**1. Introduction**

Soybean sauces are commonly used as seasonings and sauces in Asia. Among them, black bean-fermented soybean sauce which uses black beans (*Glycine max* (L.) Merr.) as the major raw material is one of the most favored. During the soybean sauce manufacturing process, raw soybean steaming is an important step before yeast inoculation [1]. However, steamed soybean liquid waste, particularly by-products, may contain nutrients and other phytochemicals. These by-products may not only provide nutrient and active compounds for supplements but also possess sustainable and circular economic characteristics if they can be developed into a healthy food material. Considering global sustainability, the circular economy is a new alternative approach to the traditional economy [2]. Therefore, recycling certain by-products from food manufacturing processes as new food and pharmaceutical materials is not only a solution for food waste and supply issues, but also a new preferred resource for human health [2].

**Citation:** Hsieh, S.-L.; Shih, Y.-W.; Chiu, Y.-M.; Tseng, S.-F.; Li, C.-C.; Wu, C.-C. By-Products of the Black Soybean Sauce Manufacturing Process as Potential Antioxidant and Anti-Inflammatory Materials for Use as Functional Foods. *Plants* **2021**, *10*, 2579. https://doi.org/10.3390/ plants10122579

Academic Editors: Juei-Tang Cheng, I-Min Liu and Szu-Chuan Shen

Received: 27 October 2021 Accepted: 23 November 2021 Published: 25 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The black soybean cultivar has abundant polyphenols in its seed coating [3]. During the black soybean sauce manufacturing process, the beans are steamed at a high temperature and pressure [4]. This process is similar to soybean extraction with high-temperature steam under high pressure. These steamed soybean extracts contain polyphenols and some of the active components of black soybeans [5]. Previous studies have shown that black soybeans can reduce cardiovascular disease [6], regulate blood sugar [7] and have anticancer effects, [8,9], improve bone resorption in menopause [10], and possess antioxidative [11] and anti-inflammatory [12] properties. Kim et al. [13] showed that raw black soybeans have abundant total free polyphenols, flavonoids and phenolic acids. Anthocyanidin is a type of isoflavone that is present in black soybean coats [9]. Recycling this black soybean steamed liquid (BBSL) has the potential to providing an effective physiological material for functional food development.

The inflammatory response is a physiological protective mechanism of the body that occurs in response to infection; however, the occurrence of chronic inflammation with chronic inflammatory cells, including macrophages, lymphocytes and plasma cells, among others, is increasing [14]. During the inflammatory response process, reactive oxygen species (ROS), nitric oxide (NO) and prostaglandin E<sup>2</sup> (PGE2) act as messengers for different physiological functions and pathological processes [15]. Conversely, intracellular cytokines, such as IL-1β, IL-6, IL-10 and TNF-α, are secreted from macrophages and lymphocytes [16]. These cytokines mediate the immune response and influence the macrophage microenvironment [17]. Excess or long-term chronic inflammation will lead to chronic diseases, such as cardiovascular disease (CVD), sepsis, diabetes mellitus (DM), and chronic kidney disease (CKD), increasing human health risks [18].

Some inflammatory response factors, such as IL-1β, IL-6, and TNF-α, and inflammationmediated molecular enzymes, including inducible nitroxide synthase (iNOS) and cyclooxygenase-2 (COX-2), are primarily controlled by nuclear factor kappa B (NF-κB), which is well recognized to play an important role in inflammation [15,19–22].

Currently, the food supply chain has many challenges due to decreased natural resources and increased food waste [23]. Thus, recycling reusable by-products of plant foods manufacturing process as new foods or components with active physiological effects is a forward-looking issue. For further more study and assessment the potential of this by-product of the soybean sauce manufacturing process on functional food material. The present study aims to investigate the capability of antioxidation and anti-inflammation of black bean steamed liquid lyophilized product (BBSLP).

### **2. Results**

### *2.1. pH Value, Flavonoids, Total Phenol, and Protein Contents in BBSL and BBSLP*

To know the application of fresh BBSL, the pH was measured in this study. As shown in Table 1, the pH of fresh BBSL was 5.84 ± 0.12. The flavonoids, total phenol, and protein contents of BBSL were 11.5 ± 1.5 mg rutin equivalents (RUE)/mL, 3.83 ± 0.3 mg gallic acid equivalents (GAE)/mL and 3.5 ± 0.8 mg/mL, respectively (Table 1). In addition, after fresh BBSL was concentrated by a rotary vacuum dryer and frozen dry by a frozen dryer, the flavonoids, total phenol, and protein contents of BBSLP were 0.1 ± 0.01 mg RUE/mg, 0.03 ± 0.01 mg GAE/mg and 0.31 ± 0.04 mg/mg, respectively (Table 1).

**Table 1.** pH, Flavonoids, total polyphenols and crude protein levels of BBSL and BBSLP.


\* BBSL: black bean steamed liquid; BBSLP: black bean steamed liquid lyophilized product; GAE: gallic acid equivalents; RUE: rutin equivalents. Values are presented as means ± SD (*n* = 3–5).

#### *2.2. BBSLP Showed Antioxidant Effects In Vitro Conditions* 20.5 ± 6.3% and 50.2 ± 8.3%, respectively, and showed a dose-dependent increase (*p* < 0.05).

