Preparation method of plant embryo leavening and application of plant embryo leavening in blocking of trans-generation delivery metabolic diseases

Abstract

The invention belongs to the field of biotechnology and nutritional medicine, relates to a maternal obesity intervention preparation, and particularly relates to a preparation method of a plant embryo fermentation product and application of the plant embryo fermentation product in blocking of trans-generation delivery metabolic diseases. FWG is prepared through a probiotic fermentation technology, obesity, glucose and lipid metabolism disorder and oxidative stress induced by high-fat diet of a parent body can be remarkably improved, and cross-generation transmission of metabolic diseases is blocked by adjusting composition of intestinal flora of the parent body and offspring and a hypothalamic PI3K-Akt signal channel. The invention reveals that maternal FWG intervention passes through a mechanism that a PI3K-Akt pathway is dominated and multiple pathways are coordinated on the transcriptome level for the first time, the HFD-induced offspring hypothalamus metabolism programming abnormality is reversed, and a new central regulation target is provided for cross-generation metabolic disease prevention. The FWG is a wheat processing byproduct, is free of chemical additives, is suitable for long-term dietary supplement, is a natural and safe nutrition intervention scheme, and is suitable for the field of maternal and infant health.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and nutritional medicine, and relates to a maternal obesity intervention preparation. BACKGROUND

[0002] A large number of studies have clearly confirmed that maternal obesity is a key and independent risk factor for offspring to suffer from metabolic syndrome and related diseases (including obesity, type 2 diabetes, non-alcoholic fatty liver disease, increased risk of cardiovascular disease, etc.). This adverse effect not only manifests in childhood of the offspring, but is more likely to continue into adulthood, forming a "metabolic imprint" and increasing the lifelong health risk. The existing intervention strategies for maternal obesity (especially during pregnancy) mainly include limiting caloric intake, increasing physical activity (exercise intervention) and using specific drugs. However, these methods face significant challenges in effectiveness and safety, and have the following problems: 1. Traditional dietary adjustment is difficult to maintain for a long time, and has limited effect on improving cross-generation metabolic disorders; 2. Chemical drugs can have side effects on the mother and fetus; 3. Existing probiotics or dietary fiber interventions lack systematic mechanism research (such as integrated analysis of microbiota-metabolism-neural regulation network).

[0003] Strict caloric restriction and regular exercise programs are difficult for pregnant women to adhere to for a long time in terms of physiology (such as pregnancy reactions, increased physical burden) and psychology (emotional fluctuations, stress), resulting in a significant reduction in intervention effectiveness. The cross-generation protection effect is limited, and even if the maternal metabolic indicators are improved in the short term, existing evidence shows that relying solely on maternal dietary adjustment and exercise often fails to effectively prevent or significantly reverse the metabolic disorders (such as insulin resistance, abnormal adipose tissue function, liver lipid metabolism disorders) of the offspring programmed by maternal obesity. This suggests that the programming effect of maternal obesity on the offspring may involve more complex mechanisms beyond simple energy balance. The safety requirements for drugs during pregnancy are extremely high. Many chemical drugs used for adult obesity or metabolic diseases have not been fully verified for their potential teratogenicity, long-term developmental effects on developing fetuses, and safety during the maternal pregnancy process, or have known risks, which greatly limit their application during pregnancy.

[0004] Only through such multi-dimensional, systematic, and mechanism-driven in-depth research can we provide a solid scientific foundation and innovative intervention strategies for effectively preventing and blocking the cross-generation metabolic syndrome risk caused by maternal obesity. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a preparation method of plant embryo fermentation and application thereof in blocking transgenerational transmission of metabolic diseases, aiming to provide a safe and natural nutritional intervention scheme, and blocking transgenerational transmission of obesity and related metabolic diseases by regulating maternal and offspring intestinal flora and metabolic pathways.

[0006] The technical scheme of the present application is implemented as follows: The present application takes plant embryo fermentation as the core active ingredient, and the preparation method thereof includes probiotic fermentation of defatted wheat germ powder and optimization of fermentation conditions to enrich γ-aminobutyric acid, polyphenols and dietary fiber.

[0007] In one aspect, the present application discloses a preparation method of plant embryo fermentation, taking fermented wheat germ as an example, and the steps are as follows: The steps are as follows: defatted wheat germ powder is mixed with water at a ratio of 1:15-20 and sterilized at 121℃ for 15 min. After cooling, inoculate the seed solution of bread yeast (1.5-2% v / v) and incubate at 25-30℃ and 150-180 rpm for 3-3.5 h; then add the seed solution of lactobacillus plantarum (2.5-4% v / v) and glucose (3-7% v / v), and incubate at 35-37℃ and 180-220 rpm for 14-15 h. After fermentation, centrifuge at 8000 rpm for 10 min to obtain the supernatant, freeze-dry, and store at -80℃ for use, which is recorded as FWG.

[0008] The volume ratio of defatted wheat germ powder to water in the above mixed solution is 1:10.

[0009] The preparation method of the above bread yeast seed solution is as follows: the bread yeast solution with a concentration of 1×10 8 ~1×10 10 CFU / mL is incubated at 25-30℃ for 20-25 h; and the preparation method of the lactobacillus plantarum seed solution is as follows: the lactobacillus plantarum solution with a concentration of 2.5×10 10 CFU / mL is incubated at 37℃ for 24 h. In another aspect, the present application also provides fermented wheat germ prepared by the above method.

[0010] In a third aspect, the present application provides a composition with the above fermented wheat germ as the active ingredient.

[0011] In a fourth aspect, the present application provides application of the above fermented wheat germ or the above composition in functional products, and the functional products have one or more of the following purposes: (1) reducing maternal high-fat diet (HFD) induced weight gain, fat deposition and liver lipid accumulation; (2) improving maternal and offspring glucose intolerance, insulin resistance and leptin sensitivity; (3) Regulating the composition of maternal and offspring intestinal flora, reducing the Firmicutes / Bacteroidetes (F / B) ratio, and inhibiting the proliferation of obesity-related genera (Lactobacillus, Dorea); (4) Inhibiting appetite disorders and metabolic disorders in offspring by regulating hypothalamic PI3K-Akt signaling pathway gene expression.

