**1. Introduction**

Okara is a by-product of soybean processing. Over 20 million tons of wet soybean dregs (okara) are produced each year in China [1], which is one of the world-leading producers and consumers of soybean. This huge sum of okara produced during soybean processing is evidenced to be packed with a significant amount of nutritional and nonnutritional constituents. However, these are usually used as animal feeds, fertilizers, landfills, or discarded as waste due to their high susceptibility to spoilage, which exerts serious economic and socio-environmental problems [2]. Hence, its valorization will be important to help utilize the untapped nutrients and minimize the environmental problems caused by this waste disposal. Okara is high in nutrients, with insoluble fiber fraction accounting for 70% of the total dietary fiber [1]. Besides, the functional characteristics of insoluble dietary fiber have recently been discovered and gained recognition. Dietary fiber is tagged as "The Seventh Nutrient" [3], and is necessary for a balanced diet [4]. Hence, can be utilized in shaping the gut microbiota [5]. The consumption of a high dietary fiber diet is evidenced by decreases in the bioavailability of some important nutritional components, such as some vitamins and minerals [6], and may also impact the rate of digestion of food

**Citation:** Fan, H.; Zhang, Y.; Swallah, M.S.; Wang, S.; Zhang, J.; Fang, J.; Lu, J.; Yu, H. Structural Characteristics of Insoluble Dietary Fiber from Okara with Different Particle Sizes and Their Prebiotic Effects in Rats Fed High-Fat Diet. *Foods* **2022**, *11*, 1298. https://doi.org/10.3390/ foods11091298

Academic Editor: Antonella Pasqualone

Received: 1 April 2022 Accepted: 26 April 2022 Published: 29 April 2022

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substances as well as energy metabolism. The intake of a high-fiber diet can also improve the activities of the gut microbial composition [7].

The gut ecosystem is a home for over 1014 microbial cells, and collectively comprise of at least 150 times more genes than their host [8]. In both humans and animals, the gastrointestinal tract is host to a complex community of varied microorganisms, whose activities considerably impact host nutrition and health [9]. The composition of the gut microbiota is said to vary based on genetic characteristics, sex, age, and diet [10]. The association between the host gut-microbiota are dynamic and are highly susceptible to several environmental conditions, mainly diet [8], and the interactive relationship between prebiotics and dietary fiber is reliant on variations in the gut microflora as well as their colonic degradation [7]. Numerous studies indicates that the gut microorganisms can directly impact the physiological conditions of the host organism by encouraging the immune system through stimulating the defensive mechanisms against pathogens and inflammatory bowel diseases, as well as improving the roles of the intestinal barrier, regulating autoimmunity, producing biological metabolites, destroying cancer cells, regulating diabetes and preventing obesity. The host gut-microbiota interactions are highly influenced by several environmental conditions, buy mainly by diet [3,10]. Dietary fiber intake is reported to impact gastrointestinal health via encouraging the gastrointestinal barrier function, nutrient absorption, as well as selectively stimulating the composition and activities of the gut microbes, that can confer health benefits to the host, including lowering the risk of cardiovascular disease, cancer, diabetes, and other ailments [11,12]. For instance, dietary fiber alters the gastrointestinal barrier function through increasing cells and the mucins that produce the "goblet cells" [11]. Mucins are large glycoproteins that, along with antibodies, bacteria, lipids, proteins, ions, antimicrobial peptides, and water, form what is known as mucus. Mucus shields the gut epithelium from mechanical stress, to inhibit the translocation of toxic substances and to lubricate the intestine as well as encourage smooth transportation of digested material [3,12]. In addition, dietary fiber may absorb and excrete toxic chemicals in the intestines, promote intestinal flora, and offer energy and nutrients for probiotic growth [13]. The majority of studies on okara's insoluble dietary fiber focus on changing their fundamental physical and chemical characteristics [14,15], and only a few studies have been conducted on the functional qualities of okara due to their unpleasant taste [16]. The prolonged intake of a high-fat diet poses a major hazard to human/animal health. Emerging evidence has indicated that high-fat-diet can alter the composition of the microbiota, by inducing dysbiosis or imbalance of the gut microbial ecosystem. Dysbiosis is associated with diseases and metabolic health ranging from insulin resistance, glucose intolerance and to metabolic syndrome [17]. Furthermore, the gut microbiota can influence lipid metabolism via short-chain fatty acid (SCFA) metabolites [18]. SCFAs are primarily generated in the large intestine by anaerobic bacteria fermenting indigestible carbohydrates. They provide the material foundation for the gut flora's "conversation" with the host [13]. They have the ability to stimulate cell development, enhance intestinal function, and have an impact on cardiovascular metabolism, as well as anti-inflammatory, anti-tumor, and immunological regulatory activities [19].

In this work, high-pressure microfluidic technology was employed to extract three insoluble dietary fibers (IDFs) with varying particle sizes from okara (purity > 90%) to investigate the effects of insoluble dietary fiber from okara (OIDF) on blood lipid levels and intestinal flora in rats fed a high-fat diet. Okara can obtain high-purity OIDF and increase its value addition.

#### **2. Materials and Methods**

#### *2.1. Preparation of OIDFs with Three Different Particle Sizes*

Okara, a by-product of producing soybean protein isolate with a protein level of 10–15% (Liaocheng, China), was generously provided by Shandong Jiahua Health Care Products Co., Ltd. The IDF from okara was further processed into three different particle sizes using High Pressure Microfluidic Technology to obtain the OIDFs. The steps comprise of α-amylase treatment at 95 ◦C for 35 min, being starch glucosidase treated at 60 ◦C for 30 min, being neutral protease treated 60 ◦C for 30 min, 70 ◦C water precipitation for 1.5 h, centrifugation at 3500 rpm for 10 min, alcohol precipitation until colorless, and being freeze-dried for standby. According to the Chinese national standard GB 5009.88-2014, OIDF has a purity of 90.50 percent [20]. The OIDF (purity higher than 90.50%) extracted by biological enzymatic method was processed by dynamic high pressure microfluidization (DHPM). OIDF weighing 20 g was mixed with 800 mL of deionized water, and treated at 10 MPa, 50 MPa, and 150 MPa for 2 min, respectively. Three distinct particle sizes of OIDFs were named OIDF-10 (μm), OIDF-50 (μm), and OIDF-100 (μm).

#### *2.2. Determination of the Basic Components of the Three OIDFs*

The protein content was determine according to the Kjeldahl's method in GB5009.5-2016 "Determination of Protein in Food"; Ash content was determine based on GB5009.4-2016 "Determination of ash in food; Moisture content was determined by MB35 Ohaus moisture analyzer; SDF (soluble dietary fiber), IDF and TDF content were determined using GB5009.88-2014 "Determination of Dietary Fiber in Food".
