**1. Introduction**

Hyperlipidemia is a chronic systemic metabolic disease with lower levels of highdensity lipoprotein cholesterol (HDL-C) and higher levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) due to abnormal fat transport or metabolism [1]. It is considered as one of the risk factors of cardiovascular diseases, including atherosclerosis [2], coronary heart disease [3], and diabetes [4]. Intestinal microbes, the "second genome" of the human body [5], are inextricably linked to these diseases, and the intestinal microbiota of such patients is significantly different from that of healthy people [6]. The gut microbiome in hyperlipidemic subjects is also characterized by low microbial diversity, such as a high abundance of some taxa from the phylum Actinobacteria and lower abundance of many taxa from phyla Proteobacteria and Bacteroidetes [7].

**Citation:** Zhang, Z.; Wang, Y.; Zhang, Y.; Chen, K.; Chang, H.; Ma, C.; Jiang, S.; Huo, D.; Liu, W.; Jha, R.; et al. Synergistic Effects of the Jackfruit Seed Sourced Resistant Starch and *Bifidobacterium pseudolongum* subsp. *globosum* on Suppression of Hyperlipidemia in Mice. *Foods* **2021**, *10*, 1431. https://doi.org/10.3390/ foods10061431

Academic Editor: Robert G. Gilbert

Received: 25 April 2021 Accepted: 30 May 2021 Published: 21 June 2021

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Li et.al [8] found that *Alistipes*, *Intestinibacter*, *Subdoligranulum,* and *unidentified Ruminococcaceae* in the gut were significantly negatively correlated to TG, which was an important indicator of hyperlipidemia.

Resistant starch (RS) is defined as "the sum of starch and starch degradation products not absorbed by the small intestine of a healthy individual" [9], which is naturally found in cereal grains, seeds, heated starch, or starch-containing food [10]. RS has been widely recognized for its beneficial effects, such as improving insulin resistance and glucose homeostasis [11,12], maintaining colon health [13], controlling body weight [14], elevating large-bowel short-chain fatty acids (SCFAs) [15], and especially lowering blood lipid [16,17]. It has been suggested that a high dosage of RS administration in hamsters could increase HDL-C concentration and decrease TG, TC, and LDL-C concentrations to ameliorate hyperlipidemia [18], and *Bifidobacteria* and *Lactobacillus* in the gut were dramatically increased and positively correlated with blood lipid levels. Some of these functions of RS are linked to their fermentation characteristics, thus labeled as prebiotic. However, there are also inconsistent reports. Zhang et al. [19] tested the effect of RS on 19 volunteers and found no significant difference in body weight and HDL-C. However, LDL-C decreased significantly after four weeks of treatment. This was similar to a study reporting that body weight and liver triglycerides of C57BL/6J mice did not change after being fed a 45%-fat diet with 20% high-amylose-maize RS [20]. This might be because different types of RS are affected differently due to their fermentation characteristics, thereby affecting the gut physiology and health differently [21], which is affected by the gut microbial ecology [22]. Thus, these inconsistencies might be due to neglecting the interaction between RS and the key bacteria in the intestine. Therefore, we wanted to explore the crucial role of microbiota during the digestion of RS. Generally, jackfruit seed is a good source of RS [23]. However, there is limited or no information about the effect of jackfruit seed sourced resistant starch (JSRS) on the intestinal microbiota and hyperlipidemia, requiring exploration.

To address the problem, a two-stage experiment was conducted. In Stage I, we revealed the effect of JSRS on hyperlipidemia-related indexes and gut microbiome in mice. Meanwhile, *Bifidobacterium pseudolongum* was identified as the key microorganism that can utilize RS in the gut through 16s rRNA and shotgun metagenomic data. Based on this result, in vitro and in vivo validation experiments in Stage II were conducted to determine the ability of *B. pseudolongum (Bifidobacterium pseudolongum* subsp. *globosum)* to utilize JSRS and to explore the synergistic effects between the two for the prevention and treatment of hyperlipidemia in mice. This study highlights the irreplaceable role of intestinal microbes in the utilization of RS and a new idea that intervention studies of functional macromolecules, including RS, need to consider the involvement of exogenous microorganisms.

#### **2. Methods**

#### *2.1. Animal Feeding and Diet Formula*

Four-week-old C57BL/6 J male mice (*n* = 40) were sourced from Hunan SJA Laboratory Animal Co. Ltd. China, which were bred and housed in specific pathogen-free conditions with 12-h day and night light cycles at 22 ± 2 ◦C temperature and 55 ± 10% relative humidity. The normal-fat diet (NFD) was made up of 41% corn, 26% bran, 29% bean cake, 1% salt, 1% bone meal, 1% lysine, and 1% other. The high-fat diet (HFD) was made up of 1% cholesterol, 10% egg yolk, 10% lard, 0.2% sodium cholate, and 78.8% NFD. The proximate nutrient content of the NFD was as follows: crude protein, 18%; crude fat, 4%; crude fiber, 5%; calcium, 1.0–1.8%; and phosphorus, 0.6–1.2%. The proximate nutrient content of the HFD was as follows: crude protein, 17.6%; crude fat, 19.7%; crude fiber, 4%; calcium, 0.81–1.45%; and phosphorus, 0.56–1.04%. JSRS was provided by the Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China. The *B. pseudolongum* was provided by Inner Mongolia Agricultural University, China.