*Plants* **2021**, *10*, x FOR PEER REVIEW 3 of 14

**Table 1.** pH, Flavonoids, total polyphenols and crude protein levels of BBSL and BBSLP.  **pH Value Flavonoids Total Polyphenols Crude Protein**  BBSL \* 5.84 ± 0.12 11.5 ± 1.5 mg RUE mL−1 3.83 ± 0.3 mg GAE mL−1 3.5 ± 0.8 mg mL−<sup>1</sup> BBSLP - 0.1 ± 0.01 mg RUE mg−1 0.03 ± 0.01 mg GAE mg−1 0.31 ± 0.04 mg mg−<sup>1</sup> \* BBSL: black bean steamed liquid; BBSLP: black bean steamed liquid lyophilized product; GAE: gallic acid equivalents; RUE:

ent manner.

rutin equivalents. Values are presented as means ± SD (*n* = 3–5).

In the 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) radical-reducing ability test the scavenging abilities of 0.5, 1 and 2 mg/mL BBSLP were 42.5 ± 8.7%, 54.4 ± 0.4% and 79.6 ± 0.2%, respectively. The observed scavenging ability in the vitamin C-treated group was 88.0 ± 5.8% (Figure 1A). Although the DPPH radical-reducing ability in the BBSLP groups was lower than that in the vitamin C group, it increased in a dose-dependent manner. The effect concentration (EC50) value of BBSLP for ABTS+ radical scavenging ability was 1.50 mg/mL. In addition, the reducing power of 0.5, 1 and 2 mg/mL BBSLP reached 42.5 ± 84.6% in a dose-dependent manner (*p* < 0.05). The vitamin C-treated group showed 88.7 ± 9.6% reducing power (Figure 1C). The EC50 value of BBSLP for reducing power was 0.80 mg/mL.

The EC50 value of BBSLP for DPPH radical-scavenging ability was 0.81 mg/mL. Figure 1B shows that the Trolox-treated group demonstrated 95.5 ± 4.3% 2,2′-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS+) radical scavenging. The ABTS+ radical scavenging abilities in the 0.5, 1 and 2 mg/mL BBSL groups were 7.8 ± 2.3%,

BBSLP groups was lower than that in the vitamin C group, it increased in a dose-depend-

**Figure 1.** In vitro antioxidative ability of the BBSLP. DPPH radical scavenging activity (**A**), ABTS+ radical scavenging activity (**B**) and reducing power (**C**). Vitamin C was used as the positive control in the DPPH radical scavenging assay and reducing power ability assay, Trolox was used as the positive control in the ABTS+ radical scavenging. Values are presented as means ± SD (*n* = 3–5). abc Values are significantly different from the other groups as determined by Duncan's test (*p* < 0.05). Black bean steamed liquid lyophilized product (BBSLP). **Figure 1.** In vitro antioxidative ability of the BBSLP. DPPH radical scavenging activity (**A**), ABTS<sup>+</sup> radical scavenging activity (**B**) and reducing power (**C**). Vitamin C was used as the positive control in the DPPH radical scavenging assay and reducing power ability assay, Trolox was used as the positive control in the ABTS<sup>+</sup> radical scavenging. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 3–5). abc Values are significantly different from the other groups as determined by Duncan's test (*p* < 0.05). Black bean steamed liquid lyophilized product (BBSLP).

*2.3. BBSLP Maintained the Viability of RAW264.7 Macrophages after Lipopolysaccharide (LPS) Induction*  Our preliminary experimental results showed that the viability of RAW264.7 macrophages treated with 0.1 to 5 μg/mL BBSLP was not significantly different compared with that of the control cells (data not shown). For reasons related to BBSLP yield and solubility, we used 0.1, 0.5 and 1 μg/mL BBSLP as the experimental doses in the following study. In the present study, the cell viability of RAW264.7 macrophages did not significantly differ between the 0.1, 0.5 or 1 μg/mL BBSLP (approximately 97–101%) under LPS induction or the control group (100%) (Figure 2A). Based on morphological examination results The EC<sup>50</sup> value of BBSLP for DPPH radical-scavenging ability was 0.81 mg/mL. Figure 1B shows that the Trolox-treated group demonstrated 95.5 ± 4.3% 2,2<sup>0</sup> -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS<sup>+</sup> ) radical scavenging. The ABTS<sup>+</sup> radical scavenging abilities in the 0.5, 1 and 2 mg/mL BBSL groups were 7.8 ± 2.3%, 20.5 ± 6.3% and 50.2 ± 8.3%, respectively, and showed a dose-dependent increase (*p* < 0.05). The effect concentration (EC50) value of BBSLP for ABTS<sup>+</sup> radical scavenging ability was 1.50 mg/mL. In addition, the reducing power of 0.5, 1 and 2 mg/mL BBSLP reached 42.5 ± 84.6% in a dose-dependent manner (*p* < 0.05). The vitamin C-treated group showed 88.7 ± 9.6% reducing power (Figure 1C). The EC<sup>50</sup> value of BBSLP for reducing power was 0.80 mg/mL.