[0012] The dosage of the fermented wheat germ used in the functional product is 800-1000 mg / kg of body weight.

[0013] The functional product is administered orally, and the mother takes FWG (884 mg / kg) orally every day before pregnancy, during pregnancy and lactation period; the intervention is continued until the end of the lactation period, and the offspring maintains a normal diet from weaning to adulthood without additional intervention.

[0014] The present application has the following beneficial effects: 1. The present application first confirms that FWG can improve the offspring's glucose and lipid metabolism, liver function and hypothalamic gene expression through maternal intervention, and block the transgenerational programming effect of obesity. Through multi-target mechanism, its effects are: ① regulating intestinal flora: reducing the F / B ratio, inhibiting obesity-promoting genera (such as Lactobacillus, Dorea), and restoring the balance of flora; ② antioxidant / anti-inflammatory: reducing the oxidative stress markers (MDA, NO) and inflammatory factors (TNF-α, IL-6) of the mother and offspring; ③ neural regulation: inhibiting appetite disorders in offspring through the PI3K-Akt signaling pathway.

[0015] 2. The present application discloses a maternal obesity intervention preparation based on fermented wheat germ (FWG) and its application. FWG is prepared by probiotic fermentation technology, which can significantly improve maternal obesity, glucose and lipid metabolism disorders and oxidative stress induced by high-fat diet, and block the transgenerational transmission of metabolic diseases by regulating the composition of maternal-offspring intestinal flora and hypothalamic PI3K-Akt signaling pathway. For the first time, the present application reveals that maternal FWG intervention reverses hypothalamic metabolic programming abnormalities induced by HFD through the mechanism of "PI3K-Akt pathway as the main one, multiple pathways synergistically", providing a new central control target for the prevention of transgenerational metabolic diseases. The FWG of the present application is a byproduct of wheat processing without chemical additives, which is suitable for long-term dietary supplementation, and is a natural and safe nutritional intervention program suitable for the field of maternal and infant health. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0017] Figure 1 Process flow chart for FWG preparation.

[0018] Figure 2 Figure for maternal body weight, fat index and liver pathological changes after intervention; wherein (A) experimental design schematic diagram; (B) body mass index (BMI); (C) gestational weight gain; (D) 10th week oral glucose tolerance test (OGTT); (E) 16.5th day of pregnancy (GD 16.5) oral glucose tolerance test (OGTT); (F) caloric intake; (G) inguinal white adipose tissue (iWAT) index; (H) gonadal white adipose tissue (gWAT) index; (I) liver index; (J) liver gross morphology; (K) liver tissue hematoxylin-eosin (H&E) staining; data are expressed as mean ± standard deviation (n=8 per group). Statistical analysis was performed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0019] Figure 3 Figure for offspring metabolic parameters (OGTT, blood lipids, liver enzymes) and intestinal flora α / β diversity analysis; wherein (A) Chao1 index, (B) observed species, (C) Shannon index and (D) Simpson index. (E) β diversity was evaluated by principal coordinate analysis (PCoA) based on weighted UniFrac distance. Intestinal flora composition distribution at (F) door level and (H) genus level. (G) Firmicutes / Bacteroidetes ratio. (I) Relative abundance of Lactobacillus and (J) Dorea. Data are expressed as mean ± standard deviation (n=6 per group). Statistical significance was determined by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0020] Figure 4Intestinal microbiota composition of 3-week-old offspring supplemented with FWG to mothers; (A) Chao1 index, (B) observed species, (C) Shannon index, and (D) Simpson index. (E) Beta diversity was assessed by principal coordinate analysis (PCoA) based on weighted UniFrac distance. Distribution of intestinal microbiota composition at (F) phylum level and (G) genus level. (H) Firmicutes / Bacteroidetes ratio. (I) Relative abundance of Lactobacillus genus and (J) Dorea genus. Data are presented as mean ± SD (n = 6 per group). Statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0021] Figure 5 Intestinal microbiota composition of 13-week-old offspring supplemented with FWG to mothers; (A) Chao1 index, (B) observed species, (C) Shannon index, and (D) Simpson index. (E) Beta diversity was assessed by principal coordinate analysis (PCoA) based on weighted UniFrac distance. (F) Overall PCoA plot comparing the structure of intestinal microbiota communities of mothers and their offspring at weaning (3 weeks) and adulthood (13 weeks) using weighted UniFrac. Distribution of intestinal microbiota composition at (G) phylum level and (I) genus level. (H) Firmicutes / Bacteroidetes ratio. (J) Relative abundance of Lactobacillus genus. (K) Spearman correlation analysis of the common genera among the top 20 dominant genera of mothers and 3- and 13-week-old offspring. Data are presented as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0022] Figure 6 Analysis of the effects of maternal FWG intake on offspring body weight and metabolic outcomes; (A) body weight; (B) body mass index (BMI); (C) gonadal white adipose tissue (gWAT) index; (D) inguinal white adipose tissue (iWAT) index; (E) respiratory exchange ratio (RER); (F) area under the curve (AUC) of respiratory exchange ratio; (G) daily activity distance; (H) daily activity frequency; (I) food intake; (J) water intake; (K) 3-week-old offspring liver hematoxylin-eosin (H&E) staining, (L) oil red O staining; (M) 13-week-old offspring liver hematoxylin-eosin (H&E) staining, (N) oil red O staining (O) alanine aminotransferase (ALT); (P) aspartate aminotransferase (AST); (Q) liver lipoprotein lipase (LPL) activity; data are presented as mean ± SD (n = 6 per group). Figure 6 A-D and O-Q, n = 8 per group; Figure 6K and L, n = 3 for each group; Figure 6 E-J, n = 6 for each group). Statistical significance was assessed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0023] Figure 7 Figure 6. Fermented wheat germ (FWG) supplementation to dams alleviates offspring metabolic disorders induced by high-fat diet (HFD); (A) 3-week-old offspring oral glucose tolerance test (OGTT); (B) 13-week-old offspring oral glucose tolerance test (OGTT); (C) insulin resistance homeostasis model assessment (HOMA-IR); (D) serum leptin, (E) total cholesterol (TC), (F) triglyceride (TG), (G) low-density lipoprotein cholesterol (LDL-C), (H) high-density lipoprotein cholesterol (HDL-C); (I) liver nitric oxide (NO), (J) malondialdehyde (MDA), (K) glutathione (GSH); (L) serum tumor necrosis factor-α (TNF-α) levels. Data are expressed as mean ± standard deviation (n = 8 for each group). Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0024] Figure 8 Figure 7. Hypothalamic transcriptome KEGG pathway enrichment and PI3K-Akt gene expression heat map; (A) hierarchical clustering heat map of gene expression profiles of normal diet group (NCD), high-fat diet group (HFD), and FWG intervention group; (B) volcano plot of differentially expressed genes in FWG group and HFD group; (C) KEGG pathway enrichment analysis of differentially expressed genes in HFD group and FWG group (n = 3 for each group). DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0026] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0027] The application discloses a preparation method of fermented wheat germ, which comprises the following steps: mixing defatted wheat germ powder and water in a proportion of 1:15-20, sterilizing at 121 DEG C for 15 min, inoculating baker's yeast seed liquid (1.5-2% v / v) after cooling, and incubating at 25-30 DEG C and 150-180 rpm for 3-3.5 h; then adding lactobacillus plantarum seed liquid (2.5-4% v / v) and glucose (3-7% v / v), and incubating at 35-37 DEG C and 180-220 rpm for 14-15 h. After fermentation, the supernatant is obtained by centrifugation at 8000 rpm for 10 min, and then freeze-drying and storing at-80 DEG C for use, and is recorded as FWG.

