree. The pre-processed ingredients were mixed according to the following ratios and kneaded into dough: 40.8% flour (low-gluten wheat flour:corn starch=7:3), 25.1% maltitol, 33.3% medicinal and edible ingredients (G. elata Bl:P. cocos:Coix seed:black sesame seed:walnut:jujube=3:10:10:10:10:10), 0.8% baking soda. Subsequently, the dough was molded, baked, and cooled, and the final product was obtained. For specific processes and components, refer to the cited literature [24].

Test kits

NE, GABA, and acetylcholine transferase (ChAT) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Kenuodi Biotechnology Co., Ltd. (Quanzhou, China) Malondialdehyde (MDA) biochemical assay kits were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Preparation of a functional biscuit suspension

The developed functional biscuits were powdered, and a certain amount of biscuit powder was weighed and placed in clean beakers. Pure water was added to prepare three different concentrations (low, medium, and high doses) of biscuit suspensions, which were then kept at room temperature for later use.

Animals grouping

After acclimating for 4 days in a suitable environment (temperature 23–25°C, relative humidity 50–70%, clean, and quiet), 40 C57BL/6J mice were randomly divided into the blank control group (NG, 10 mice), low-dose functional biscuit group (GLG, 10 mice), medium-dose functional biscuit group (GMG, 10 mice), and high-dose functional biscuit group (GHG, 10 mice). All mice were numbered, with 5 mice per cage.

Feeding and administration methods

The blank control group was gavaged with pure water, while the low-, medium-, and high-dose functional biscuit groups were respectively gavaged with biscuit suspensions at doses of 2.295 g/kg·d, 4.595 g/kg·d, and 6.890 g/kg·d. Apart from the administration period, the mice in each group had ad libitum access to feed and water throughout the experiment, which lasted for 42 days.

Observation of general condition

During the experimental process, the general conditions of the mice in each group were observed, including food intake, activity level, and mental status.

Detection of neurotransmitters and oxidative stress markers in mice

Quantitative determination of NE, ChAT, and GABA in the hippocampus of mice was conducted using ELISA. The specific procedures were carried out following the instructions provided with the assay kits. The absorbance (OD values) of each sample was measured using an enzyme reader, and the NE, ChAT, and GABA content in each sample was calculated by generating standard curves. The liver MDA levels in the mice were assessed using visible spectrophotometry, following the instructions provided in the assay kit.

Collection of intestinal mucosal samples

The collection of intestinal mucosal samples was conducted using a previously established method [25]. Under aseptic conditions, the intestines were longitudinally cut from the pylorus to the cecum. The intestinal contents were rinsed with sterile physiological saline, and the mucosa of each mouse was individually scraped with a sterile glass slide. The collected mucosa was then preserved in EP tubes and stored at −80°C.

High-throughput sequencing of intestinal mucosal microbiota

Total DNA extraction from the intestinal mucosal samples of each mouse group was performed using a DNA extraction kit (MN NucleoSpin 96 Soil), following the kit’s protocol. The concentration of the extracted DNA was determined using a NanoDrop, and the purity of the DNA was verified by 1.8% agarose gel electrophoresis. The V3+V4 hypervariable regions of the 16S rRNA gene were selected for polymerase chain reaction (PCR) amplification, utilizing universal 16S primers (338F/806R): forward primer 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The qualified DNA was amplified, and the resulting products were purified, quantified, and normalized to create sequencing libraries. The prepared libraries underwent quality control before being sequenced on an Illumina NovaSeq 6000 platform. Extraction, amplification, library preparation, and sequencing services were provided by Beijing Biomarker Technologies Co., Ltd. (Beijing, China).

Data processing and bioinformatics statistical analysis

The sequences were clustered using the USEARCH software platform (version 10.0) at a 97% similarity level, and a threshold of 0.005% of the total sequencing reads was used as the default filtering for operational taxonomic units (OTUs). Dilution curves and Shannon index curves were generated to assess the sequencing depth of the 16S rRNA gene of intestinal mucosal microbiota. Alpha and β diversity were evaluated through the Chao1, Shannon, ACE, Simpson indices, principal coordinates analysis (PCoA), non-metric multidimensional scaling (NMDS), and the unweighted pair-group method with arithmetic mean (UPGMA). Abundances of microbial taxa at different levels were calculated based on OTUs and presented in histogram form. Linear discriminant analysis effect size (LEfSe) was employed to identify biomarkers for different groups. To further analyze the regulatory effects of functional biscuits on the intestinal mucosal microbiota of mice, a correlation heatmap was generated using Spearman correlation analysis.

Statistical analysis and histogram plotting were performed using the GraphPad Prism 8.0 software. The data from each group are presented as the mean ± standard deviation. When the data met the assumption of a normal distribution, one-way analysis of variance (ANOVA) followed by the Tukey method was utilized for multiple-group comparisons. Alternatively, in cases where the data did not follow a normal distribution, the Kruskal–Wallis H test was employed for multiple-group comparisons. Results were considered statistically significant at p<0.05 and highly significant at p<0.01.