### cantly differ between any BBSLP-treated group under LPS induction and the control group. Under LPS induction, RAW264.7 macrophage treatment with 0.1, 0.5, or 1 μg/mL *2.3. BBSLP Maintained the Viability of RAW264.7 Macrophages after Lipopolysaccharide (LPS) Induction*

BBSLP did not affect viability. Our preliminary experimental results showed that the viability of RAW264.7 macrophages treated with 0.1 to 5 µg/mL BBSLP was not significantly different compared with that of the control cells (data not shown). For reasons related to BBSLP yield and solubility, we used 0.1, 0.5 and 1 µg/mL BBSLP as the experimental doses in the following study.

using inverted microscopy (Figure 2B), the cell number and morphology did not signifi-

In the present study, the cell viability of RAW264.7 macrophages did not significantly differ between the 0.1, 0.5 or 1 µg/mL BBSLP (approximately 97–101%) under LPS induction or the control group (100%) (Figure 2A). Based on morphological examination results using inverted microscopy (Figure 2B), the cell number and morphology did not significantly differ between any BBSLP-treated group under LPS induction and the control group. Under LPS induction, RAW264.7 macrophage treatment with 0.1, 0.5, or 1 µg/mL BBSLP did not affect viability.

**Figure 2.** Effects of BBSLP on the viability of LPS-induced RAW264.7 cells. RAW264.7 cells (1 × 105 cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS for another 24 h. The group treated with 1 μg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 μg/mL BBSLP without LPS treatment for 48 h were used as another control group. Cell viability (**A**) and morphological changes (**B**) were examined. Values are presented as means ± SD (*n* = 3–5). abc Values are significantly different from the other groups as **Figure 2.** Effects of BBSLP on the viability of LPS-induced RAW264.7 cells. RAW264.7 cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 µg/mL BBSLP for 24 h and then induced or not with 1 µg/mL LPS for another 24 h. The group treated with 1 µg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 µg/mL BBSLP without LPS treatment for 48 h were used as another control group. Cell viability (**A**) and morphological changes (**B**) were examined. Values are presented as means ± SD (*n* = 3–5). **Figure 2.** Effects of BBSLP on the viability of LPS-induced RAW264.7 cells. RAW264.7 cells (1 × 105 cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS for another 24 h. The group treated with 1 μg/mL LPS alone served as an induced control group. BBSLP was diluted in

#### determined by Tukey's test (*p* < 0.05). *2.4. BBSLP Reduced Oxidative Stress in RAW264.7 Macrophages after LPS Induction* without LPS treatment for 48 h were used as another control group. Cell viability (**A**) and morphological changes (**B**) were examined. Values are presented as means ± SD (*n* = 3–5). abc Values are significantly different from the other groups as

sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 μg/mL BBSLP

*2.4. BBSLP Reduced Oxidative Stress in RAW264.7 Macrophages after LPS Induction*  When RAW264.7 macrophages were treated with LPS alone, the thiobarbituric acid reactive substances (TBARS) level was significantly increased by 229% (*p* < 0.05) (Figure 3A). However, when RAW264.7 macrophages were treated with 0.1, 0.5 or 1 μg/mL BBSLP and then stimulated with LPS, the TBARS levels were significantly decreased by 38 to 70% compared with those in the LPS alone-treated group (*p* < 0.05) (Figure 3A). The TBARS levels in the group treated with only 5 μg/mL BBSLP did not differ from those in the control group. The ROS levels (100%) of RAW264.7 macrophages treated with only LPS were significantly higher than those in control cells (23.1 ± 5.3%) (*p* < 0.05) (Figure 3B). However, the ROS levels in RAW264.7 macrophages did significantly decrease in the groups treated When RAW264.7 macrophages were treated with LPS alone, the thiobarbituric acid reactive substances (TBARS) level was significantly increased by 229% (*p* < 0.05) (Figure 3A). However, when RAW264.7 macrophages were treated with 0.1, 0.5 or 1 µg/mL BBSLP and then stimulated with LPS, the TBARS levels were significantly decreased by 38 to 70% compared with those in the LPS alone-treated group (*p* < 0.05) (Figure 3A). The TBARS levels in the group treated with only 5 µg/mL BBSLP did not differ from those in the control group. The ROS levels (100%) of RAW264.7 macrophages treated with only LPS were significantly higher than those in control cells (23.1 ± 5.3%) (*p* < 0.05) (Figure 3B). However, the ROS levels in RAW264.7 macrophages did significantly decrease in the groups treated with 0.1, 0.5 or 1 µg/mL BBSLP after stimulation with LPS (approximately 21.2–66.5%) (*p* < 0.05). determined by Tukey's test (*p* < 0.05). *2.4. BBSLP Reduced Oxidative Stress in RAW264.7 Macrophages after LPS Induction*  When RAW264.7 macrophages were treated with LPS alone, the thiobarbituric acid reactive substances (TBARS) level was significantly increased by 229% (*p* < 0.05) (Figure 3A). However, when RAW264.7 macrophages were treated with 0.1, 0.5 or 1 μg/mL BBSLP and then stimulated with LPS, the TBARS levels were significantly decreased by 38 to 70% compared with those in the LPS alone-treated group (*p* < 0.05) (Figure 3A). The TBARS levels in the group treated with only 5 μg/mL BBSLP did not differ from those in the control group. The ROS levels (100%) of RAW264.7 macrophages treated with only LPS were significantly higher than those in control cells (23.1 ± 5.3%) (*p* < 0.05) (Figure 3B). However, the ROS levels in RAW264.7 macrophages did significantly decrease in the groups treated with 0.1, 0.5 or 1 μg/mL BBSLP after stimulation with LPS (approximately 21.2–66.5%) (*p*  < 0.05).