[0028] The preparation method of the above-mentioned baker's yeast seed liquid is as follows: incubating a baker's yeast solution with a concentration of 1x10 8 ~1x10 10 CFU / mL at 25-30 DEG C for 20-25 h; and the preparation method of the lactobacillus plantarum seed liquid is as follows: incubating a lactobacillus plantarum solution with a concentration of 2.5x10 10 CFU / mL at 37 DEG C for 24 h.

[0029] The fermented wheat germ of the application can be prepared in the above-mentioned ranges, and the following points in the above-mentioned ranges are selected for experimental verification: Embodiment

[0030] The preparation method of a kind of fermented wheat germ (FWG) of this embodiment, its technological process is as shown in Figure 1 The steps are as follows: First, prepare seed liquid, 5 g lactobacillus plantarum powder (Lactobacillus plantarum LP-G18, 5*10 11 CFU / g) is incubated in 100 mL sterile water at 37 DEG C for 24 h, and 5 g baker's yeast (1.5x10 9 CFU / mL is incubated in 100 mL sterile water at 30 DEG C for 24 h. Defatted wheat germ powder (Anyang Man Tianxue Food Manufacturing Co., Ltd., WG) is mixed with water in a proportion of 1:18, sterilized at 121 DEG C for 15 min. After cooling, inoculate baker's yeast seed liquid (1.6% v / v), incubate at 30 DEG C and 150 rpm for 3.2 h; then add lactobacillus plantarum seed liquid (3.4% v / v) and glucose (6% v / v), incubate at 37 DEG C and 200 rpm for 14.8 h. After fermentation, the supernatant is obtained by centrifugation at 8000 rpm for 10 min, and then freeze-drying and storing at-80 DEG C for use, and is recorded as FWG.

[0031] The components of FWG were analyzed as follows: ash determination refers to GB 5009.4-2016 Food safety national standard Determination of ash in food; dietary fiber determination refers to GB 5009.88-2023 Food safety national standard Determination of dietary fiber in food; fat content determination GB 5009.6-2016 Food safety national standard Determination of fat in food; protein content determination refers to GB 5009.5-2016 Food safety national standard Determination of protein in food; total sugar determination uses a commercial kit. The results are shown in Table 1: Table 1 Component analysis of FWG From Table 1, it can be seen that after fermentation, the total dietary fiber content in wheat germ increased by 1.34 times, and the total polyphenol content increased by 0.5 times.

[0032] Further, non-targeted metabolomics was used to analyze the changes in wheat germ during fermentation. Simply put, 700 μL of pre-cooled (-40℃) methanol / water (3:1, v / v) containing internal standards was used to extract 50 mg of sample, and then vortexed, homogenized and ultrasonicated repeatedly in an ice water bath. After extraction at 4℃ overnight, the sample was centrifuged (12000 rpm, 15 minutes, 4℃), filtered (0.22 μm), diluted, and QC was combined. UHPLC-MS analysis was performed using an EXION LC system (Sciex) coupled with a QTrap 6500+ mass spectrometer, with 0.1% formic acid in water and acetonitrile as the mobile phase. Data collection was performed in MRM mode, and peak detection / annotation was performed using internal R programs and databases. The results are shown in Table 2: Table 2 Differential metabolites of wheat germ and wheat germ fermentation product From Table 2, it can be seen that creatinine, arachidonic acid, 1-isocryptoxanthin hydrate, phenylacetyl glycine, shikimic acid and 5-uracil nucleotide, the content of these components is 1.46, 38.44, 2.43, 1.32, 8.9 and 5.87 times respectively in unfermented wheat germ.

