Soybean Meal Extract Preserves Memory Ability by Increasing Presynaptic Function and Modulating Gut Microbiota in Rats

Age-related degenerative brain diseases frequently manifest as memory deficits. Dietary interventions or nutraceuticals may provide efficacious treatments through prevention and cure. Soybean meal, a byproduct of soy oil refining, has health benefits, but its effect on memory function is unknown. Therefore, we evaluated the effect of the oral administration of soybean meal extract (SME) for 2 weeks on memory function using the Morris water maze (MWM) test in healthy rats and investigated the possible underlying mechanisms. First, analysis of the composition revealed that SME is rich in isoflavones; SME did not exhibit hepatotoxicity or renal toxicity at the different doses tested. The MWM results revealed that the escape latency and movement distance of rats were significantly shorter in the SME group than in the control group, indicating that SME can help in memory preservation. In addition, SME increased the levels of presynaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, syntaxin, synapsin I, and 25-kDa synaptosome-associated protein as well as protein kinases and their phosphorylated expression, including extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal nerve terminals (synaptosomes). Transmission electron microscopy also indicated that SME increased the number of synaptic vesicles in hippocampal synaptosomes. Furthermore, SME rats exhibited altered microbiota composition compared with control rats. Therefore, our data suggest that SME can increase presynaptic function and modulate gut microbiota, thus aiding in memory preservation in rats.


Introduction
With the aging of society worldwide, the incidence of degenerative brain diseases, including Parkinson's disease, Alzheimer's disease (AD), Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis, is increasing [1]. Memory impairment is one of the main symptoms of agerelated degenerative brain diseases [2,3]. Because no effective therapies exist for memory impairment, preventive approaches, such as dietary interventions or nutraceuticals, have been receiving increasing attention [4][5][6][7]. Consumption of foods rich in polyphenols is associated with improved cognitive performance and a reduced risk of cognitive impairment in humans [8][9][10].
In the present study, we focused on the soybean meal, which is a byproduct of soybean oil extraction and is widely used in feed and food industries [22]. It contains functional bioactive compounds similar to soybeans, such as phenolics and isoflavones [23]. Similar to soybean, soybean meal has diverse biological effects, including antioxidative, anti-inflammatory, anticancer, and antiphotodamage effects [24][25][26][27]. However, no study has focused on the role of soybean meal in memory function. Here, we (i) determined whether the oral administration of soybean meal extract (SME) for 2 weeks affected memory in healthy rats using the Morris water maze (MWM) test, (ii) elucidated the mechanisms of action of SME and (iii) determined whether SME produced any side effects in rats. In addition, the feces of rats were collected to analyze gut microbiota, which is associated with memory function [28].

Preparation of SME
The extract was prepared as described in previous studies [26,27]. Briefly, 50 g of soybean meal was dissolved in 150 mL of ethanol/water (1:1 v/v) by continuously stirring for 2 h at 4°C. Then, the supernatants were obtained by centrifugation (6000 rpm for 20 min at 25 °C) and lyophilized to obtain a powder. The extract powder was then stored at −20 °C for further use in the biological assays.

LC-MS/MS Analysis
Isoflavone and phenolic acid of SME were separated on an Agilent Eclipse plus C18 column (100 mm × 4.6 mm, 3.5 μm) at a flow rate of 0.35 mLmin −1 by using an Agilent 1200 series binary pump . Gradient elution was performed with mobile phase A (5% methanol with 0.1% formic acid) and mobile phase B (methanol with 0.1% formic acid). The initial condition was 50:50 mobile phase A/mobile phase B (v/v) for 2.5 min, after which is was changed to 80% mobile phase B in 0.1 min that was maintained for 2 min. Finally, the solvent composition was quickly reverted to the initial conditions and equilibrated for 11 min. Mass spectrometry was operated in multiple ion-monitoring mode (MRM) and negative polarity at −4200 V by using API 3000 (MDS SCIEX, Applied Biosystems, Ontario, Canada).

Experimental Animals
The International Guidelines for Care and Use of Laboratory Animals were followed for all experiments, and the experimental protocol was approved by the Animal Care Committee of Fu Jen Catholic University (approval number: A11018). Thirty male Sprague-Dawley rats (Taiwan Bio-LASCO) weighing 160-200 g were used. They were housed in plastic cages and were fed on pellets with free access to tap water. Room temperature was controlled at 22 ± 2°C with a 12-h light:12-h dark cycle. After 3 days of training with MWM, rats were divided into SME and tap water (control) groups; they were orally administered SME solution or an equal volume of 0.9% normal saline, respectively, daily for 2 consecutive weeks. After 2 weeks, the behavioral test was conducted 30 min by using the MWM video analysis system. Next, the rat's body weight was measured, and stool samples from 15 rats were collected and immediately stored at −80°C for gut microbiota analysis. Finally, 15 rats were deeply anesthetized and killed, and the hippocampus was collected to prepare synaptosomes for transmission electron microscopy (TEM) and western blotting. In addition, the liver and kidney were collected from the rats after sacrifice for hematoxylin-eosin (H&E) staining.

