1. Introduction
Hemp seed meal (HSM) is a high-value byproduct obtained after oil extraction from hemp seeds. Hemp seeds are rich in protein, carbohydrates, oil, insoluble fiber, and minerals [
1]. The are also a good source of phytonutrients, such as tocopherols, carotenoids, and sterols [
2]. HSM contains a full complement of recommended amino acids required for the development of infants and children, and the protein content of HSM is not only abundant, but also highly digestible [
3,
4]. Traditionally, HSM was added directly to animal feed, causing a waste of valuable nutritional resources [
5]. In previous studies, researchers have attempted to add HSM during the preparation of bread and biscuits to produce foods suitable for a wider range of people [
6,
7]. However, recent studies have shown that HSM can be used as a substrate or supplement in solid-state fermentation (SSF), providing the nutritional support of carbon and nitrogen sources [
8]. Nissen et al. found that HSM could be used as a substrate for beneficial lactic acid bacteria, producing fermented HSM with more bioactive substances, such as organic acids and terpenes, which could be applied as a prebiotic [
9].
Fermented foods are popular in Asian countries due primarily to their unique flavors. Nattokinase (NK) is a fibrinolytic protease found in the traditional Japanese fermented food natto, made from soybeans fermented with
Bacillus [
10]. Research has demonstrated that the strong fibrinolytic activity of NK is retained after intestinal absorption with high enzymatic specificity and minimal side effects [
11]. NK is a potential thrombolytic drug with high efficiency, safety, and economy, leading to widespread interest in its application. Soybeans, chickpeas, and wheat bran are commonly used as fermentation substrates for NK [
12,
13,
14]. In addition to traditional fermentation sources, current NK research has been focused on the ongoing quest for low-cost fermentation substrates with rich flavor and higher nutritional quality. Wang et al. used shrimp shells as a substrate and inoculated
Pseudomonas sp. to produce NK, effectively reducing the production cost of NK [
15]. Li et al. replaced the nitrogen source in the fermentation medium with tofu processing wastewater and observed a 47.89% increase in NK activity after optimizing the culture conditions [
16]. Guo et al. used Ginkgo seeds as a substrate and achieved a fibrinolytic activity of 3682 ± 43 U/g, and the fermented Ginkgo seeds had higher total flavonoid and lower ginkgolic acid contents [
17]. Dong et al. fermented chestnuts with
Bacillus natto and obtained highly active NK. Furthermore, the antioxidant activity and α-glucosidase inhibition activity were also increased [
18].
Solid-state fermentation is a simpler and less costly and energy-intensive method of producing NK, with the advantages of high production efficiency, good product stability, and environmental friendliness [
19]. Another advantage is that beneficial byproducts, such as valuable fermentation substrates, are more readily produced by solid-state fermentation.
B. subtilis, the strain most commonly used for NK production, is biosafe and has a high protein secretion capacity [
20,
21]. The rich protease system of B. subtilis is able to break down large protein molecules in the substrate into small polypeptides, facilitating the uptake and use of the nitrogen source by the bacterium. B. subtilis strain 13,932 was previously shown to produce highly active NK in the medium when tofu processing wastewater was used as the nitrogen source [
16]. Numerous studies have demonstrated that peptides derived from hemp protein exhibit many beneficial properties, including regulating hypertension, antioxidation, preventing platelet aggregation, modulating the immune system, reducing cholesterol, and possessing antibacterial properties [
22]. Furthermore, the fermentation of HSM using B.subtilis is anticipated to produce active polypeptides alongside NK.
The aim of this study was to investigate the feasibility of using HSM as a novel solid-state fermentation substrate and to explore the optimum conditions for NK production via solid-state fermentation of HSM by B. subtilis 13,932. At the same time, we wanted to obtain HSM peptides with antioxidant activity and reduce the anti-nutritional factors of HSM via solid-state fermentation. This innovative method aims to create a novel, highly active NK substrate, reducing the overall manufacturing cost while simultaneously providing an efficient and eco-friendly means of utilizing HSM for high-value applications.
2. Materials and Methods
2.1. Materials and Reagents
The HSM used in this study was obtained by crushing leftover hemp seed waste after shelling and oil extraction (particle size range: 0.6–4.0 mm), which was purchased from Hongtian Jiali Co., Ltd. (Jinzhong, China). Thrombin (1000 U) and fibrinogen were purchased from Beijing Zhongke Quality Control Biotechnology Co., Ltd. (Beijing, China). Agarose was purchased from Sigma-Aldrich (St Louis, MO, USA), and other analytical reagents were purchased from Nanjing WANQING Chemical Glassware and Instrument Co., Ltd. (Nanjing, China).
2.2. Microorganism and Culture
B. subtilis 13,932 (Conservation No. CGMCC13932) was maintained in our laboratory. The strain was activated using LB medium (10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl, pH 7.4). The activated strain was then transferred to a seed medium and incubated at 37 °C for 16 h prior to being transferred onto a solid medium. The seed medium included 20 g/L glucose, 20 g/L soybean protein, 0.2 g/L CaCl2, 0.5 g/L MgSO4·7H2O, 1 g/L K2HPO4, and 1 g/L KH2PO4, with the pH adjusted to 7.4.