[0033] Application Example 1 I. Animal experiment 24 female Wistar rats were divided into 3 groups: normal diet group (NCD), high-fat diet group (HFD), HFD+FWG intervention group; SPF Wistar rats, 6 weeks old, were purchased from Beijing Sbiopharm Co., Ltd. [SCXK(JING) 2019-0010]. The rats were allowed free access to food and water and were housed in the animal room under the following conditions: 12 h light / dark cycle (8:00-20:00), humidity 50±15%, temperature 23±2℃. All animal experiments were conducted in accordance with the National Guidelines for Laboratory Animal Welfare and were approved by the Ethics Review Committee on Animal Experiments of the College of Food Science and Engineering, Henan Polytechnic University (HAUTETHI-2023-0629) The female rats were divided into 3 groups, 10 in each group, and were given normal diet (NCD) group: NCD + normal saline; high-fat diet (HFD) group: HFD + normal saline; FWG group: high-fat diet + FWG (884 mg / kg). Once a day by gavage.

[0034] Metabolic indicators (body weight, OGTT, blood lipids) of the mother and offspring, liver pathology, intestinal flora (16S rRNA sequencing), and hypothalamic transcriptome (RNA-Seq) were detected.

[0035] OGTT: Female rats fasted overnight were given oral glucose (2 g / kg) at 10 weeks and 15.5 days of pregnancy. Offspring were treated the same way at 3 and 13 weeks after birth. Blood glucose was measured from the tail vein at 0, 30, 60, 90, and 120 minutes after oral glucose.

[0036] HE staining: Liver tissue samples were fixed in 4% paraformaldehyde solution overnight, then cut into 5 μm thick sections. These tissue sections were stained with hematoxylin and eosin (H&E), observed under a vertical optical microscope and photographed.

[0037] Oil red staining: Liver tissue samples were fixed in 4% paraformaldehyde solution overnight, cryoprotected in graded sucrose solution, and subjected to OTC embedding and frozen sectioning. After ice-cold sectioning (10 μm thick), the sections were stained with oil red O for 10 min, differentiated in 75% ethanol, and rinsed with distilled water. After staining, the sections were counterstained with hematoxylin for 3-5 min, washed under tap water, mounted with glycerol gelatin, and observed under an optical microscope and photographed.

[0038] Intestinal microbiota analysis: Intestinal microbial analysis was performed by 16S rRNA gene sequencing. First, total genomic DNA of the sample was extracted using a total DNA kit. Subsequently, the V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using a forward primer (5'-actcctacggggaggcagca - 3') and a reverse primer (5' - ggactachvgggtwtctaat - 3'). The amplified PCR product was subjected to double-end high-throughput sequencing on the Illumina NovaSeq-PE250 platform. Microbiome bioinformatics analysis was performed using QIIME2 (2019.4). Sequence quality trimming and amplicon sequence variant calling were performed using the DADA2 plugin. Alpha diversity indices (Chao1, Observed Species, Simpson, and Shannon indices) and beta diversity indices were calculated by the diversity plugin. The characteristic microorganisms of the difference between groups were determined by linear discriminant analysis effect size (LEfSe).

[0039] Biochemical analysis: Blood glucose, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected using commercial kits. Insulin, tumor necrosis factor alpha (TNF-a), interleukin (IL)-6, adiponectin, and leptin were detected using ELISA kits.

[0040] Hypothalamic transcriptome sequencing: Hypothalamic RNA was extracted using TRIzol, and its quality was evaluated by NanoDrop (ThermoScientific, USA) and Agilent 2100 Bioanalyzer. Libraries were prepared and sequenced on an Illumina NovaSeq 6000 at Shanghai Biotechnology Co., Ltd. Gene counts were generated using HTSeq (v0.9.1) and normalized to FPKM. Differential expression was analyzed using DESeq (v1.38.3) with thresholds of |log2FC|>2 and p<0.05 II. Experimental conclusions 1. Association study of maternal BMI, dietary structure, and metabolic indicators: The effect of pre-pregnancy dietary structure on maternal body mass index (BMI), weight gain during pregnancy, glucose homeostasis, and postpartum metabolic status was investigated. It was found that a high-fat diet (HFD) can induce maternal metabolic abnormalities, while FWG supplements can significantly improve related indicators.

[0041] Maternal supplementation with fermented wheat germ (FWG) reduces HFD-induced maternal obesity and liver morphological changes, such as Figure 2 as shown by Figure 2 it can be seen that: ①Effect of pre-pregnancy diet on maternal BMI and weight gain during pregnancy BMI changes Figure 2 Medium B): High-fat diet (HFD group): Pre-pregnancy BMI was 17.32% higher than that of the normal control diet (NCD group, p<0.01). FWG supplement group: Compared with the HFD group, the BMI decreased by 10.83%, indicating that FWG could alleviate the excessive weight gain induced by HFD. Weight gain during pregnancy Figure 2 Medium C): The maternal weight gain during pregnancy in the HFD group was 13.8% and 11.89% higher than that in the NCD group and the FWG group, respectively (p<0.01), suggesting that HFD might exacerbate the difficulty of weight management during pregnancy by promoting fat accumulation.

[0042] ②Maternal glucose homeostasis and OGTT results Impaired glucose tolerance Figure 2 Medium D, E): After 10 weeks of HFD feeding, the maternal oral glucose tolerance test (OGTT) showed significant glucose intolerance, and this metabolic abnormality persisted until late pregnancy. FWG intervention effect: FWG supplements could significantly improve the glucose tolerance of the HFD group, indicating that they had a reversing effect on glucose metabolism disorders.

[0043] ③Diet-dependent regulation of energy intake Energy intake before and after lactation Figure 2 Medium F): Before lactation: There was no significant difference in energy intake among the groups (p>0.05).

[0044] During lactation: The energy intake of the HFD group increased by 19.73% compared with the NCD group, while the FWG supplement reduced the energy intake of the HFD group by 9.8% (p<0.01), suggesting that FWG might affect energy balance during lactation by regulating appetite or metabolic efficiency.