MWM Test
The MWM test was conducted to evaluate the performance of spatial learning and memory, as described by previous study [29]. A circular pool with a diameter of 55 cm and height of 25 cm was filled with opacified water (20 cm depth) at 25 ± 1°C. The pool was divided into four quadrants, and the platform was placed at the center of one fixed quadrant for all trials. Training (days 1-4) was conducted four times a day, and the escape latency time for each rat to go to the platform was measured for 120 s. Rats reaching the platform were allowed to be remain there for 15 s. Rats that failed to locate the platform were guided to the platform and allowed to stay for 30 s. The latency period of the failed rats was recorded within 120 s. The swimming path from the entry to the hidden platform, escape latency, and movement distance in the coverage zone was recorded using a video-tracking system (Version 1.17, SINGA Technology Corporation, Taipei, Taiwan).

H&E Staining
Liver or kidney tissues were fixed in 4% PFA, dehydrated with graded alcohol, and embedded in paraffin wax. A series of paraffin sections (5 μm) were cut using a Leica rotation microtome and stained with H&E, and images were captured under a microscope with ×400 magnification. Histological changes in the liver and kidney sections were determined in terms of cytoplasmic color fading, vacuolization, nuclear condensation, nuclear fragmentation, nuclear fading, and erythrocyte stasis [30].

Preparation of Synaptosomes and TEM
Synaptosomes were prepared as previously reported [29]. Briefly, rats were killed by cervical dislocation and decapitation. The hippocampus was rapidly removed and homogenized in an ice-cold HEPES-buffered medium containing 0.32 M sucrose (pH 7.4). The homogenate was centrifuged at 3000×g for 10 min at 4 °C. The supernatant was retained and centrifuged at 14 500 ×g for 12 min at 4 °C. The pellet was resuspended and layered on top of a discontinuous Percoll gradient before being centrifuged at 32 500 ×g for 7 min at 4 °C. The protein concentration was determined using the Bradford assay. The synaptosomes were centrifuged in the final wash to obtain synaptosomal pellets containing 0.5 mg protein. For TEM, rat hippocampal synaptosomes from each group were placed in an electron microscope fixative solution for 1 day. Samples were then postfixed in 1% osmium tetraoxide for 2 h, followed by gradient ethanol dehydration, soaking, and embedding in pure epoxy resin. Samples were cut into 70-nm-thick sections and stained with uranium and lead. Finally, sections were observed under a TEM (JEM-1400, JEOL, Japan).

Western Blotting Analysis
Western blotting was performed as described by previous reported [29]. Briefly, hippocampal synaptosomes were homogenized and the concentrations of proteins were determined using Bradford's method, with bovine serum albumin (BSA) as a standard. Equal protein amounts (20 μg) were subjected to sodium dodecylsulfate polyacrylamide (SDS-PAGE) gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1h at room temperature and incubated overnight at 4°C with primary antibodies. The antibodies used were anti-synaptophysin ( , and anti-β-actin (1:1000). Next, the membrane was washed with TBST three times and incubated with a secondary horseradish peroxidase-conjugated antibody (1:5000) at room temperature for 1 h. Protein bands were visualized using a chemiluminescence reagent (Amersham, Buckinghamshire, UK). The intensity of the protein bands was analyzed using the ImageJ software (Synoptics, Cambridge, UK).

Gut Microbiota
Gut microbiota analysis was conducted by the Biotools Microbiome Research Center (Taipei, Taiwan). Briefly, DNA was extracted from fecal samples of rats using the QIAamp PowerFecal DNA kit (Qiagen, CA, USA). The 16s rDNA amplicon sequencing of the V4 hypervariable region was performed with an Illumina HiSeq (paired-end 250 bp). Primers was designed to target the V4 region of the 16S rDNA (position 319 of the bacterial 16s rRNA gene to position 806). Each reaction was denatured at 95°C for 3 min followed by 25 cycles of (95°C for 30 s, 55°C for 30 s, 72°C for 30 s), followed by a final extension at 72°C for 5 min [31]. Reactions each contained a unique sequence index to enable pooling. Pools were purified with the AMPure XP beads and sequenced on an Illumina HiSeq platform. The 16S rDNA data were analyzed with the open-source bioinformatics pipeline Quantitative Insights into Microbial Ecology (QIIME). The sequences were grouped into operational taxonomic units (OTUs) by UCLUST at a minimum of 97% sequence similarity. Representative sequences from each OTU were aligned using the PyNAST software (v.1.2). Taxonomy was assigned using the Silva database (v.132).