#### *2.2. Stage I, the Exploration Experiment*

After a 7-day adaptation period, mice were randomly divided into 4 groups: NFD (*n* = 10); NFD plus JSRS (90% NFD plus 10% JSRS, *n* = 10); HFD (*n* = 10); HFD plus JSRS (90% HFD plus 10% JSRS, *n* = 10). Body weight was assessed at weeks 1, 2, 4, and 8. Feces were collected at weeks 2, 4, 8, and stored at −80 ◦C until further microbiota analysis. After 8 weeks of intervention, all mice were subjected to a 16-h fast and then euthanized and dissected. Blood was collected, and serum was isolated to determine TG as a blood lipid indicator. All fecal samples from week 2 and week 4 were used for high-throughput sequencing of the V3-V4 region of the bacterial 16s rRNA gene [24], and fecal samples from week 8 were processed for deep metagenomic sequencing [25]. The experimental design is presented graphically in Figure 1A.

**Figure 1.** Experimental design. (**A**) In stage I, mice were randomly divided into four groups: NFD (normal-fat diet, *n* = 5); NFD plus JSRS (90% normal-fat diet plus 10% jackfruit seed sourced resistant starch, *n* = 6); HFD (high-fat diet, *n* = 6); HFD plus JSRS (90% high-fat diet plus 10% jackfruit seed sourced resistant starch, *n* = 6). The mice were kept for eight weeks. (**B**) The 4 groups that were treated for 3 weeks in order to make nutritionally obese mice model: NFD (normal-fat diet, *n* = 10); TR (HFD was chosen for the first 3 weeks for the development of obese mice model, then, 90% NFD plus 10% JSRSplus 8Log CFU *B. pseudolongum* infusions done later *n* = 10); HFD (high-fat diet, *n* = 10); PR (90% HFD plus 10% JSRS plus 8Log *B. pseudolongum* infusions, *n* = 10). After 3 weeks, three mice in each group were euthanized to observe abdominal fat accumulation. Then a 3-week intervention was continued on respective dietary treatments until the end of the experiment.

#### *2.3. Stage II, the Validation Experiment*

In vitro validation. *B. pseudolongum* was inoculated into MRS agar medium (agar 20 g, glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, C6H5O7(NH4)3 2 g, Tween-801 mL, CH3COONa 5 g, K2HPO4 2 g, MgSO4 0.58 g, MnSO4 0.25 g, water 1 L) as the control and JSRS agar medium (agar 20 g, JSRS 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, C6H5O7(NH4)3 2 g, Tween-80 1 mL, CH3COONa 5 g, K2HPO4 2 g, MgSO4 0.58 g, MnSO4 0.25 g, water 1 L) anaerobic culture for 48 h, and the total number of microbial colonies were calculated. To determine the extent of starch utilization by *B. pseudolongum*, the plate was treated with iodine plus potassium iodide solution, and diameter of the colony and transparent circle was calculated.

In vivo validation. After 2 weeks of acclimatization on an NFD to the laboratory environment, mice (*n* = 40) were randomly subdivided equally into 4 groups [NFD, (*n* = 10); TR (HFD was fed for the first 3 weeks for making the obese mice model, then, 90% NFD plus 10% JSRS plus 8Log CFU *B. pseudolongum* infusions were done later, *n* = 10); HFD (*n* = 10); PR (90% HFD plus 10% JSRS plus 8Log CFU *B. pseudolongum* infusions, *n* = 10)] and were treated for 3 weeks to make the nutritionally obese mice model. After 3 weeks, three mice from each group were euthanized to determine abdominal fat accumulation. The rest of the mice were continued on the respective dietary treatment for another 3 weeks. The NFD and HFD groups were gavaged with an equal volume of normal saline as controls (Figure 1B). Body weight was recorded weekly. Feces were collected weekly and stored at −80 ◦C. After 8 weeks of intervention, mice were subjected to a 16-h fast and then were euthanized. Livers and abdominal fat were excised and weighed quickly. Then, livers were washed by phosphate buffer saline and processed for further analysis.

### *2.4. Scanning Electron Microscopy*

The JSRS particle structure (granule form) analysis was done using scanning electron microscopy (SEM) as described by Zhang et al. [26] with some modifications. The samples were fixed on a sample holder with a silver plate and coated with a platinum film. The obtained samples were observed under a scanning electron microscope (Verios G4 UC, Thermo Fisher Scientific, Brno, South Moravia, Czech Republic).

#### *2.5. Serum Lipid Levels Determination*

The blood from the eyeball was collected and centrifuged at 571× *g* for 15 min at 4 ◦C. Commercial assay kits (Jian Cheng Biotechnology Co., Ltd., Nanjing, China) were used to measure blood lipid indices, including TG, TC, HDL-C, and LDL-C. The results of hepatic lipids were corrected for total protein concentration.