[0045] ④Postpartum metabolic status: fat accumulation and liver damage Fat tissue index Figure 2 Medium G, H): The inguinal white adipose tissue (iWAT) index of the HFD group was 130% and 71.04% higher than that of the NCD group and the FWG group, respectively; the gonadal white adipose tissue (gWAT) index was 92.2% and 93.69% higher than that of the NCD group and the FWG group, respectively (p<0.05), confirming that HFD induced excessive fat accumulation. FWG supplementation could reduce the fat tissue index, suggesting its anti-obesity effect.

[0046] Liver morphology and lipid deposition Figure 2Mid I-K): HFD group developed hepatomegaly, elevated liver index and significant lipid accumulation (characteristic of fatty liver) as shown by H&E staining. FWG intervention reduced liver index and intrahepatic lipid deposition, indicating its protective effect on HFD-induced liver injury.

[0047] Conclusion: This study showed that pre-pregnancy HFD could increase maternal metabolic risk by inducing obesity, glucose metabolism disorder and liver fat accumulation, while FWG supplement could significantly alleviate HFD-related metabolic abnormalities by regulating BMI, improving glucose tolerance, suppressing excessive food intake during lactation and reducing fat / liver injury. These results provide experimental evidence for dietary intervention of metabolic diseases during pregnancy, suggesting that FWG may become a potential nutritional supplement to prevent pregnancy complications such as macrosomia risk.

[0048] 2, the maternal supplementation of FWG reshaped the composition of the intestinal flora of the post-weaning maternal mice, the maternal supplementation of FWG reshaped the composition of the intestinal flora of the offspring at 3 weeks of age, and the maternal supplementation of FWG reshaped the composition of the intestinal flora of the offspring at 13 weeks of age, and the alpha diversity was evaluated by the indicators shown in Figures 3-5 .

[0049] From Figures 3-5 Analysis of the effect of maternal supplementation of FWG on the intestinal flora composition of maternal mice and their offspring: The intestinal flora is often regarded as a "metabolic organ" and plays a key role in host energy homeostasis and body weight regulation. To evaluate the effect of maternal diet on the composition of intestinal flora, the researchers performed 16S rRNA sequencing analysis on the cecal contents of maternal mice at the end of lactation. High-fat diet (HFD) significantly changed the composition of intestinal microorganisms, with a decrease in alpha diversity. Specifically, compared with the normal diet group, the Chao1 index, observed species index, Shannon index and Simpson index of HFD maternal mice were significantly lower (P<0.05) (Fig. 2A-D). Similar decrease in alpha diversity was also observed in the offspring of HFD maternal mice at 3 weeks of age, with all four indices being significantly lower than those of the offspring of the normal diet group (P<0.05) (Fig. 2A-D, p<0.05). Interestingly, at 13 weeks of age, there was no significant difference in the alpha diversity of the offspring of each group (Fig. 2A-D), suggesting that long-term exposure to a standard diet may partially restore the alpha diversity of the intestinal microorganisms of the offspring to normal, regardless of maternal dietary intervention. Figure 3 Figure 4 Figure 5

[0050] To further explore the effect of maternal diet on the composition of intestinal microorganisms, principal coordinate analysis (PCoA) based on weighted UniFrac distance was used to evaluate the beta diversity (reflecting the difference in composition of microbial communities between samples). As shown in Figure 3 ​​​As shown in FIG. E, the gut microbiota of HFD dams (MHFD group) and normal diet dams (MNCD group) formed two independent clusters, indicating that HFD significantly changed the microbial community structure. The FWG supplemented group (MFWG group) was located between the two, suggesting that FWG supplementation can partially reverse the HFD-induced beta diversity shift, pushing the microbial profile closer to the normal diet group. The 3-week-old offspring showed a similar pattern: their intestinal microbial beta diversity was highly similar to that of the dams ( Figure 4 FIG. E). The offspring of FWG supplemented dams were closer to the normal diet group cluster on the PCoAl axis, indicating that maternal diet can shape the early gut microbiota of offspring and has intergenerational transmission. By adulthood (13 weeks of age), consistent with the alpha diversity results, the beta diversity of the HFD group offspring was not significantly different from the normal diet group ( Figure 5 FIG. E), suggesting that the microbial community partially recovered after long-term exposure to a normal diet. In addition, the overall PCoA analysis including all time points showed that the microbial community was mainly clustered by developmental stage ( Figure 5 FIG. F), rather than maternal dietary intervention, indicating that age-related factors (such as gender differences, standard chow introduction, physiological maturation) play a dominant role in shaping the composition of the gut microbiota. These findings also highlight the significant differences in microbial profiles between maternal and offspring.

[0051] When analyzing the microbial community structure at different taxonomic levels, the researchers compared the relative abundance of bacterial phyla and genera in each group. At the phylum level, the dominant phylum of the dams at the end of lactation was Firmicutes, Verrucomicrobiota, Actinobacteriota, Desulfobacterota, and Proteobacteria ( Figure 3 FIG. F). The dominant phylum of the 3-week-old offspring was Firmicutes, Verrucomicrobiota, Bacteroidota, Proteobacteria, and Desulfobacterota ( Figure 4 FIG. F), while the dominant phylum of the adult offspring shifted to Firmicutes, Bacteroidota, Actinobacteriota, Verrucomicrobiota, and Desulfobacterota ( Figure 5G). Compared to the normal diet group, HFD dams had reduced Verrucomicrobia and Actinobacteria, and increased Proteobacteria; similar changes were observed in the 3-week-old offspring of HFD dams, including reduced Verrucomicrobia and Actinobacteria, and increased Proteobacteria and Desulfovibrio. These consistent changes between dams and offspring suggest that the microbiome composition at early developmental stages is heritable. Notably, maternal supplementation with FWG reversed the aforementioned changes in the level of phyla at 3 weeks of age in the offspring exposed to HFD. In adult offspring, the HFD group had lower Bacteroidetes and higher Actinobacteria than the normal diet group; whereas maternal intake of FWG significantly increased the relative abundance of Bacteroidetes and decreased the level of Actinobacteria (compared to the HFD group). The ratio of Firmicutes to Bacteroidetes (F / B ratio) is often used as an indicator of gut microbial dysbiosis, and is associated with obesity, insulin resistance, inflammatory bowel disease, and increased intestinal permeability. Although there was no significant difference in the F / B ratio between dams at the end of lactation (likely due to individual variation, Figure 3 G), the F / B ratio was significantly higher in the 3-week-old and adult offspring of the HFD group than in the normal diet and FWG groups ( Figure 4 H, and Figure 5 H). Interestingly, Bacteroidetes was the least abundant phylum in the lactating dams, but significantly increased in the offspring and became the second most abundant phylum in adulthood, next to Firmicutes. This developmental shift can be influenced by the hormonal changes in the dams during lactation (e.g., elevated prolactin levels), which in turn alter the maternal microbiome composition.