Statistical Analysis
Statistical analysis was done using the SPSS.16.0 software. The data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was run followed by Tukey post hoc comparisons test. The criterion for the statistical significance was p < 0.05. Figure 1 indicates that isoflavone content is higher than the phenolic acid content. The major compounds in SME are glucoside and malonyl types of isoflavone ( Table 1). The content of different functional groups can be ranked as glucoside >malonyl>> acetyl >aglycone, and the basic structure of isoflavone as daidzein>genistein>>glycitein. Unlike isoflavones, the phenolic acid content is very low; among the phenoic acids, p-coumaric acid content is significantly the highest (Table 1).

Body Weight and Hepatic and Renal Toxicity
The experimental design is presented in Fig. 2A. SME was orally administered to rats once daily for 14 days. The effects of SME on the body weights and hepatic and renal toxicity were investigated at the end of the experimental period, and the results are shown in Fig. 2B and C. Compared with the control group, the body weight of rats in the SME group was not significantly different [F(2, 27) = 0.03, p= 0.9]. In addition, no obvious morphological changes were observed in the liver and kidney between the two groups ( Fig. 2C), indicating that chronic administration of SME does not cause liver and kidney damage.

Memory Retention in Rats Administered SME
To determine the effect of SME on spatial learning and memory, we analyzed the rat performance in the MWM test  1 3 (Fig. 3A). In the MWM test, rats in the SME group had a shorter time for finding the platform than the control group [F(2, 21) = 54.9, p< 0.001, Fig. 3B]. Similarly, the movement distance of rats in the SME group was significantly shorter than that of the control group [F(2, 21) = 77.9, p< 0.001; Fig. 3C]. No significant difference was observed between rats administered SME 50 mg/kg and SME 100 mg/ kg groups (p = 0.9). These results suggest that the memory retention of the SME group was superior to that of the control group.

Increased Synaptic Protein Expression in the Hippocampal Synaptosomes of Rats Administered SME
Synaptic function, often measured in terms of presynaptic protein levels, indicates cognitive brain reserve [32]. To confirm that synaptic function was involved in the effects of SME, the levels of presynaptic proteins including synaptophysin, synaptotagmin, synaptobrevin, syntaxin, synapsin-1, and SNAP-25 in the hippocampal synaptosomes were detected. As shown in the Fig. 4 Fig. 4). No significant difference was observed between the effects of SME 50 mg/kg and SME 100 mg/kg groups (p > 0.05).

Increased Synaptic Vesicles in the Hippocampal Synaptosomes of Rats Administered SME
We observed the changes in synaptic vesicles in the hippocampal synaptosomes of rats under a transmission electron microscope (Fig. 6A). The number of synaptic vesicles in the hippocampal synaptosomes of the SME group was higher in the SME group than in the control group [F(2, 6) = 45.3, p< 0.001; Fig. 6B]. No significant difference was observed between rats administered SME 50 mg/ kg and SME 100 mg/kg (p = 0.9).