RESULTS

Observation of the general states of the mice

During the adaptation feeding phase, the mice exhibited normal eating habits, responsive behavior, and healthy coats. In the gavage administration of the functional biscuits, mice consuming biscuits showed a gradual reduction in food intake and an increase in water intake, along with an increase in urine output corresponding to the escalating gavage dosage.

Impacts of different dosages of the functional biscuits on NE, ChAT, GABA, and MDA in the mice

As shown in Fig. 1A, compared with the NG group, the average levels of NE in the hippocampus were significantly elevated in all groups fed the functional biscuits (p<0.05, p<0.01). Among the groups, the hippocampal NE levels were highest in the GHG group, followed by the GMG group and the GLG group.

Fig. 1.

Fig. 1.

Open in a new tab

Levels of neurotransmitters and antioxidant factors in the mice. (A) NE content in the mouse hippocampus. (B) ChAT content in the mouse hippocampus. (C) GABA content in the mouse hippocampus. (D) MDA content in the mouse liver. *p<0.05; **p<0.01. NE: norepinephrine; ChAT: acetylcholine transferase; GABA: gamma-aminobutyric acid; MDA: malondialdehyde.

As shown in Fig. 1B, compared with the NG group, the levels of ChAT in the hippocampus of mice were significantly increased in the GMG and GHG groups (p<0.05). Among all the groups, the hippocampal ChAT levels were highest in the GHG group, followed by the GMG group and the GLG group.

As shown in Fig. 1C, compared with the NG group, the levels of GABA in the hippocampus of mice fed the functional biscuits were increased on average in all groups, although not significantly (p>0.05). Among the groups, the GABA levels in the hippocampus were highest in the GHG group, followed by the GMG group and the GLG group.

As shown in Fig. 1D, compared with the NG group, the levels of MDA in the liver of mice fed the functional biscuits were significantly decreased on average in all groups, exhibiting statistical differences (p<0.05, p<0.01). Among the groups, the MDA levels in the liver were lowest in the GHG group, followed by the GMG group and the GLG group. This indicates that the functional biscuits could increase the levels of neurotransmitters in the hippocampus of mice and enhance the antioxidant capacity of the organism.

Impact of the functional biscuits on the intestinal mucosal microbiota in the mice

Impact of the functional biscuits on alpha and beta diversity of the intestinal microbiota in the mice

As shown in Fig. 2A and 2B, rarefaction curves and Shannon index curves were analyzed to assess whether the sequencing data volume was sufficient to reflect the species diversity in the samples and to suggest the species richness in the samples. With the increase in the number of sequencing reads, the rarefaction curves and Shannon index curves of the samples in this experiment gradually tended to flatten, suggesting that the sequencing depth of each sample was adequate. The microbial species information did not increase significantly with the increase in sequencing reads, indicating that the sample sequences in this experiment were sufficient for subsequent data analysis. As depicted in Fig. 2C–2E, the abundance-based coverage estimator (ACE) index, Chao1 index, and Shannon index were highest in the GLG group, followed by the GHG, GMG, and NG groups, indicating that the functional biscuits could increase species richness and the evenness of intestinal mucosal microbiota in mice. As illustrated in Fig. 2F, the Simpson index was highest in the GMG group, followed by the GHG, NG, and GLG groups; a higher Simpson index indicates a more concentrated distribution of different bacterial species in the intestinal mucosa. Therefore, the functional biscuits were capable of altering the alpha diversity of the intestinal mucosal microbiota in the mice, increasing the species diversity and evenness.

Fig. 2.

Fig. 2.

Open in a new tab

Impact of the functional biscuits on the alpha diversity of the intestinal mucosal microbiota in the mice. (A) Dilution curves. (B) Shannon index curves. (C) ACE index. (D) Chao1 index. (E) Shannon index. (F) Simpson index. ACE: Abundance-based Coverage Estimator.

Beta diversity was analyzed using PCoA, NMDS, and UPGMA methods, with the differences among multidimensional data samples visualized in a two-dimensional space through the Bray–Curtis distance algorithm. Figure 3A displays the phylogenetic tree of samples at the phylum level along with the top 10 most abundant species. The figure reveals two major clades, with the four samples from the NG group belonging to one clade and the four samples from each of the GHG and GLG groups, as well as all samples from the GMG group, belonging to the other clade. Figure 3B depicts the phylogenetic tree of samples at the genus level along with the top 10 most abundant species. Within this figure, the four samples from the NG group exhibit clustering based on species composition, while the clustering within each of the other intervention groups is not pronounced. The branch lengths of the sample phylogenetic tree are relatively short, indicating a high similarity in species composition among the samples. The PCoA analysis indicated a p-value of 0.138, and visualization of the individual samples in the two-dimensional plot similarly confirmed the findings of the UPGMA analysis (Fig. 3C). The NMDS analysis result exhibited a stress value of 0.1314, indicating a certain degree of reliability for the analysis (Fig. 3D). Most samples from the NG group exhibited separation from the intervention groups, albeit not significantly. Consequently, this indicates that the functional biscuits could modify the intestinal mucosal microbial community structure of the mice to some degree, yet without reaching statistical significance.