[0052] Further analysis of the top 20 most abundant genera revealed that Faecousia, CAG-273, CAG-269, and Desulfovibrio_R were less abundant in HFD dams compared to the normal diet group, and their abundance was restored to the normal diet group level by FWG supplementation; in contrast, HFD increased the abundance of Lactobacillus, Clostridium_T, Streptococcus, Allobaculum, Romboutsia_B, Dorea_A, Oliverpabstia, Blautia_A, and Faecalimonas, which were suppressed by FWG intervention ( Figure 3In 3-week-old offspring, the relative abundance of Blautia_A, Bilophila, Lactobacillus, Dorea_A, and Escherichia in the HFD group was significantly higher than that in the normal diet group. Maternal FWG supplementation reduced the abundance of Lactobacillus and Dorea_A. Meanwhile, the abundance of Bifidobacterium, Allobaculum, 14-2, Eubacterium_R, CAG-485, Akkermansia, and Muribaculum in the HFD group was significantly reduced. Maternal FWG supplementation restored the abundance of Bifidobacterium, 14-2, and Allobaculum. Figure 4 (G). By 13 weeks of age, the levels of Romboutsia_B, UBA3282, and Onthenecus in offspring of the HFD group were lower than those in the normal diet group. Romboutsia_B abundance was partially restored by maternal FWG supplementation. In contrast, the abundance of Lactobacillus, CAG-273, Faecousia, Muribaculum, CAG-488, Staphylococcus, and Corynebacterium in the HFD group was higher than in the normal diet group. FWG intervention effectively reversed the increase in Lactobacillus, Muribaculum, CAG-488, Staphylococcus, and Corynebacterium. Figure 5 (I). Notably, the abundance of *Lactobacillus* remained consistently high across all three stages of the HFD group (mothers, 3-week-old offspring, and adult offspring). Figure 3 Middle I, Figure 4 Middle I, Figure 5 (J), while maternal supplementation with FWG significantly reduced its level at all stages. These results indicate that maternal diet has a significant impact on the establishment of the offspring's gut microbiota, and diet-induced changes in microbial composition can be passed from the mother to the offspring and persist into adulthood.

[0053] To investigate the correlation between the gut microbiota of female mice and their male offspring from weaning to adulthood, researchers performed Spearman correlation analysis on the common genera among the top 20 most abundant bacterial genera in both mothers and offspring. For example... Figure 5As shown in Figure K, the abundance of maternal microbiota at the end of lactation was highly correlated with that of 3-week-old offspring, but the correlation weakened in adulthood. Notably, the abundance of *Allobaculum* in maternal mice was significantly negatively correlated with that of offspring, while the abundance of *Blautia_A*, *Limosilactobacillus*, *Romboutsia_B*, *Dorea_A*, and *UBA3282* in maternal mice was significantly positively correlated with that of 3-week-old offspring. These findings suggest a high degree of microbial transmissibility between maternal mice and their offspring in early life stages, but this transmissibility may weaken as offspring transition to a normal diet.

[0054] Analysis of the Effects of Maternal FWG Intake on Offspring Body Weight and Metabolic Outcomes: Maternal obesity or high-fat diet (HFD) exposure has been shown to increase the risk of altered body composition and metabolic syndrome in offspring. To investigate the potential protective effects of FWG, we evaluated the effects of maternal HFD and FWG intake on offspring growth and metabolism. Compared with the normal control diet (NCD), offspring of HFD-fed mothers showed significantly higher body weight, BMI, inguinal white adipose tissue (iWAT), and gonadal white adipose tissue (gWAT) indices at weaning and into adulthood. Figure 6 C, Figure 6 The results showed that the changes were statistically significant (p<0.05), and this phenomenon persisted into adulthood. Maternal FWG supplementation reversed these changes, particularly reducing BMI and iWAT index, and these effects continued from weaning into adulthood.

[0055] To assess metabolic function, a metabolic cage system was used to monitor adult offspring. Substrate utilization was assessed using respiratory exchange ratio (RER, VCO2 / VO2), an indicator reflecting the balance between carbohydrate and lipid oxidation. As expected, RER exhibited a diurnal rhythm, with lower values ​​during the daytime (8:00–18:00) and higher values ​​at night, consistent with the normal diurnal activity of rats. Figure 4 E, F). Notably, the area under the 24-hour RER curve (AUC) was significantly lower in the HFD group, indicating a shift in metabolism towards lipid oxidation. There were no differences in physical activity (including the number of exercise sessions and the distance exercised) among the groups. Figure 6 China G, Figure 6 The results (H) indicate that the altered substrate utilization in HFD offspring was not due to changes in physical activity. Furthermore, the study recorded water and food intake: offspring of HFD mothers consumed 24.94% more food than the NCD group, but drank less water; FWG had no significant effect on food intake, but restored water intake to the NCD group level. Figure 6 Middle I, Figure 6 (J).