Changes in Gut Microbiota in Rats Administered SME
In addition, increasing evidence has demonstrated that the gut microbiota is associated with memory function [28].
To explore gut microbiota composition and alterations, we used the next-generation sequencing technology to measure the bacterial community in fecal samples of rats. Fig. 7A presents the data of the operational taxonomic unit (OTU), and the results indicated that control, SME 50 mg/ kg, and SME 100 mg/kg had common OTUs of 428. Interestingly, SME 50 mg/kg had 34 numbers of OTUs, and SME 100 mg/kg had 27 number of OTUs which were different from control group. In Fig. 7B, the alpha-diversity analysis, including the Chao 1, ACE, Shannon and Simpson index, for gut microbiomes indicated that the overall microbial assortments in the gut microbiota of the SME group were not significant different from the control group (p > 0.05). At the phylum level, the relative abundance of three main phyla-Bacteroidates, Firmicutes, and Proteobacteria was approximately 97% (Fig. 8A). The relative abundance of Firmicutes in the SME 100 mg/kg group was significantly lower than that in the control group [F(2, 11) = 4.9, p< 0.05; Fig. 8B], whereas that of Bacteroidates was not significantly different between the two groups [F(2, 12) = 0.3, p> 0.05; Fig. 8B]. The Firmicutes-to-Bacteroidates (F/B) ratio in the SME 100 mg/kg group was reduced compared with that in the control group [F(2, 9) = 4.3, p< 0.05; Fig. 8B, inset]. By contrast, the relative abundance of Proteobacteria and Actinobacteria was higher in the SME group than in the control group [Proteobacteria, F(2, 12) = 4.3, p< 0.05; Actinobacteria, F(2, 7) = 5.3, p< 0.05; Fig. 8B]. At the genus level, Bacteroides, Lactobacillus, and other unclassified bacterial strains were the main bacteria in the control and SME groups (Fig. 8C). The relative abundance of Lactobacillus, Prevotellaceae_ UGG_001, Romboutsia, Turicibacter, and Parabacteroides decreased in the SME group, whereas that of Akkermansia, Prevotellaceae_NK3B31_group, Parasutterella, and other unclassified bacterial strains was nonsignificantly higher increased in the SME group. However, most of these differences were not statistically significant (p > 0.05; Fig. 8D), except the between-group differences in the relative abundance of other unclassified bacteria [F(2, 9) = 4.8, p< 0.05; Fig. 8D]. In addition, the results of linear discriminant analysis effect size (LEfSe) analysis (LDA score > 4.0) showed that SME group significantly had a higher abundance of Proteobacteria compared with the control group. The control group had a higher abundance of s_Lactobacil-lus_murinus compared with the SME group (Fig. 9).

Discussion
Considerable research attention has been directed to factors, particularly natural products that can enhance the Fig. 4 Effects of orally administration of SME on the expression levels of synaptic proteins in the hippocampal synaptosomes of rats. Western blot showing the expression levels of synaptophysin, synaptotagmin, synaptobrevin, syntaxin, synapsin-1, and SNAP-25 in the hippocampal synaptosomes for each group. Relative protein levels were quantified. Data are presented as mean ± SEM (n = 5 per group). *p < 0.01, **p < 0.01, ***p < 0.001 compared with the control group memory of older adults or prevent the development of cognitive deficits. Soybean meal is a cheap, readily available source of bioactive health-promoting compounds [24][25][26][27].
We investigated the effect of SME on memory function in rats through the MWM test and evaluated its possible underlying mechanisms. The MWM test is the most widely accepted model for evaluating hippocampal-dependent spatial learning and memory in rodents [34,35]. In the MWM test, a lower score in the escape latency is used as an index of enhanced spatial learning and memory [36]. In the present experiments, orally administered 50 or 100 mg/kg/day of SME for 14 days led to significantly decreased escape latencies compared with that in the control group. The shorter escape latencies suggest that the oral administration of SME plays a significant role in memory retention.
Memory formation and storage are tightly linked to synaptic plasticity [37,38], which is regulated by numerous neurochemical alterations, including changes in neurotransmitter release [39,40]. In synaptic terminals, neurotransmitter release can be regulated by multiple synaptic proteins, including SNAP-25, synaptobrevin, synapsin I, and syntaxin, which are involved in vesicle docking, priming, and triggering fast neurotransmitter exocytosis [41,42]. The phosphorylation of these proteins by various protein kinases, such as ERK1/2, PKC, and CaMKII [43][44][45], increases the availability of vesicles in the active zone and, thus, increases neurotransmitter release [33,46,47], Relative protein levels were quantified. Data are presented as mean ± SEM (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group 1 3 contributing to synaptic plasticity and memory formation and retention in the hippocampus [48][49][50]. In the present study, in the hippocampal nerve terminals of the SME group, (i) the protein levels of presynaptic proteins (synaptophysin, synaptotagmin, synaptobrevin, synapsin-1, and SNAP-25) and protein kinases (ERK1/2, PKC, CaMKII) were higher; (ii) the phosphorylation of these protein kinases was higher; (iii) the phosphorylation of synapsin I at ERK1/2-specific sites 4 and 5 and CMKII-specific sites 3 was higher; and (iv) number of synaptic vesicles was higher than the corresponding values of the control group. These findings imply that the increases in the levels of synaptic proteins and vesicles in the hippocampal nerve terminals may have contributed to increased neurotransmitter release and memory retention in rats in the MWM tasks. This speculation is supported by evidence showing that high levels of presynaptic proteins, including synaptophysin, SNAP-25, syntaxin, and synaptobrevin, are associated with higher cognitive performance and lower risk of dementia in older adults [51][52][53]. Nevertheless, how SME-induced increases in synaptic proteins and vesicles in the hippocampal nerve terminals lead to memory preservation requires further research. In fact, synaptobrevin, synapsin-1, and SNAP-25 are synaptic vesicle-associated proteins that regulate the release of neurotransmitters. Decreases in these proteins and consequent decreases in the release of glutamate have been reported to