Fig. 3.

Fig. 3.

Open in a new tab

Impact of the functional biscuits on the beta diversity of the intestinal mucosal microbiota in the mice. (A) Phylum-level UPGMA dendrogram columns. (B) Genus-level UPGMA dendrogram columns. (C) PCoA analysis. (D) NMDS analysis. UPGMA: unweighted pair-group method with arithmetic mean; PCoA: principal coordinates analysis; NMDS: non-metric multidimensional scaling.

Impact of the functional biscuits on the species composition of the intestinal mucosal microbiota in the mice

As shown in Fig. 4A and 4B, the NG group contained a total of 5,160 OTUs, among which 3,963 were unique OTUs. The GLG group had 8,890 OTUs in total, among which 7,182 were unique. Similarly, the GMG group presented with 8,450 OTUs, 6,756 of which were unique. Finally, the GHG group exhibited the highest number of OTUs, 9,805, with 8,082 of them being unique. Clearly, the GHG group exhibited the greatest total number of OTUs, closely followed by the GLG, GMG, and NG groups. This observation is consistent with the patterns depicted in the rank abundance curves (Fig. 4C). Subsequently, we identified the top 10 phyla and 15 genera with the highest relative abundances in the intestinal mucosal microbiota and displayed them in a bar chart. At the phylum level, Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteriota were the dominant phylum. Compared with the NG group, the relative abundances of Firmicutes were increased in the GLG and GMG groups but decreased in the GHG group. The relative abundances of Bacteroidota were reduced in all three treatment groups: GLG, GMG, and GHG. Furthermore, the GLG, GMG, and GHG groups all showed an increase in the relative abundances of both Proteobacteria and Actinobacteriota (Fig. 4D). At the genus level, the predominant genera included unclassified_Muribaculaceae, Ligilactobacillus, Lactobacillus, uncultured_Bacteroidales_bacterium, Candidatus_Arthromitus, and Bacillus. Compared with the NG group, the GLG, GMG, and GHG groups exhibited decreased relative abundances of unclassified_Muribaculaceae, Ligilactobacillus, Lactobacillus, and uncultured_Bacteroidales_bacterium. The relative abundances of Candidatus_Arthromitus and Bacillus increased in the GLG, GMG, and GHG groups (Fig. 4E). The results indicate changes in the relative abundances of intestinal mucosal microbiota in mice following consumption of the functional biscuits.

Fig. 4.

Fig. 4.

Open in a new tab

Impact of the functional biscuits on the species composition of the intestinal mucosal microbiota in the mice. (A) Venn diagram of the intestinal microbiota. (B) Distribution of OTU numbers. (C) Rank abundance curve analysis. (D) Bar chart depicting species distribution at the phylum level. (E) Bar chart depicting species distribution at the genus level. OTU: operational taxonomic unit.

Analysis of inter-group species differences in the intestinal mucosal microbiota of the mice after consumption of the functional biscuits

We further employed the LEfSe analysis method, using a linear discriminant analysis (LDA) score greater than 2.5 as the screening criterion, to compare the differences in bacterial genera between the NG group and the GLG, GMG, and GHG groups. The analysis of species differences between the NG group and the GLG group revealed (Fig. 5A) that Ligilactobacillus, uncultured_Bacteroidales_bacterium, Parasutterella, and Muribaculum were the characteristic bacterial genera enriched in the NG group, whereas Corynebacterium, Tyzzerella, Lachnospiraceae_NK4A136_group, unclassified_Atopobiaceae, Lawsonella, and 23 other major bacterial genera were the characteristic genera enriched in the GLG group. The analysis of species differences between the NG group and the GMG group revealed (Fig. 5B) that Bifidobacterium and Eubacterium_brachy_group were the characteristic bacterial genera enriched in the NG group, whereas Staphylococcus, Corynebacterium, Tyzzerella, Clostridium_sensu_stricto_1, Ruminococcus_torques_group, and six other major bacterial genera were the characteristic genera enriched in the GMG group. The analysis of species differences between the NG group and the GHG group revealed distinct bacterial profiles (Fig. 5C). Specifically, Muribaculum was identified as a characteristic bacterial genus enriched in the NG group, whereas 22 major bacterial genera, including Staphylococcus, Corynebacterium, Tyzzerella, Lactococcus, and unclassified_Gemmatimonadaceae, were enriched as characteristic genera in the GHG group.

Fig. 5.

Fig. 5.

Open in a new tab

Impact of the functional biscuits on the characteristics of the mouse intestinal mucosal microbiota. (A) NG group and GLG group; (B) NG group and GMG group; (C) NG group and GHG group. NG: blank control group; GLG: low-dose functional biscuit group; GMG: medium-dose functional biscuit group; GHG: high-dose functional biscuit group.