[0056] Maternal obesity increases the risk of non-alcoholic fatty liver disease in offspring. Histological analysis showed that the livers of post-weaning offspring of HFD dams had obvious lipid accumulation after H&E and oil red O staining, and although fat deposition decreased in adulthood, some accumulation persisted Figure 6 K-N). Notably, maternal supplementation with FWG inhibited liver lipid accumulation in post-weaning and adult offspring. To further assess liver function, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected: consistent with the histological results, the ALT and AST levels of post-weaning offspring of HFD dams were 14.77% and 29.89% higher than those of the NCD group (p < 0.05), indicating liver damage; but these differences were no longer apparent in adulthood Figure 6 P). Similarly, the liver lipase activity of post-weaning offspring of the HFD group was significantly increased, but returned to normal in adulthood Figure 6 Q). Maternal intake of FWG effectively alleviated the increase in liver enzyme and lipase activity, returning these indicators to levels comparable to the NCD group The results of maternal supplementation with fermented wheat germ (FWG) to alleviate high-fat diet (HFD)-induced results are shown in Figure 7 Improved glucose metabolism: OGTT results Figure 7 A, Figure 7 B): The glucose clearance capacity of offspring in the HFD group was significantly decreased (the area under the blood glucose curve increased) at both 3 and 13 weeks of age, indicating persistent glucose intolerance.

[0057] FWG intervention significantly reduced the blood glucose peak and the area under the curve at both time points, indicating that maternal FWG supplementation can improve the glucose metabolism function of offspring across generations.

[0058] HOMA-IR Figure 7 C): The insulin resistance index of offspring in the HFD group was 6.48 times higher than that of the NCD group, while the HOMA-IR of the FWG group was close to that of the normal diet (NCD) group, confirming that FWG can alleviate insulin resistance.

[0059] Reversal of abnormal lipid metabolism: serum lipid levels Figure 7 D-H): The serum leptin Figure 7 D), TC Figure 7 E), TG Figure 7 F), LDL-C Figure 7 G) of offspring in the HFD group were significantly increased, and HDL-C Figure 7 H) was decreased, reflecting typical hyperlipidemia and leptin resistance.

[0060] ​FWG intervention significantly reduced leptin, TG, and LDL-C levels, while increasing HDL-C, indicating its multidimensional improvement effect on lipid metabolism disorders.

[0061] Oxidative stress and the suppression of inflammation: liver oxidative markers ( Figure 7 IK): Progenitors of the HFD group showed increased levels of hepatic prooxidant NO (NO2-) Figure 7 (I), MDA ( Figure 7 The level of J (in the middle) was significantly increased, and the antioxidant GSH (in the middle) was significantly increased. Figure 7 Decreased K levels indicate an oxidative imbalance.

[0062] FWG intervention significantly reduced NO and MDA levels and restored GSH levels, indicating that it alleviates liver damage by enhancing antioxidant capacity.

[0063] Inflammatory factors ( Figure 7 (L) In the HFD group, serum TNF-α levels were significantly elevated in offspring, reflecting a chronic inflammatory state; FWG intervention significantly reduced TNF-α and suppressed systemic inflammatory responses. To assess the intergenerational impact on glucose and lipid metabolism, the study performed an oral glucose tolerance test (OGTT) and a homeostasis model assessment of insulin resistance (HOMA-IR) on offspring. Offspring of mothers on a high-fat diet (HFD) showed impaired glucose tolerance and significantly elevated HOMA-IR at weaning, and these abnormalities persisted into adulthood. Figure 7 In A and B, p<0.05). These offspring also exhibited leptin resistance, which is closely related to insulin resistance (…). Figure 7 (C, D). Notably, maternal supplementation with fermented wheat germ (FWG) significantly improved OGTT performance in weaning and adult offspring and reduced leptin resistance.

[0064] Regarding lipid metabolism, compared with the normal diet (NCD) group, the HFD group showed significantly higher serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels and lower high-density lipoprotein cholesterol (HDL-C) levels in weaned offspring (p<0.05). Except for TC, most of these lipid abnormalities persisted into adulthood. Figure 7 (E–H). Maternal intake of FWG significantly improved the lipid profile of offspring, indicating that it has a protective effect against maternal HFD-induced dyslipidemia.

[0065] It is known that maternal HFD can induce oxidative stress and inflammation in offspring. For example... Figure 8MDA and nitric oxide (NO) levels in the liver of weaned offspring of HFD dams were significantly increased compared to the NCD group, and glutathione (GSH) levels in the liver of adult offspring were significantly decreased (p<0.05). Maternal supplementation with FWG significantly reduced the levels of MDA and NO in the liver of weaned offspring, indicating an improvement in the oxidative state. At the same time, the levels of tumor necrosis factor-a (TNF-a) were increased in weaned offspring of the HFD group, reflecting an increase in inflammation Figure 8 L). Although this pro-inflammatory response was attenuated in adulthood, maternal FWG intake effectively alleviated the HFD-induced inflammatory response throughout development.

[0066] Maternal intake of fermented wheat germ (FWG) modulates hypothalamic transcriptome profile in adult offspring: The hypothalamus is a central regulator of whole-body metabolism and energy homeostasis, and is particularly susceptible to developmental programming by maternal high-fat diet (HFD). To explore the potential regulatory effects of maternal supplementation with FWG on hypothalamic function in adult offspring of HFD-fed dams, the hypothalamic tissue was subjected to transcriptome analysis. Cluster analysis showed that the gene expression profile of the FWG group was similar to that of the normal diet (NCD) group, indicating that FWG attenuated the HFD-induced transcriptome changes Figure 8 A). Differential expression analysis showed that 36 genes were upregulated and 763 genes were downregulated in the FWG group compared to the HFD group (|log2 fold change| > 2 and p<0.05) Figure 2 B). KEGG enrichment analysis showed that these differentially expressed genes were significantly enriched in extracellular matrix-receptor interaction, complement and coagulation cascade, protein digestion and absorption, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathway Figure 7 C). Among them, the PI3K-Akt pathway, which is closely related to metabolic regulation, was the most enriched, involving 33 differentially expressed genes. These findings suggest that maternal supplementation with FWG may help restore metabolic homeostasis in the hypothalamus of offspring by regulating multiple genes and signaling pathways. Normalization trend of transcriptome remodeling: FWG intervention brought the gene expression pattern of the offspring hypothalamus closer to that of the NCD group, suggesting that it reversed the transcriptional abnormalities induced by HFD, especially the core pathways related to metabolic regulation. Biological significance of cluster analysis: The similarity of gene co-expression patterns directly reflects the differential effects of different dietary groups on the molecular network of the hypothalamus.