2.3. Determination of HSM Composition
The primary components of HSM were determined using the Chinese national standard analytical techniques, protein (GB 5009.5-2016), fat (GB 5009.6-2016), dietary fiber (GB 5009.88-2014), moisture (GB 5009.3-2016), and amylum (starch) (GB 5009.9-2016).
2.4. Solid-State Fermentation of Nattokinase by HSM
2.4.1. One-Factor-at-a-Time Experiments (OFAT)
A 100 g sample of HSM was soaked in distilled water for 8 h and sterilized at 121 °C for 20 min, according to the initial ratio of HSM to water (1:1, 1:1.5, 1:2, 1:2.5, 1:3) (w:v). After cooling, the fermentation substrate at a prescribed thickness (1 cm, 2 cm, 3 cm, 4 cm, 5 cm) was prepared, and the B. subtilis 13,932 seed medium was added according to the tested bacterial inoculum volumes (4%, 6%, 8%, 10%, 12%) (w:v). The mixture was evenly mixed and spread onto the sterilization tray. The relative humidity of the fermentation tank was set at a precise level (60%, 65%, 70%, 75%, 80%), and the temperature was controlled constantly (25 °C, 30 °C, 35 °C, 40 °C, 45 °C) throughout the fermentation. The mixture was mixed every 4 h, and the fermentation was carried out for a defined length of time (12 h, 16 h, 20 h, 24 h, 28 h).
2.4.2. Plackett–Burman Design (PB Design)
Based on the results of our OFAT experiments, the PB design sought to determine the optimal conditions for the fermentation process. Specifically, it selected six factors and investigated the initial ratio of HSM to water (A), thickness of the substrate (B), bacterial inoculum volume (C), relative humidity (D), temperature (E), and fermentation time (F) (coding levels shown in
Table 1). The response value chosen for investigation was the NK activity, and the experiment aimed to examine the impact of each factor on the fermentation results.
2.4.3. Box–Behnken Design (BBD)
Based upon the results of our PB design, the four factors A, B, D, and E were identified as the key factors impacting fermentation. Acknowledging these factors, BBD was carried out with NK activity as the response (coding levels shown in
Table 2).
2.5. Extraction of NK and Determination of Enzyme Activity
To prepare the sample, 10 g of fermented hemp seed meal (FHSM) and 40 mL of 0.9% NaCl solution were mixed in a 250 mL flask and mixed at 100 rpm for 1 h. After centrifugation at 10,000 rpm for 10 min, the supernatant was collected for analysis of NK enzyme activity using the fibrinolytic plate method [
16]. To prepare the agarose solution, solid agarose was dissolved in 100 mL of a mixed solution containing phosphate buffer solution (pH = 7.8) and 0.9% NaCl solution at a ratio of 1:17. The solution was then sterilized at high temperature and kept at 60 °C. To prepare the solid plates, 68.67 mL of 1.5 mg/mL fibrinogen solution and 5.28 mL of 1 bp/mL thrombin solution (dissolved in 0.9% NaCl solution) were preheated and added to the agarose solution. The mixture was then thoroughly mixed and prepared in solid plates, which were then punched after coagulation. For analysis, 15 μL of the supernatant was added into the well of the solid plate, which was then cultured at 37 °C for 16–18 h. The diameter of the fibrinolytic ring was then measured. The enzyme activity of the urokinase standard solution was taken as the ordinate, and the product of the diameter of the fibrinolytic ring produced by urokinase under the same operation was taken as the abscissa, in order to create a standard curve.
2.6. Determination of Soluble Peptide Content
The method for the determination of soluble peptide content is based upon the method of Benjakul et al. [
23]. A 1 g sample of FHSM was thoroughly homogenized with 10 mL of water. The homogenization solution was mixed with an equal volume of 10% (
w/
v) trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 10 min after allowing it to stand for 30 min to remove macromolecular proteins. The nitrogen content was measured using a total nitrogen analyzer. The TCA soluble peptide content percentage was calculated as follows:
TCA soluble peptide content (%) = nitrogen content in supernatant after TCA precipitation/nitrogen content in homogenization solution × 100
2.7. Detection of Antioxidant Activity
The hydroxyl and DPPH radical scavenging abilities were analyzed using kits produced by Jiangsu Addison Biotechnology Co., Ltd. (Yancheng, China).
2.8. Determination of Anti-Nutritional Factors
Urease levels were measured using a Keming Biotechnology Co., Ltd. kit. Urease enzyme activity was defined as 1 μg NH3-N per minute produced per g of tissue is one enzyme activity unit. Trypsin inhibitor activity was determined using an ELISA kit purchased from Jiangsu Meimian Industrial Co., Ltd. (Yancheng, China).
2.9. Sensory Characteristics
The sensory properties of FHSM have been modified from a protocol established by Feng et al. [
24]. The sensory evaluation team was composed of 10 people with sensory evaluation experience. The prepared FHSM was scored from 1 to 5 points for its appearance, smell, texture, taste, and stringiness (5 = very good, 4 = good, 3 = acceptable, 2 = dislike, 1 = highly dislike).
2.10. Statistical Analysis
All tests were performed in triplicate. The data are presented as mean ± standard deviation (SD). Design-Expert 8.0.6 software was used in the PB Design and BBD, as well as for corresponding data analysis, while Origin 2018 software was used for graph construction. The significance threshold values of the obtained data were set at p < 0.05.