Analysis of the correlation between characteristic bacterial genera and NE, ChAT, GABA, and MDA

We further adopted the Spearman correlation analysis method to explore the associations between the characteristic bacterial genera selected in Section 3.3.3 and NE, ChAT, GABA, and MDA. This analysis aimed to investigate the correlation between the functional biscuit’s impact on the intestinal mucosal microbiota and cytokines in mice. The top 10 characteristic bacterial genera with the highest correlation were selected and presented. Comparing the NG group and the GLG group, the correlation heatmap (Fig. 6A) revealed significant positive correlation of Synechococcus_CC9902 and GCA_900066575 with NE. Additionally, GCA_900066575, Lachnospiraceae_NK4A136_group, Actinomyces, Macrococcus, and Sulfurovum showed significant negative correlation with MDA. Comparing the NG group with the GMG group, the correlation heatmap (Fig. 6B) revealed significant positive correlation between Leuconostoc and NE. Fusicatenibacter and Ruminococcus_torques_group also exhibited significant positive correlation with ChAT. On the other hand, Bifidobacterium showed significant negative correlation with both NE and ChAT. Furthermore, both unclassified_Clostridia_vadinBB60_group and Leuconostoc demonstrated significant negative correlation with MDA. Comparing the NG group with the GHG group, the correlation heatmap (Fig. 6C) revealed significant positive correlations. Specifically, Candidatus_Actinomarina, Megamonas, unclassified_Atopobiaceae, Tyzzerella, Cyanobium_PCC_6307, and Lachnospira were positively correlated with NE. Additionally, Lachnospira, Ruminococcus_torques_group, Romboutsia, and Roseomonas exhibited significant positive correlations with ChAT, whereas Muribaculum showed significant negative correlation with ChAT. Furthermore, Candidatus_Actinomarina, unclassified_Atopobiaceae, Megamonas, Tyzzerella, and Cyanobium_PCC_6307 demonstrated significant negative correlations with MDA. Based on the correlation analysis conducted above, changes in the characteristic intestinal mucosal bacterial genera following consumption of the functional biscuits by the mice were found to be associated with NE, ChAT, and MDA. However, the specific underlying mechanisms await further experimental investigation.

Fig. 6.

Fig. 6.

Open in a new tab

Correlation analysis among representative bacterial genera, NE, ChAT, GABA, and MDA. (A) NG group and GLG group; (B) NG group and GMG group; (C) NG group and GHG group. Note: The legend displays the correlation coefficient values. Red indicates positive correlation, blue indicates negative correlation, and the color gradient represents the strength of the correlation. *p<0.05, **p<0.01. NE: norepinephrine; ChAT: acetylcholine transferase; GABA: gamma-aminobutyric acid; MDA: malondialdehyde; NG: blank control group; GLG: low-dose functional biscuit group; GMG: medium-dose functional biscuit group; GHG: high-dose functional biscuit group.

DISCUSSION

The balanced and diverse state of the intestinal microbiota establishes a beneficial symbiotic relationship with the host through complex molecular communication; on the other hand, intestinal microbiota dysbiosis has also been identified as a determining factor in many diseases [26]. Throughout the lifecycle, various factors, including age-related changes, geographical environment, lifestyle, dietary preferences, and medication use, can potentially affect the composition of an individual’s intestinal microbiota [27, 28]. G. elata Bl is a crucial functional food ingredient that can be utilized for neuroprotection and neurogenesis promotion, enhancing resistance, delaying aging, and benefiting brain health [29, 30]. Sesamin, the primary active component of sesame seeds, can alter the intestinal microbiota, inhibit damage to the intestinal barrier integrity induced by stress, and suppress neuroinflammatory responses [31]. Jujube polysaccharides can regulate the intestinal microbiota composition, exerting potential regulatory effects on both the digestive and nervous systems [32]. Additionally, walnuts [33], Coix seeds [34], and P. cocos [35] all possess the ability to modulate the intestinal microbiota composition and microbial diversity. Hence, the present experiment examined the effects of functional biscuits, primarily composed of the aforementioned medicinal foods, on the intestinal mucosal microbiota, brain function, and antioxidant capacity in mice.