3
be involved in cognitive dysfunction [48,54]. Glutamate, a major excitatory neurotransmitter in the central nervous system, plays an important role maintaining cognition [55]. In the present study, we did not examine the effect of SME on glutamate release. However, the possible involvement of increasing glutamate exocytotic processes in the preservation of memory function by SME observed in the present study should be considered.
Several studies have indicated that the memory ability is associated with alterations in gut microbiota [56,57]. With respect to the gut microbiota composition, phyla Bacteroidetes and Firmicutes are the predominant divisions in the gut flora [58]. In our study, the number of phyla Bacteroidetes was not significantly changed, but that of Firmicutes was decreased in the SME rats, thereby decreasing the F/B ratio. This was accompanied by an increase in the phylum Actinobacteria. Previous preclinical and clinical studies have demonstrated a decrease in the microbiota diversity and Actinobacteria content and a increase in the F/B ratio in AD and aging; thus, these alterations are Fig. 7 Effects of orally administration of SME on gut microbiota in rats. A Venn diagram, B alpha diversity. Data are presented as mean ± SEM (n = 5 per group). Fig. 8 Effects of orally administration of SME on the abundance of gut microbiota at the phylum and genus level. Relative abundances of OTUs at the phylum (A) and genus (B) level. Relative abundance of major bacterial OTUs in phylum (C) and genus (D) level. The ratio of Firmicutes to Bacteroidates (inset B). Data are presented as mean ± SEM (n = 5 per group). *p < 0.05 compared with the control group 1 3 related to cognitive decline [57,[59][60][61][62][63][64][65]. In our study, SME decreased the F/B ratio, leading to functional profile changes in the microbial community, which may have contributed to the improved memory retention. Further research is required to determine how intestinal flora alterations by SME affect memory function. In fact, it has been reported that communication between the gut and the brain involves multiple pathways including the enteric nervous system, the neuroimmune and the neuroendocrine systems. By regulating the nervous, endocrine, and immune system, gut microbiota can influence brain functions [66,67]. In addition, it has been reported that gut microboita are able to produce certain chemicals such as short-chain fatty acids, brain-derived neurotrophic factor, and neurotransmitters (GABA and serotonin), which can penetrate the blood-brain barrier (BBB) and affect neuronal function [67]. Whether the preservation of memory function by SME is related to the metabolites of intestinal bacteria remains to be clarified.

3
High concentrations of isoflavones and low concentrations of phenolic acids were detected in SME rats in the present study, which is consistent with previous findings [24,26,27]. A higher intake of polyphenols, including isoflavones and phenolic acids, has been linked to higher cognitive function in both animals and humans [68][69][70][71][72]. In the current study, polyphenols might have been played a role in memory retention in SME rats. Polyphenols exert a direct action on the brain by crossing the BBB, and they also affect brain function by modifying the gut microbiota composition and functions [66,73]. However, the interactions and relationships between polyphenols and gut microbiota and between gut microbiota and memory function are complex and warrant further research. In addition, SME is water-solubility, and our results revealed that it did not produce significant changes in the morphology of the liver and kidney in rats; this implies that SME administration may be safe. Thus, it has potential for use as a functional food ingredient. Fig. 9 Effect of orally administration of SME on gut communties by linear discriminant analysis (LDA) effect size (LEfSe) for SME versus control group. The threshold on the logarithmic LDA score for discriminative features was set to 4.0.

Conclusion
Our study is the first to demonstrate that SME helps in memory preservation in rats. This beneficial effect might be due to the enhancement of presynaptic integrity and the modulation of microbiota composition, as summarized in the schematic (Fig. 10). Our findings imply that soybean meal has potential as a food ingredient or supplement for preventing memory impairment. Future studies should investigate the effects of soybean meal or SME on human cognitive function. Fig. 10 Suggested graphical representation of the possible mechanisms of memory retention by SME in rats. The effect of oral administration of SME on memory might result from the increased expression of synaptic proteins, phosphorylation of protein kinases, and number of synaptic vesicles in the hippocampal nerve terminals, thus enhancing neurotransmitter release and synaptic plasticity in the hippocampus of these rats. Furthermore, the preservation of memory in the SME rats might be related to its modulatory effects on gut microbiota composition