**Figure 3.** Effects of BBSLP on LPS-induced RAW264.7 cell oxidative stress. RAW264.7 cells (1 × 105 cells/30-mm plate) were **Figure 3.** Effects of BBSLP on LPS-induced RAW264.7 cell oxidative stress. RAW264.7 cells (1 × 105 cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS for another 24 h. The group treated with 1 μg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 μg/mL BBSLP **Figure 3.** Effects of BBSLP on LPS-induced RAW264.7 cell oxidative stress. RAW264.7 cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 µg/mL BBSLP for 24 h and then induced or not with 1 µg/mL LPS for another 24 h. The group treated with 1 µg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 µg/mL BBSLP without LPS treatment for 48 h were used as another control group. TBARS levels (**A**) and ROS levels (**B**) were examined. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 3–5). abc Values are significantly different from the other groups as determined by Tukey's test (*p* < 0.05).

seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS

Tukey's test (*p* < 0.05).

### *2.5. BBSLP Reduced NO and PGE<sup>2</sup> Production in RAW264.7 Macrophages after LPS Induction 2.5. BBSLP Reduced NO and PGE2 Production in RAW264.7 Macrophages after LPS Induction*

*Plants* **2021**, *10*, x FOR PEER REVIEW 5 of 14

Values are presented as means ± SD (*n* = 3–5). abc Values are significantly different from the other groups as determined by

NO and PGE<sup>2</sup> production was significantly increased after RAW264.7 macrophages were induced with LPS compared with the control group (*p* < 0.05, Figure 4A,B). However, when RAW264.7 macrophages were cotreated with 1 µg/mL BBSLP and LPS, NO levels were decreased by 15% compared with those in the LPS group (*p* < 0.05). A 60–68% decrease in PGE<sup>2</sup> was noted in cells cotreated with 0.1 to 1 µg/mL BBSLP and LPS (*p* < 0.05). Immunoblot analysis showed that the iNOS levels in RAW264.7 macrophages treated with 0.1, 0.5 and 1 µg/mL BBSLP were 82%, 77% and 69% that of cells treated with LPS alone, respectively (*p* < 0.05, Figure 4C,D). When RAW264.7 macrophages were treated with 0.1, 0.5 and 1 µg/mL BBSLP, the COX-2 protein levels were significantly reduced by 84%, 84% and 82%, respectively, compared with those in the LPS-treated group (*p* < 0.05, Figure 4C,D). NO and PGE2 production was significantly increased after RAW264.7 macrophages were induced with LPS compared with the control group (*p* < 0.05, Figure 4A,B). However, when RAW264.7 macrophages were cotreated with 1 μg/mL BBSLP and LPS, NO levels were decreased by 15% compared with those in the LPS group (*p* < 0.05). A 60–68% decrease in PGE2 was noted in cells cotreated with 0.1 to 1 μg/mL BBSLP and LPS (*p* < 0.05). Immunoblot analysis showed that the iNOS levels in RAW264.7 macrophages treated with 0.1, 0.5 and 1 μg/mL BBSLP were 82%, 77% and 69% that of cells treated with LPS alone, respectively (*p* < 0.05, Figure 4C,D). When RAW264.7 macrophages were treated with 0.1, 0.5 and 1 μg/mL BBSLP, the COX-2 protein levels were significantly reduced by 84%, 84% and 82%, respectively, compared with those in the LPS-treated group (*p* < 0.05, Figure 4C,D).

**Figure 4.** Effects of BBSLP on NO, PGE2, iNOS and COX-2 levels in RAW264.7 cells induced by LPS. RAW264.7 cells (1 × 105 cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS for another 24 h. The group treated with 1 μg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 μg/mL BBSLP without LPS treatment for 48 h were used as another control group. NO (**A**), PGE2 (**B**), iNOS and COX-2 (**C**) protein expression and quantified iNOS and COX-2 levels (**D**) were examined. Values are presented as means ± SD (*n* = 3–5). abc Values are significantly different from the other groups as determined by Tukey's test (*p* < 0.05). **Figure 4.** Effects of BBSLP on NO, PGE<sup>2</sup> , iNOS and COX-2 levels in RAW264.7 cells induced by LPS. RAW264.7 cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 µg/mL BBSLP for 24 h and then induced or not with 1 µg/mL LPS for another 24 h. The group treated with 1 µg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 µg/mL BBSLP without LPS treatment for 48 h were used as another control group. NO (**A**), PGE<sup>2</sup> (**B**), iNOS and COX-2 (**C**) protein expression and quantified iNOS and COX-2 levels (**D**) were examined. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 3–5). abc Values are significantly different from the other groups as determined by Tukey's test (*p* < 0.05).

#### *2.6. BBSLP Decreased IL-1β, IL-6 and TNF-α Levels in RAW264.7 Macrophages after LPS Induction 2.6. BBSLP Decreased IL-1β, IL-6 and TNF-α Levels in RAW264.7 Macrophages after LPS Induction*

Figure 5A shows that the IL-1β level was significantly increased after an inflammatory response was induced in RAW264.7 macrophages by LPS compared with the control group (*p* < 0.05); however, when RAW264.7 macrophages were treated with 0.5 or 1 μg/mL BBSLP combined with LPS, the IL-1β levels were 59.9 ± 8.7% and 32.0 ± 8.7%, they were significantly lower than the LPS induction alone group (100%, *p* < 0.05, Figure 5A). The IL-6 levels in RAW264.7 cells decreased significantly by 7% after treatment with 1 μg/mL BBSLP compared with those after LPS induction alone (*p* < 0.05, Figure 5B). Figure 5C also Figure 5A shows that the IL-1β level was significantly increased after an inflammatory response was induced in RAW264.7 macrophages by LPS compared with the control group (*p* < 0.05); however, when RAW264.7 macrophages were treated with 0.5 or 1 µg/mL BBSLP combined with LPS, the IL-1β levels were 59.9 ± 8.7% and 32.0 ± 8.7%, they were significantly lower than the LPS induction alone group (100%, *p* < 0.05, Figure 5A). The IL-6 levels in RAW264.7 cells decreased significantly by 7% after treatment with 1 µg/mL BBSLP compared with those after LPS induction alone (*p* < 0.05, Figure 5B). Figure 5C also shows that the TNF-α level in the group treated with only 1 µg/mL BBSLP decreased significantly compared with the LPS alone group (*p* < 0.05, Figure 5B). Notably, IL-10 production did not differ among the control group, LPS-treated group, the group treated