Neurotransmitters play a crucial role in the development of the nervous system and brain function. NE participates in the shaping and connectivity of the nervous system during critical periods of development, exerting long-lasting and permanent effects on the developmental trajectory and function of the brain [36]. In this experiment, the NE content in the brains of normal mice increased significantly after being fed the functional biscuits, and there was a positive correlation between the NE content and the administered dose. Cholinergic dysfunction is one of the primary causes of cognitive impairments, and ChAT is the biosynthetic enzyme responsible for the production of acetylcholine [37]. Studies have indicated that neural stem cells and microglia can successfully ameliorate intracerebral acetylcholine (ACh) levels through overexpression of the functional gene ChAT, thereby restoring memory function [38]. We observed that the functional biscuits could elevate intracerebral ChAT levels, with significant differences observed in the GMG and GHG groups compared with the control group (p<0.05). GABA is widely distributed in the central nervous systems of mammals, and GABAergic neurons can influence neural circuits through inhibitory, excitatory, or modulatory synaptic actions [39]. In this experiment, the functional biscuits demonstrated a certain enhancing effect on GABA in normal mice, but there was no significant difference (p>0.05). This suggests that under the conditions of this experiment, the regulatory effect of the functional biscuits on GABA was relatively small. However, it is possible that with further extension of the experimental duration, a more pronounced regulatory effect may be observed, which warrants further experimental investigation.

Due to the high oxygen consumption, relatively low antioxidant defense capacity, and high fat content of the brain, reducing oxidative stress levels is beneficial for brain health [40]. The liver functions as a critical organ for lipid metabolism and redox reactions in the body, holding a pivotal role in safeguarding tissue cells from oxidative stress and other potential injuries [41]. MDA is one of the commonly used biomarkers for evaluating intracellular lipid peroxidation, and higher concentrations of MDA are often detected in biological samples from individuals with various diseases compared with healthy individuals [42]. G. elata polysaccharide, one of the active ingredients of G. elata Bl, has been found in animal experiments to inhibit monoamine oxidase (MAO) activity in the brain and reduce MDA levels in aging brain tissue [43]. In addition, the main components of Coix seeds [44], P. cocos [45], and walnuts [46] also exhibit good antioxidant activity. In this experiment, the functional biscuits were found to significantly reduce liver MDA levels in normal mice. A negative correlation was observed between the reduction in MDA levels and the dose of functional biscuits administered. These findings suggest that functional biscuits possess significant antioxidant properties in the body. However, the underlying mechanism remains unclear and warrants further investigation.

Through the evaluation of the OTU counts, alpha diversity, and beta diversity, we found that the functional biscuits can alter the diversity of the gut mucosal microbiota in mice, increase the OTU counts of intestinal mucosal microbiota, change the microbial community structure of the intestinal mucosa in mice, and regulate species diversity and evenness. By comparing relative abundances, we found that at the phylum level, the abundance of Firmicutes increased in the GLG and GMG groups compared with the NG group; the abundances of Proteobacteria and Actinobacteriota increased in the GLG, GMG, and GHG groups; and the abundance of Bacteroidota decreased in the GLG, GMG, and GHG groups. At the genus level, compared with the NG group, mice fed the different doses of the functional biscuits exhibited decreased relative abundances of bacteria such as unclassified_Muribaculaceae and uncultured_Bacteroidales_bacterium in their intestinal mucosal microbiota, while the relative abundances of Candidatus_Arthromitus and Bacillus increased. The LEfSe analysis revealed enrichment of four bacterial genera, Corynebacterium, Romboutsia, Tyzzerella, and Turicibacter, in the biscuit intervention groups. Finally, our Spearman correlation analysis revealed the presence of multiple bacterial genera that correlated with hippocampal NE, ChAT, and hepatic MDA levels across the three biscuit-intervention groups. Specifically, Ruminococcus_torques_group and Tyzzerella were commonly associated with the GMG and GHG groups, while Romboutsia was shared between the GLG and GHG groups.

Romboutsia possesses extensive metabolic capabilities in carbohydrate utilization, single amino acid fermentation, and metabolite end-product formation, playing a crucial role in maintaining host metabolic stability. It has the ability to produce short chain fatty acids (SCFAs) and exhibits potential correlations with inflammation and oxidative stress [47, 48]. Turicibacter exhibits immunomodulatory and anti-inflammatory effects, potentially serving as a key probiotic for ameliorating metabolic endotoxemia [49]. Tyzzerella is a bacterium closely associated with dietary fatty acid intake [50]. In this experiment, the enrichment of Tyzzerella may be related to the nut-based medicinal foods abundant in the functional biscuits. Consequently, we infer that the bacterial genera with common characteristics identified in this experiment may be key players in the biological effects of the functional biscuits. Nonetheless, in this study, we did not observe concentration-dependent changes in the intestinal mucosal microbiota associated with the functional biscuits. Given the complexity of active ingredients in medicinal foods and the varying dosage patterns observed in different individuals [51], as well as the intricate and dynamic microbial communities on the intestinal mucosal surface, which exhibit a highly complex symbiotic relationship with host health [52], we contemplate adopting a more optimized experimental design in the future to explore the regulatory effects of functional biscuits on the intestinal microbiota.

In summary, functional biscuits can modulate the structure and abundance of the intestinal mucosal microbiota in mice, increase the levels of NE and ChAT in the hippocampus, and inhibit the levels of MDA in the liver. Thus, functional biscuits exhibit effects on modulating the intestinal mucosal microbiota, improving brain function, and antioxidant activity. However, further longitudinal studies are required to investigate the microecological mechanisms underlying the effects of characteristic bacterial genera in the intestinal mucosal microbiota on the gut-brain axis and to clarify the functional mechanisms of functional biscuits.

DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online repositories. The name of the repository and accession number are as follows: NCBI (accession: PRJNA1046237).

FUNDING

This work was supported by the Key Discipline Project on Chinese Pharmacology of Hunan University of Chinese Medicine (202302).

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

REFERENCES

  • 1.Baker MT, Lu P, Parrella JA, Leggette HR. 2022. Consumer acceptance toward functional foods: a scoping review. Int J Environ Res Public Health 19: 1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Sharma S, Singh A, Sharma S, Kant A, Sevda S, Taherzadeh MJ, Garlapati VK. 2021. Functional foods as a formulation ingredients in beverages: technological advancements and constraints. Bioengineered 12: 11055–11075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Alkhatib A, Tsang C, Tiss A, Bahorun T, Arefanian H, Barake R, Khadir A, Tuomilehto J. 2017. Functional foods and lifestyle approaches for diabetes prevention and management. Nutrients 9: 1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Soares JM, Teixeira F, Oliveira ML, Amaral LAD, Almeida TDSF, Souza GHO, Hokama LM, Menegassi B, Santos EFD, Novello D. 2022. Eggplant flour addition in cookie: nutritional enrichment alternative for children. Foods 11: 1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Nguyen NDT, Phan TTH, Tran TTT, Ton NMN, Vo DLT, Man Le VV. 2022. Enzymatic treatment of spent green tea leaves and their use in high-fibre cookie production. Food Technol Biotechnol 60: 396–405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Sawashita J, Onitsuka S, Gen-no H, Ishikawa S, Iino F, Tateishi N, Murakami T, Seki Y, Nagaiwa T, Hanaoka M, et al. 2009. Effects of mild calorie restriction and high-intensity interval walking in middle-aged and older overweight Japanese. Exp Gerontol 44: 666–675. [DOI] [PubMed] [Google Scholar]
  • 7.Filgueiras AR, Pires de Almeida VB, Koch Nogueira PC, Alvares Domene SM, Eduardo da Silva C, Sesso R, Sawaya AL. 2019. Exploring the consumption of ultra-processed foods and its association with food addiction in overweight children. Appetite 135: 137–145. [DOI] [PubMed] [Google Scholar]
  • 8.Yildiz E, Gocmen D. 2021. Use of almond flour and stevia in rice-based gluten-free cookie production. J Food Sci Technol 58: 940–951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Sun X, Jia B, Sun J, Lin J, Lu B, Duan J, Li C, Wang Q, Zhang X, Tan M, et al. 2023. Gastrodia elata Blume: a review of its mechanisms and functions on cardiovascular systems. Fitoterapia 167: 105511. [DOI] [PubMed] [Google Scholar]
  • 10.Gong MQ, Lai FF, Chen JZ, Li XH, Chen YJ, He Y. 2024. Traditional uses, phytochemistry, pharmacology, applications, and quality control of Gastrodia elata Blume: a comprehensive review. J Ethnopharmacol 319: 117128. [DOI] [PubMed] [Google Scholar]
  • 11.Gao M, Wu Y, Yang L, Chen F, Li L, Li Q, Wang Y, Li L, Peng M, Yan Y, et al. 2023. Anti-depressant-like effect of fermented Gastrodia elata Bl. by regulating monoamine levels and BDNF/NMDAR pathways in mice. J Ethnopharmacol 301: 115832. [DOI] [PubMed] [Google Scholar]
  • 12.Dodevska M, Kukic Markovic J, Sofrenic I, Tesevic V, Jankovic M, Djordjevic B, Ivanovic ND. 2022. Similarities and differences in the nutritional composition of nuts and seeds in Serbia. Front Nutr 9: 1003125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wei P, Zhao F, Wang Z, Wang Q, Chai X, Hou G, Meng Q. 2022. Sesame (Sesamum indicum L.): a comprehensive review of nutritional value, phytochemical composition, health benefits, development of food, and industrial applications. Nutrients 14: 4079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ni ZJ, Zhang YG, Chen SX, Thakur K, Wang S, Zhang JG, Shang YF, Wei ZJ. 2022. Exploration of walnut components and their association with health effects. Crit Rev Food Sci Nutr 62: 5113–5129. [DOI] [PubMed] [Google Scholar]
  • 15.Lu Y, Bao T, Mo J, Ni J, Chen W. 2021. Research advances in bioactive components and health benefits of jujube (Ziziphus jujuba Mill.) fruit. J Zhejiang Univ Sci B 22: 431–449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Lam CT, Gong AG, Lam KY, Zhang LM, Chen JP, Dong TT, Lin HQ, Tsim KW. 2016. Jujube-containing herbal decoctions induce neuronal differentiation and the expression of anti-oxidant enzymes in cultured PC12 cells. J Ethnopharmacol 188: 275–283. [DOI] [PubMed] [Google Scholar]
