*3.4. Maternal Nutrient Intake and Infant Gut Microbiome*

Heatmaps of the correlation between maternal nutrient intake during pregnancy and infant gut microbiome at 2 months of age are displayed in Figure 3. A high-fructose maternal diet was negatively associated with *Erysipelatoclostridium*. A high-glucose maternal diet was significantly associated with an enrichment of *Isobaculum* in the infant gut microbiome. The Lachnospiraceae count was lower among infants with higher maternal consumption of dietary fiber (Figure 3A). Betaproteobacteria, Burkholderiaceae, and *Sutterella* were strongly negatively correlated with folic acid, and Betaproteobacteria and Burkholderiaceae were negatively correlated with ascorbic acid (Figure 3B). Clostridia, Clostridiales, and Lachnospiraceae were negatively correlated with both magnesium and potassium, and *Lachnoclostridium* was negatively correlated with potassium. *Nutrients* **2021**, *13*, 1559 6 of 12 Propionibacteriaceae, *Cutibacterium*, Tannerellaceae, *Parabacteroides*, and *Lactococcus* were higher in the microbiome of 2-month-old infants with high maternal vegetable and fruit consumption. However, the counts of *Prevotella\_2*, *Prevotella\_9*, *Isobaculum*, Clostridia, Clostridiales, Lachnospiraceae, *Hungatella*, *Lachnoclostridium*, Ruminococcaceae, *Flavonifractor*, *Erysipelatoclostridium*, Acidaminococcaceae*, Phascolarctobacterium*, *Megamonas*, Betaproteobacteriales, Burkholderiaceae, and Sutterella were higher in the microbiome of 2-months-old infants with low maternal fruit and vegetable consumption.

Adonis *p* = 0.001; Betadisper; *p* = 0.90.

**Figure 2.** *Cont*.

(**A**)

*Nutrients* **2021**, *13*, 1559 7 of 12

(**B**)

**Figure 2.** Variations in the infant gut microbiome at 2 months of age according to maternal fruit and vegetable consumption during pregnancy. (**A**) Principal coordinates analysis (PCoA) on unweighted unique fraction (UniFrac). (**B**) linear discriminant analysis effect size (LEfSe). The ordination is from A to W in a tree diagram (Figure S3). Definition of abbreviation: H, high maternal consumption of fruits and vegetables during pregnancy; L, low maternal consumption of fruits and vegetables during pregnancy; O, order; F, family; G, genus. **Figure 2.** Variations in the infant gut microbiome at 2 months of age according to maternal fruit and vegetable consumption during pregnancy. (**A**) Principal coordinates analysis (PCoA) on unweighted unique fraction (UniFrac). (**B**) linear discriminant analysis effect size (LEfSe). The ordination is from A to W in a tree diagram (Figure S3). Definition of abbreviation: H, high maternal consumption of fruits and vegetables during pregnancy; L, low maternal consumption of fruits and vegetables during pregnancy; O, order; F, family; G, genus.

*3.4. Maternal Nutrient Intake and Infant Gut Microbiome*  Heatmaps of the correlation between maternal nutrient intake during pregnancy and infant gut microbiome at 2 months of age are displayed in Figure 3. A high-fructose maternal diet was negatively associated with *Erysipelatoclostridium*. A high-glucose maternal diet was significantly associated with an enrichment of *Isobaculum* in the infant gut microbiome. The Lachnospiraceae count was lower among infants with higher maternal consumption of dietary fiber (Figure 3A). Betaproteobacteria, Burkholderiaceae, and *Sutter-*As shown in Figure S4, counts of *Hungatella* and *Megamonas* were lower among infants with higher maternal consumption of vegetables. Moreover, the count of *Erysipelatoclostridium* was lower among those with higher maternal consumption of fruits, and the count of *Megamonas* was higher among those with higher maternal consumption of dairy. The count of *Isobaculum* was lower among those with higher maternal consumption of grains, whereas the count of *Flavonifractor* was higher. The counts of both Lachnospiraceae and *Lachnoclostridium* were lower among those with higher maternal consumption of meat.

*ella* were strongly negatively correlated with folic acid, and Betaproteobacteria and Burkholderiaceae were negatively correlated with ascorbic acid (Figure 3B). Clostridia, Clostridiales, and Lachnospiraceae were negatively correlated with both magnesium and

As shown in Figure S4, counts of *Hungatella* and *Megamonas* were lower among infants with higher maternal consumption of vegetables. Moreover, the count of *Erysipelatoclostridium* was lower among those with higher maternal consumption of fruits, and the count of *Megamonas* was higher among those with higher maternal consumption of dairy.

potassium, and *Lachnoclostridium* was negatively correlated with potassium.

**Figure 3.** Heatmaps showing the correlation between maternal nutrient intake during pregnancy and the infant gut microbiome at 2 months of age (\* Significant association). (**A**) Macronutrients. (**B**) Vitamins. (**C**) Minerals. **Figure 3.** Heatmaps showing the correlation between maternal nutrient intake during pregnancy and the infant gut microbiome at 2 months of age (\* Significant association). (**A**) Macronutrients. (**B**) Vitamins. (**C**) Minerals.