with various concentrations of BBSLP combined with LPS, and the group treated with BBSLP alone (Figure 5D). alone (Figure 5D).

shows that the TNF-α level in the group treated with only 1 μg/mL BBSLP decreased significantly compared with the LPS alone group (*p* < 0.05, Figure 5B). Notably, IL-10 production did not differ among the control group, LPS-treated group, the group treated with various concentrations of BBSLP combined with LPS, and the group treated with BBSLP

*Plants* **2021**, *10*, x FOR PEER REVIEW 6 of 14

**Figure 5.** Effects of BBSLP on the inflammatory response in RAW264.7 cells induced by LPS. RAW264.7 cells (1 × 105 cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS for another 24 h. The group treated with 1 μg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 μg/mL BBSLP without LPS treatment for 48 h were used as another control group. Levels of NOIL-1β (**A**), IL-6 (**B**), IL-10 (**C**), and TNF-α (**D**) were examined. Values are presented as means ± SD (*n* = 3–5). abcValues are significantly different from the other groups as determined by Tukey's test (*p* < 0.05). **Figure 5.** Effects of BBSLP on the inflammatory response in RAW264.7 cells induced by LPS. RAW264.7 cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 µg/mL BBSLP for 24 h and then induced or not with 1 µg/mL LPS for another 24 h. The group treated with 1 µg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 µg/mL BBSLP without LPS treatment for 48 h were used as another control group. Levels of NOIL-1β (**A**), IL-6 (**B**), IL-10 (**C**), and TNF-α (**D**) were examined. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 3–5). abc Values are significantly different from the other groups as determined by Tukey's test (*p* < 0.05).

### *2.7. BBSLP Reduced the Activation of NF-kB Signalling in RAW264.7 Cells after LPS Induction*

*2.7. BBSLP Reduced the Activation of NF-ĸB Signalling in RAW264.7 Cells after LPS Induction*  Figures 6A,B show that IκB phosphorylation was significantly reduced by 20–50% after 0, 1, 0.5 or 1 μg/mL BBSLP treatment in RAW264.7 cells induced with LPS (*p* < 0.05). However, BBSLP did not affect the protein contents of cytosolic I-κB in EA.hy926 cells (Figures 6A,B). The nuclear NF-κB levels were significantly decreased by 24% and 16%, respectively, after 0.5 or 1 μg/mL BBELP treatment in RAW264.7 cells induced by LPS (*p*  < 0.05, Figure 6A,B). The DNA-binding activity of nuclear NF-κB was significantly sup-Figure 6A,B show that IκB phosphorylation was significantly reduced by 20–50% after 0, 1, 0.5 or 1 µg/mL BBSLP treatment in RAW264.7 cells induced with LPS (*p* < 0.05). However, BBSLP did not affect the protein contents of cytosolic I-κB in EA.hy926 cells (Figure 6A,B). The nuclear NF-κB levels were significantly decreased by 24% and 16%, respectively, after 0.5 or 1 µg/mL BBELP treatment in RAW264.7 cells induced by LPS (*p* < 0.05, Figure 6A,B). The DNA-binding activity of nuclear NF-κB was significantly suppressed by 49% in cells treated with 100 µg/mL BBSLP (Figure 6C).

pressed by 49% in cells treated with 100 μg/mL BBSLP (Figure 6C).

**Figure 6.** Effects of BBSLP on NF-κB signalling activation in LPS-induced RAW264.7 cells. RAW264.7 cells (1 × 105 cells/30 mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 μg/mL BBSLP for 24 h and then induced or not with 1 μg/mL LPS for another 24 h. The group treated with 1 μg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 μg/mL BBSLP without LPS treatment for 48 h were used as another control group. Phosphorylated IκB (p-IκB), IκB and nuclear p65 expression (**A**), quantified p-IκB, IκB and nuclear p65 levels (**B**) and NF-κB-DNA binding activity (**C**) were examined. Values are presented as means ± SD (*n* = 3–5). abc Values are significantly different from the other groups as determined by Tukey's test (*p* < 0.05). **Figure 6.** Effects of BBSLP on NF-κB signalling activation in LPS-induced RAW264.7 cells. RAW264.7 cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/30-mm plate) were seeded and cultured overnight, treated with 0.1, 0.5 or 1 µg/mL BBSLP for 24 h and then induced or not with 1 µg/mL LPS for another 24 h. The group treated with 1 µg/mL LPS alone served as an induced control group. BBSLP was diluted in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. Cells treated with 1 µg/mL BBSLP without LPS treatment for 48 h were used as another control group. Phosphorylated IκB (p-IκB), IκB and nuclear p65 expression (**A**), quantified p-IκB, IκB and nuclear p65 levels (**B**) and NF-κB-DNA binding activity (**C**) were examined. Values are presented as means <sup>±</sup> SD (*<sup>n</sup>* = 3–5). abc Values are significantly different from the other groups as determined by Tukey's test (*p* < 0.05).