  • 17.Nie A, Chao Y, Zhang X, Jia W, Zhou Z, Zhu C. 2020. Phytochemistry and pharmacological activities of Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb. Front Pharmacol 11: 505249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Zeng Y, Yang J, Chen J, Pu X, Li X, Yang X, Yang L, Ding Y, Nong M, Zhang S, et al. 2022. Actional mechanisms of active ingredients in functional food adlay for human health. Molecules 27: 4808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Schmidt TSB, Raes J, Bork P. 2018. The human gut microbiome: from association to modulation. Cell 172: 1198–1215. [DOI] [PubMed] [Google Scholar]
  • 20.El Aidy S, Dinan TG, Cryan JF. 2015. Gut microbiota: the conductor in the orchestra of immune-neuroendocrine communication. Clin Ther 37: 954–967. [DOI] [PubMed] [Google Scholar]
  • 21.Dicks LMT. 2022. Gut bacteria and neurotransmitters. Microorganisms 10: 1838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Wu Y, Peng X, Li X, Li D, Tan Z, Yu R. 2022. Sex hormones influence the intestinal microbiota composition in mice. Front Microbiol 13: 964847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Li C, Xiao N, Deng N, Li D, Tan Z, Peng M. 2023. Dose of sucrose affects the efficacy of Qiweibaizhu powder on antibiotic-associated diarrhea: association with intestinal mucosal microbiota, short-chain fatty acids, IL-17, and MUC2. Front Microbiol 14: 1108398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Li CR, Zhou K, Peng MJ, Yuan JL, Wu YY, Tan ZJ. 2023. Development of Gastrodia elata Bl Biscuits. Baoxian Yu Jiagong 23: 50–56. [Google Scholar]
  • 25.He Y, Tang Y, Peng M, Xie G, Li W, Tan Z. 2019. Influence of Debaryomyces hansenii on bacterial lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea. PLoS One 14: e0225802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Miniello VL, Miniello A, Ficele L, Skublewska-D’Elia A, Dargenio VN, Cristofori F, Francavilla R. 2023. Gut immunobiosis and biomodulators. Nutrients 15: 2114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Shanahan F, Ghosh TS, O’Toole PW. 2021. The healthy microbiome-what is the definition of a healthy gut microbiome? Gastroenterology 160: 483–494. [DOI] [PubMed] [Google Scholar]
  • 28.Martinez JE, Kahana DD, Ghuman S, Wilson HP, Wilson J, Kim SCJ, Lagishetty V, Jacobs JP, Sinha-Hikim AP, Friedman TC. 2021. Unhealthy lifestyle and gut dysbiosis: a better understanding of the effects of poor diet and nicotine on the intestinal microbiome. Front Endocrinol (Lausanne) 12: 667066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Hsu WH, Huang NK, Shiao YJ, Lu CK, Chao YM, Huang YJ, Yeh CH, Lin YL. 2021. Gastrodiae rhizoma attenuates brain aging via promoting neuritogenesis and neurodifferentiation. Phytomedicine 87: 153576. [DOI] [PubMed] [Google Scholar]
  • 30.Li Y, Zhang E, Yang H, Chen Y, Tao L, Xu Y, Chen T, Shen X. 2022. Gastrodin ameliorates cognitive dysfunction in vascular dementia rats by suppressing ferroptosis via the regulation of the Nrf2/Keap1-GPx4 signaling pathway. Molecules 27: 6311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wang Q, Jia M, Zhao Y, Hui Y, Pan J, Yu H, Yan S, Dai X, Liu X, Liu Z. 2019. Supplementation of Sesamin alleviates stress-induced behavioral and psychological disorders via reshaping the gut microbiota structure. J Agric Food Chem 67: 12441–12451. [DOI] [PubMed] [Google Scholar]
  • 32.Li Z, Wu M, Wei W, An Y, Li Y, Wen Q, Zhang D, Zhang J, Yao C, Bi Q, et al. 2023. Fingerprinting evaluation and gut microbiota regulation of polysaccharides from Jujube (Ziziphus jujuba Mill.) fruit. Int J Mol Sci 24: 7239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ji J, Yi X, Gao X, Wang B, Zhang X, Shen X, Xia G. 2024. Synergistic effects of tilapia head protein hydrolysate and walnut protein hydrolysate on the amelioration of cognitive impairment in mice. J Sci Food Agric 104: 5419–5434. [DOI] [PubMed] [Google Scholar]
  • 34.Ge Q, Hou CL, Rao XH, Zhang AQ, Xiao GM, Wang LY, Jin KN, Sun PL, Chen LC. 2024. In vitro fermentation characteristics of polysaccharides from Coix seed and its effects on the gut microbiota. Int J Biol Macromol 262: 129994. [DOI] [PubMed] [Google Scholar]
  • 35.Ma KL, Kei N, Yang F, Lauw S, Chan PL, Chen L, Cheung PCK. 2023. In Vitro fermentation characteristics of fungal polysaccharides derived from Wolfiporia cocos and their effect on human fecal microbiota. Foods 12: 4014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Saboory E, Ghasemi M, Mehranfard N. 2020. Norepinephrine, neurodevelopment and behavior. Neurochem Int 135: 104706. [DOI] [PubMed] [Google Scholar]