[0067] Functional and pathway characteristics of differentially expressed genes: Downregulated genes (763): enriched in inflammation-related pathways (such as cytokine-receptor interaction) and complement system, consistent with the phenotype that FWG suppressed HFD-induced chronic inflammation in the hypothalamus (e.g. Figure 5 , Figure 6Key down-regulated genes (36): including components of PI3K-Akt pathway (e.g. Akt1, IRS2), directly associated with insulin sensitivity and energy metabolism regulation.

[0068] Core function of the pathway: This pathway is a key signaling axis for hypothalamic regulation of energy balance, and its activation can enhance the response of neurons to insulin and leptin, inhibit the activity of appetite-related neurons (e.g. NPY / AgRP), and promote energy consumption. Among the 33 differentially expressed genes, there may be key nodes such as IRS (insulin receptor substrate), PIK3R (PI3K regulatory subunit), and Akt (protein kinase B), forming a regulatory chain of "insulin signal input → PI3K-Akt activation → downstream metabolic gene expression change".

[0069] Potential association of cross-system regulation: The enrichment of "protein digestion and absorption" and "extracellular matrix-receptor interaction" pathways may indirectly reflect the remote regulation of gut microbiota metabolites (such as short-chain fatty acids) on the hypothalamus (microbiota-gut-brain axis), echoing Figure 7 Remodeling of gut microbiota composition by FWG.

[0070] Linkage mechanism with metabolic phenotype ① Appetite regulation: Activation of the PI3K-Akt pathway may enhance the activity of POMC neurons in the arcuate nucleus of the hypothalamus, promote the release of α-MSH (melanocyte-stimulating hormone), and inhibit feeding behavior (although ​ I showed no significant difference in offspring food intake, but energy metabolism efficiency may have improved).

[0071] Insulin sensitivity: Hypothalamic insulin resistance is an upstream driver of peripheral metabolic disorders, and FWG improves the central regulatory capacity for systemic blood glucose by restoring PI3K-Akt signaling (e.g. ​ OGTT results of A-B). ② Inflammation-metabolism interaction: Down-regulation of complement and cytokine pathway genes reduces the toxic effects of neural inflammation on metabolic neurons, forming a positive cycle of "anti-inflammatory → protection of insulin signal → improvement of metabolism".

[0072] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing plant embryo fermentation products, characterized in that, The steps are as follows: Prepare a mixed solution of defatted plant germ powder and water, then inoculate it with baker's yeast seed liquid and incubate for I, then add Lactobacillus plantarum seed liquid and glucose and incubate for II, centrifuge to collect the supernatant, freeze dry to obtain plant germ fermentation product, denoted as FWG; the defatted plant germ powder is defatted wheat germ powder.

2. The method for preparing plant embryo fermentation products according to claim 1, characterized in that: The volume ratio of defatted wheat germ powder to water in the mixed solution is 1:15~20.

3. The method for preparing plant embryo fermentation products according to claim 2, characterized in that, The method for preparing the baker's yeast seed liquid is to use a concentration of 1×10 8 ~1×10 10 The baker's yeast solution was incubated at 25-30℃ for 20-25 hours; The method for preparing the *Lactobacillus plantarum* seed solution is as follows: a concentration of 2.5 × 10⁻⁶ is used. 10 A CFU / mL solution of Lactobacillus plantarum was incubated at 37°C for 24 h.

4. The method for preparing plant embryo fermentation product according to claim 3, characterized in that: The amount of baker's yeast seed liquid added is 1.5~2% v / v; the amount of Lactobacillus plantarum seed liquid added is 2.5~4% v / v; and the amount of glucose added is 3~7% v / v.

5. The method for preparing plant embryo fermentation products according to any one of claims 1-4, characterized in that, The conditions for incubation I are: incubation at 25~30℃ and 150~180 rpm for 3~3.5h; the conditions for incubation II are: incubation at 35~37℃ and 180~220 rpm for 14~15h.

6. A method for preparing plant embryo fermentation products using the method described in claim 5.

7. A composition, characterized in that: The plant embryo fermentation product as described in claim 6 is used as the active ingredient.

8. The use of the plant embryo fermentation product of claim 6 or the composition of claim 7 in the preparation of products that block the intergenerational transmission of metabolic diseases, characterized in that, The transgenerational metabolic diseases have one or more of the following phenotypes: (1) Reduce maternal weight gain, fat deposition and liver lipid accumulation induced by a high-fat diet; (2) Improves maternal and offspring glucose intolerance, insulin resistance and sensitivity to leptin; (3) It regulates the composition of the gut microbiota of the mother and offspring, reduces the Firmicutes / Bacteroidetes F / B ratio, and inhibits the proliferation of obesity-related bacteria such as Lactobacillus and Dorea; (4) By regulating gene expression in the hypothalamic PI3K-Akt signaling pathway, abnormal appetite and metabolic disorders in offspring can be suppressed.

9. The application according to claim 8, characterized in that: The dosage of plant embryo fermentation product used in the functional product is 800-1000 mg / kg body weight.

10. The application according to claim 8, characterized in that: The functional product is administered orally.

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