#### **3. Discussion 3. Discussion**

The present study showed that BBSLP has the ability of free radical scavage and reducing power enhance in vitro. And, the potential antioxidant and anti-inflammatory effects of BBSLP in LPS-induced RAW264.7 macrophages. Because BBSLP significantly reduced oxidative stress, including an ability to decrease levels of free radicals and lipid peroxidation, and reduced pro-inflammation molecules, including IL-1β, IL-6 and TNF-α levels, in LPS-induced RAW264.7 cells. The present study showed that BBSLP has the ability of free radical scavage and reducing power enhance in vitro. And, the potential antioxidant and anti-inflammatory effects of BBSLP in LPS-induced RAW264.7 macrophages. Because BBSLP significantly reduced oxidative stress, including an ability to decrease levels of free radicals and lipid peroxidation, and reduced pro-inflammation molecules, including IL-1β, IL-6 and TNF-α levels, in LPS-induced RAW264.7 cells.

Our results found that BBSLP contained flavonoids and polyphenols, which were may be involved in its high antioxidative and anti-inflammatory properties. Glevitzky et al. [24] showed that there are a high intercorrelation between the number of phenolic groups within the basic structure of flavonoids and their antioxidant activity and between the antioxidant activity and the number of -OH phenolic groups also with a high correlation. However, polyphenols or the other components of BBSL and BBSLP whether playing a major or important role in antioxidation or not, are still need furthermore composition analysis and investigation. Inflammatory response mediators, including PGE2 and NO, and inflammatory cytokines, including IL-1β, IL-6, IL-10 and TNF-α, were all regulated, reducing inflammation in LPS-induced RAW264.7 cells. Furthermore, BBSLP could downregulate NF-κB signalling activation, which led to reduced iNOS, COX-2, IL-1β, IL-6, IL-10 and TNF-α transcription. Our results found that BBSLP contained flavonoids and polyphenols, which were may be involved in its high antioxidative and anti-inflammatory properties. Glevitzky et al. [24] showed that there are a high intercorrelation between the number of phenolic groups within the basic structure of flavonoids and their antioxidant activity and between the antioxidant activity and the number of -OH phenolic groups also with a high correlation. However, polyphenols or the other components of BBSL and BBSLP whether playing a major or important role in antioxidation or not, are still need furthermore composition analysis and investigation. Inflammatory response mediators, including PGE<sup>2</sup> and NO, and inflammatory cytokines, including IL-1β, IL-6, IL-10 and TNF-α, were all regulated, reducing inflammation in LPS-induced RAW264.7 cells. Furthermore, BBSLP could downregulate NF-κB signalling activation, which led to reduced iNOS, COX-2, IL-1β, IL-6, IL-10 and TNF-α transcription.

Oxidative stress usually triggers the inflammatory response in various cells and tissues [25]. Macrophages then use ROS production to scavenge xenobiotics, including bacteria and oxidized low-density lipoprotein (ox-LDL) [26]. Long-term inflammation, chronic inflammation and oxidative stress lead to chronic diseases, such as CKD, CVD, DM and sepsis with a poor prognosis. In the present study, BBSLP displayed excellent free radical-scavenging ability, reducing power and SOD activity in an in vitro model and significantly reduced ROS production and TBARS levels in LPS-induced RAW264.7 cells. The above excellent antioxidative effects were from the polyphenols and isoflavones contained in BBSLP. Takahashi et al. [27] showed that the seed coats of black soybeans have a higher total polyphenol content than those of yellow soybeans. Black soybeans may Oxidative stress usually triggers the inflammatory response in various cells and tissues [25]. Macrophages then use ROS production to scavenge xenobiotics, including bacteria and oxidized low-density lipoprotein (ox-LDL) [26]. Long-term inflammation, chronic inflammation and oxidative stress lead to chronic diseases, such as CKD, CVD, DM and sepsis with a poor prognosis. In the present study, BBSLP displayed excellent free radical-scavenging ability, reducing power and SOD activity in an in vitro model and significantly reduced ROS production and TBARS levels in LPS-induced RAW264.7 cells. The above excellent antioxidative effects were from the polyphenols and isoflavones contained in BBSLP. Takahashi et al. [27] showed that the seed coats of black soybeans have a higher total polyphenol content than those of yellow soybeans. Black soybeans may more effectively inhibit LDL oxidation than yellow soybeans because of the higher

total polyphenol contents in their seed coat. Additionally, cyanidin-3-glucoside, petunidin-3-glucoside and peonidin-3-glucoside, three major anthocyanins, have been detected in black soybean seed coats [3]. DPPH radical scavenging, ABTS<sup>+</sup> radical scavenging and ferric reducing antioxidant power (FRAP) analysis results have also shown that the black soybean seed coat is a more efficient reducing agent than dehulled black soybeans and yellow soybean coats [3]. In addition, black soybeans are rich in polyphenols, including isoflavones, anthocyanidins and flavan-3-ols. Moreover, black soybeans can prevent CVD risks by increasing polyphenol concentrations and decreasing oxidative stress in healthy women [28]. Previous studies in different experimental models have shown that polyphenols and isoflavones also have anti-inflammatory characteristics. Takekawa et al. [29] showed that genistein, a soybean polyphenol, can significantly suppress water immersion restraint (WIR) stress-induced gastric mucosal injury. The underlying mechanism involves a significant elevation of SOD activity and significant suppression of both TBARS levels and the production of TNF-α to protect against gastric mucosal injury [29]. Additionally, puerarin, an isoflavonoid extracted from Kudzu roots, reduces malondialdehyde levels, increases SOD activity and alleviates TNF-α, IL-1β and IL-6 protein levels in the hippocampus. Antioxidation and anti-inflammation are induced by the streptozotocin (STZ) group to protect DM rats from cognitive deficits [30].