  • 37.Granger AJ, Wang W, Robertson K, El-Rifai M, Zanello AF, Bistrong K, Saunders A, Chow BW, Nuñez V, Turrero García M, et al. 2020. Cortical ChAT+ neurons co-transmit acetylcholine and GABA in a target- and brain-region-specific manner. eLife 9: e57749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Ban YH, Park D, Choi EK, Kim TM, Joo SS, Kim YB. 2023. Effectiveness of combinational treatments for alzheimer’s disease with human neural stem cells and microglial cells over-expressing functional genes. Int J Mol Sci 24: 9561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Miller MW. 2019. GABA as a neurotransmitter in gastropod molluscs. Biol Bull 236: 144–156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.de França Silva RC, de Souza MA, da Silva JYP, Ponciano CDS, Bordin Viera V, de Menezes Santos Bertozzo CC, Guerra GC, de Souza Araújo DF, da Conceição MM, Querino Dias CC, et al. 2021. Evaluation of the effectiveness of macaíba palm seed kernel (Acrocomia intumescens drude) on anxiolytic activity, memory preservation and oxidative stress in the brain of dyslipidemic rats. PLoS One 16: e0246184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Deng M, Zhang S, Wu S, Jiang Q, Teng W, Luo T, Ouyang Y, Liu J, Gu B. 2024. Lactiplantibacillus plantarum N4 ameliorates lipid metabolism and gut microbiota structure in high fat diet-fed rats. Front Microbiol 15: 1390293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Tsikas D. 2017. Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges. Anal Biochem 524: 13–30. [DOI] [PubMed] [Google Scholar]
  • 43.Zhu H, Liu C, Hou J, Long H, Wang B, Guo D, Lei M, Wu W. 2019. Gastrodia elata Blume polysaccharides: a review of their acquisition, analysis, modification, and pharmacological activities. Molecules 24: 2436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Lee ES, Kim YI, Lee JH, Kim YG, Han KS, Yoon YH, Cho BO, Park K, Lee H, Cho JS. 2023. Comparison of quality, antioxidant capacity, and anti-inflammatory activity of Adlay [Coix lacryma-jobi L. var. ma-yuen (Rom. Caill.) Stapf.] sprout at several harvest time. Plants 12: 2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Wang S, Tan W, Zhang L, Jiang H. 2024. Pachymic acid protects against bleomycin-induced pulmonary fibrosis by suppressing fibrotic, inflammatory, and oxidative stress pathways in mice. Appl Biochem Biotechnol 196: 3344–3355. [DOI] [PubMed] [Google Scholar]
  • 46.Martínez ML, Labuckas DO, Lamarque AL, Maestri DM. 2010. Walnut (Juglans regia L.): genetic resources, chemistry, by-products. J Sci Food Agric 90: 1959–1967. [DOI] [PubMed] [Google Scholar]
  • 47.Lv Z, Zhang C, Song W, Chen Q, Wang Y. 2022. Jellyfish collagen hydrolysate alleviates inflammation and oxidative stress and improves gut microbe composition in high-fat diet-fed mice. Mediators Inflamm 2022: 5628702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Chen F, Zhang X, Bai J, Cao X, Chen L, Wang D, Guo S, Shang E, Su S, Duan J. 2024. Anti-proliferative effect and mechanisms of Peony pollen on BPH via inhibition of inflammatory factors, oxidative damage and modulation of gut microbiota and SCFAs metabolism. Pharmacol Res Mod Chin Med 12: 100472. [Google Scholar]
  • 49.Gao X, Chang S, Liu S, Peng L, Xie J, Dong W, Tian Y, Sheng J. 2020. Correlations between α-Linolenic acid-improved multitissue homeostasis and gut microbiota in mice fed a high-fat diet. mSystems 5: e00391–e20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Xu AA, Kennedy LK, Hoffman K, White DL, Kanwal F, El-Serag HB, Petrosino JF, Jiao L. 2022. Dietary fatty acid intake and the colonic gut microbiota in humans. Nutrients 14: 2722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Gao Y, Liang A, Fan X, Hu L, Hao F, Yubo L. 2019. Safety research in traditional Chinese medicine: methods, applications, and outlook. Engineering 5: 76–82. [Google Scholar]
  • 52.Duerkop BA, Vaishnava S, Hooper LV. 2009. Immune responses to the microbiota at the intestinal mucosal surface. Immunity 31: 368–376. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The name of the repository and accession number are as follows: NCBI (accession: PRJNA1046237).

Articles from Bioscience of Microbiota, Food and Health are provided here courtesy of IPEC, Inc.

RESOURCES