### **4. Discussion 4. Discussion**

meat.

We demonstrated that, in this mother–infant nutrition cohort, the infant gut microbiome at 2 months of age varied according to the level of maternal fruit and vegetable consumption during pregnancy. We identified 6 and 17 taxa in the infant gut microbiome from the high and low fruit and vegetable consumption groups, respectively. Furthermore, we have shown the detailed nutrients and gut microbiome taxonomic interactions. We demonstrated that, in this mother–infant nutrition cohort, the infant gut microbiome at 2 months of age varied according to the level of maternal fruit and vegetable consumption during pregnancy. We identified 6 and 17 taxa in the infant gut microbiome from the high and low fruit and vegetable consumption groups, respectively. Furthermore, we have shown the detailed nutrients and gut microbiome taxonomic interactions.

The count of *Isobaculum* was lower among those with higher maternal consumption of grains, whereas the count of *Flavonifractor* was higher. The counts of both Lachnospiraceae and *Lachnoclostridium* were lower among those with higher maternal consumption of

## *4.1. Maternal Fruit and Vegetable Consumption Affects Infant Microbiome 4.1. Maternal Fruit and Vegetable Consumption Affects Infant Microbiome*

High or low maternal fruit and vegetable consumption was significantly correlated with infant gut microbiome composition. Gut microbiome composition is related to the intake of dietary fiber [26], which is fermented by certain bacteria, producing short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate [27]. Animal studies have reported an association of increased maternal dietary microbiome-accessible fiber and SCFA exposure during pregnancy, with a reduced incidence of asthma in offspring [11,28], and this effect persists into adulthood [11]. A follow-up small human component (*n* = 61) of the same study indicated that an association exists between reduced dietary fiber intake and reduced serum acetate levels in pregnant women. A separate component (*n* = 40) revealed a correlation between serum acetate levels that were lower than the me-High or low maternal fruit and vegetable consumption was significantly correlated with infant gut microbiome composition. Gut microbiome composition is related to the intake of dietary fiber [26], which is fermented by certain bacteria, producing short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate [27]. Animal studies have reported an association of increased maternal dietary microbiome-accessible fiber and SCFA exposure during pregnancy, with a reduced incidence of asthma in offspring [11,28], and this effect persists into adulthood [11]. A follow-up small human component (*n* = 61) of the same study indicated that an association exists between reduced dietary fiber intake and reduced serum acetate levels in pregnant women. A separate component (*n* = 40) revealed a correlation between serum acetate levels that were lower than the median, and increased frequency of coughing/wheezing during the child's first year of life [11,28].

dian, and increased frequency of coughing/wheezing during the child's first year of life [11,28]. In a mouse model study, the plasma SCFA levels of the offspring of mice fed a high-fiber diet were higher than those of mice fed a no-fiber diet, and the frequencies of thymic regulatory T cells (Tregs) and peripheral Tregs were higher in the offspring of high-fiber-diet-fed mice [26]. During pregnancy, SCFA (such as acetate) can cross the placenta and affect the expression of fetal lung genes, such as NPPA, which encodes ANP (a molecule related to epithelial biology and immune regulation) [11]. In demonstrating associations between maternal high dietary fiber intake, antenatal exposure to SCFAs, and offspring allergic diseases, these mouse experiments have suggested a possible target for interventions to reduce the burden of allergic diseases; however, no clinical trials have investigated the protective effects of maternal microbiome against allergic diseases through a maternal high-fiber diet.

In the present study, we observed a higher relative abundance of *Cutibacterium*, *Parabacteroides*, and *Lactococcus* in the fecal microbiome of infants exposed to high vegetable and fruit consumption during gestation. However, the higher abundance of *Prevotella\_2*, *Prevotella\_9*, *Isobaculum*, *Hungatella*, *Lachnoclostridium*, *Flavonifractor*, *Erysipelatoclostridium*, *Phascolarctobacterium*, *Megamonas*, and *Sutterella* was associated with low fruit and vegetable consumption. Our findings regarding the beneficial effects of *Cutibacterium*, *Parabacteroides*, and *Lactococcus* on infant immunity are consistent with those of previous studies. *Cutibacterium* ferments hexoses through the Embden–Meyerhof pathway to produce pyruvate, which is further metabolized into propionate [29], whose consumption was reported to reduce antigen presentation on dendritic cells as a result of GPR41-dependent modulation of hematopoiesis and affected allergic diseases in a mouse model [28]. *Parabacteroides* have multiple beneficial effects on human health. P. distasonis can improve human bowel health [30] and is negatively associated with celiac disease [31]. It can reduce weight gain, hyperglycemia, and liver steatosis in ob/ob and high-fat diet mice [32] and significantly reduce the severity of intestinal inflammation in murine models of acute and chronic colitis [33]. The SCFA-producing *Parabacteroides* were richer in the cecum and colorectum, where, accordingly, more SCFAs were produced [34]. *Lactococcus* lactis activates innate immunity and protects from infections [35,36]. Moreover, some Lactobacilli can produce SCFAs [34], which can induce Tregs to modulate gut immune responses [37–39], and can shape the pulmonary immune environment and influence the severity of allergic inflammation [28].