In addition, resveratrol, a polyphenol constituent of grapes, acts as a COX suppressor, reducing the inflammatory response similarly to a nonsteroidal anti-inflammatory drug (NSAID). A molecular basis for the mutually beneficial relationship between plants and humans has been speculated [31]. Hussain et al. [32] reported the anti-inflammatory and antioxidative properties of polyphenols, the mechanisms by which polyphenols inhibit molecular signalling pathways that have been activated by oxidative stress, and the roles of polyphenols in inflammation-mediated chronic disorders. The above data and previous reports indicate that BBSLP, an extract rich in polyphenols and isoflavones, can significantly reduce oxidative stress, inhibit IL-1β, IL-6 and TNF-α and increase IL-10 as an anti-inflammatory material in RAW264.7 macrophages. BBSLP may be helpful for the development of future antioxidant therapeutics and new anti-inflammatory drugs [33].

In the present study, BBSLP significantly inhibited NF-κB signalling activity by reducing I-κB phosphorylation and NF-κB-DNA binding activity. NF-κB signalling plays an important role in iNOS, COX-2, IL-1β, IL-6 and TNF-α transcription [15,19–22]. Previous studies have shown that one of the major mechanisms of reducing inflammation is to reduce NF-κB signalling activity and the expression of proinflammatory molecules [23]. Bao et al. [34] showed that chlorogenic acid, a major polyphenol compound from coffee, can prevent diabetic nephropathy by inhibiting oxidative stress and inflammation through the reduction of NF-κB signalling activity. Singh et al. [35] also reported that polyphenols have antioxidative and anti-neuroinflammatory properties by regulating NF-κB activation in neurodegenerative diseases.

Currently, the food supply chain is facing substantial pressures, including the availability of fewer natural resources and increased food waste [23]. One important way to increase the food supply and decrease the environmental consequences of current food production is to reduce food waste levels and their economic, environmental and social implications [36]. Previous studies have shown that various by-products from the manufacturing of animals and plants for food contain various fatty acids [37], phytochemicals [38,39], and amino acids [40], all of which are beneficial to the food supply chain and/or can act as health promoters for food sustainability. In the present study, BBSLP showed potential antioxidant and anti-inflammatory effects. In the present study, BBSLP showed preliminary potential on antioxidant and anti-inflammatory effects in vitro. However, the molecular mechanisms of the physiological effects require further study in animal models or human clinical trials. On the other hand, how does BBSLP apply in function foods? Where BBSLP to be developed into a functional food material, a functional assessment of this product is needed. The above questions are an important issue for BBSLP application.

### **4. Materials and Methods**

### *4.1. Materials*

BBSL was a gift from Ta-Tung Soya Sauce Co., Ltd., located in Siluo Town (Yunlin, Taiwan). BBSL is collected from the soybean sauce manufacturing process. After fresh black beans are washed and steamed by 120 ◦C streams in a closed steam tank for 1 h. BBSL were directly collected as experimental materials. LPS was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

### *4.2. Preparation of BBSLP*

According to our previous methods [41], BBSLP was prepared by our laboratory. Fresh BBSL was concentrated in a rotary evaporator (N-1110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and then dried in a freeze dryer (Freezone 4.5, Labconco, Kansas City, MO, USA) at −43 ◦C. The BBSLP was stored at −20 ◦C until use. The percent yield of BBSLP was 1.16% (*w*/*v*).

### *4.3. Determination of the pH Value and Total Flavonoids, Phenols and Protein in BBSL and BBSLP*

The pH values of fresh BBSL were measured using a pH metre (MP220 pH meter, Mettler Toledo, Greifensee, Switzerland). The total phenol contents were analysed using a colorimetric method according to Padmavati et al. [42]. One hundred microlitres of 1 N Folin-Ciocalteu reagent (Sigma-Aldrich Co.) was added to 100 µL of diluted BBSL or BBSLP (dissolved in reverse-osmosis (RO) H2O). Then, 500 µL of 7.5% Na2CO<sup>3</sup> solution was added to react for 30 min, and the absorbance of each sample was measured at an optical density (OD) of 760 nm in a Biokinetics microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Calibration curves were constructed using 0, 0.125, 0.25 and 0.5 mg/mL gallic acid (GA). The total phenolic content is represented as mg GAE/mL BBSL or mg GAE/g BBSLP.

The flavonoid content was analysed using the colorimetric method according to Jia et al. [43]. One hundred microlitres of 5% NaNO<sup>3</sup> solution was added to 100 µL of the BBSL or BBSLP solution to react for 5 min. Then, 50 µL of 10% AlCl<sup>3</sup> solution was added. Finally, 600 µL of 4% NaOH solution was added to the mixture for 30 min. The absorbance of the mixture was measured at OD 510 nm on the Biokinetics microplate reader. Calibration curves were constructed with 0, 0.02, 0.06, 0.08 and 0.1 mg/mL rutin (RU) as the standard. The total flavonoid content is represented as mg RUE/mL BBSL or mg RUE/g BBSLP.

Crude protein contents were analysed according to Lowry et al. [44]. Fifty microlitres of each standard of BBSL or BBSLP was added to 50 µL of trichloroacetic acid and standing for reacted for 30 min at room temperature. The mixture was centrifuged at 15,000× *g* for 20 min at 4 ◦C. Then, the supernatant was discarded. The precipitate was dissolved in 1 mL of NaOH and standing for reacted for 30 min at room temperature. Then, 1.0 mL of modified Lowry Reagent (Sigma-Aldrich Co.) was added, then mixing and incubation at room temperature for 10 min. Five hundred µL Prepared 1X Folin-Ciocalteu's phenol reagent (Sigma-Aldrich Co.) was added, and the mixture was standing in a water bath at 37 ◦C for 30 min. After 30 min, the absorbance was measured at 660 nm on the Biokinetics microplate reader.

### *4.4. In Vitro Antioxidant Ability of BBSLP*

In this study, DPPH (Sigma-Aldrich Co.) radical scavenging activity by BBSLP was analysed according to the method of Shimada et al. [45]. For the measure of the DPPH radical-scavenging activity, 1.5 mL of the sample solution with varying BBSLP concentrations (0.5, 1, 2 and 4 mg/mL) were added 1.5 mL of 0.15 mM DPPH in 50% ethanol. The mixture was mixed and incubated at room temperature in the dark for 30 min. The optical density at 517 nm was measured using the Biokinetics microplate reader. In this test, 1 mg/mL vitamin C was used as a control. The scavenging activity was calculated as (1 − ABBSLP or Avitamin C/Ablank) × 100.

According to the method of Shimada et al. [45] to analyse the reducing power activity of BBSLP in vitro. Here, 0.5 mL of the 0.5, 1, 2 and 4 mg/mL BBSLP, respectively were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide, then the mixture was incubated at 50 ◦C for 20 min. A 2.5 mL aliquot of 10% trichloroacetic acid was added to the mixture, and the mixture was then centrifuged at 3000× *g* for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 2.5 mL of 0.1% ferric chloride, and the absorbance at 700 nm was read using the microplate reader. The reducing power was calculated as (ABBSLP or Avitamin C − Ablank)/Avitamin C × 100. A vitamin C (1 mg/mL) was used as a control.

The ABTS<sup>+</sup> radical scavenging ability of 0.5, 1, 2 or 4 mg/mL BBSLP was analysed according to the method described by Re et al. [46]. Use a 10 µL of the sample solution with varying BBSLP concentrations (0.5, 1, 2 and 4 mg/mL) were added 990 µL of 2 mM 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•<sup>+</sup> ) solution. The mixture was mixed and incubated at room temperature in the dark for 10 min. The optical density at 737 nm was measured using the microplate reader. The ABTS<sup>+</sup> radicalscavenging ability was calculated as (Ablank or ABBSLP − ATrolox)/Ablank × 100. In this test, 1 mg/mL Trolox was used as a control.

### *4.5. Cell Culture and Treatment*

RAW264.7 macrophages and mouse monocyte macrophages were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Dulbecco's modified Eagle's medium containing 42 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 10% (*v/v*) heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used as the culture medium. All cultured cells were incubated in an atmosphere of 5% CO2/95% air at 37 ◦C.

In this study, 1 <sup>×</sup> <sup>10</sup><sup>5</sup> RAW264.7 macrophages per 30 mm plate or 1 <sup>×</sup> <sup>10</sup><sup>6</sup> per 60 mm plate were cultured for various biochemical tests. RAW264.7 macrophages were incubated with 0.5, 1 or 5 µg/mL BBSLP for 24 h and then induced with 1 µg/mL LPS (Sigma-Aldrich Co.) for another 24 h. LPS was used to induce inflammation [47]. The induced control group was treated with 1 µg/mL LPS alone. BBSLP was soluble in sterilized H2O, and cells treated with sterilized H2O alone served as the control group. BBSLP (1 µg/mL) without LPS treatment for 48 h made up another control group.

### *4.6. Cell Viability Analysis*

To determine the optimum test concentration of BBSLP for use in this study, the cell viability of RAW264.7 macrophages was analysed according to the method of Denizot and Lang [48]. After RAW264.7 macrophages were incubated in DMEM containing 0.5 mg/mL thiazolyl blue formazan (MTT; Sigma-Aldrich Co.) for an additional 3 h, the medium was removed and extracted with isopropanol for 15 min. The isopropanol fraction was measured with the Biokinetics microplate reader at OD 570 nm. To evaluate morphological changes, a phase-contrast inverted fluorescence microscope (Olympus IX51, Olympus, Tokyo, Japan) was used.

### *4.7. Measurement of Lipid Peroxidation and ROS Levels*

The effect of BBSLP on lipid peroxidation in RAW264.7 macrophages induced by LPS was determined according to the method of Fraga et al. [49]. The lipid peroxidation indicator thiobarbituric acid reactive substances (TBARS) was extracted and measured with a fluorescence microplate reader (excitation wavelength 515 nm and emission wavelength 555 nm, Bio-Tek Instruments, Winooski, VT, USA). The protein levels were determined according to the method described by Lowry et al. [44]. The TBARS level is shown in nmol TBARs/mg protein. The levels of ROS in RAW364.7 cells were determined using a Cellular ROS Assay Kit (ab113851, Abcam Inc., Cambridge, MA, USA).
