Sulforaphane (SFN) was generated by the hydrolysis of glucoraphanin under the action of myrosinase. However, due to the instability of SFN, the bioavailability of SFN was limited. Meanwhile, the gut flora obtained the ability to synthesize myrosinase and glucoraphanin, which could be converted into SFN in the intestine. However, the ability of microorganisms to synthesize myrosinase in the gut was limited. Therefore, microorganisms with myrosinase synthesis ability need to be supplemented. With the development of research, microorganisms with high levels of myrosinase synthesis could be obtained by artificial selection and gene modification. Researchers found the SFN production rate of the transformed microorganisms could be significantly improved. However, despite applying transformation technology and regulating nutrients to microorganisms, it still could not provide the best efficiency during generating SFN and could not accomplish colonization in the intestine. Due to the great effect of microencapsulation on improving the colonization ability of microorganisms, microencapsulation is currently an important way to deliver microorganisms into the gut. This article mainly analyzed the possibility of obtaining SFN-producing microorganisms through gene modification and delivering them to the gut via microencapsulation to improve the utilization rate of SFN. It could provide a theoretical basis for expanding the application scope of SFN.
1. Introduction
Sulforaphane (1-isothiocyanate-4-(methylsulfonyl) butane, SFN) is produced by the degradation of glucoraphanin under the action of myrosinase (β-D-thioglucosidase)
[1,2,3][1][2][3]. SFN and its precursor (glucoraphanin) are important safeguard substances used by plants to defend themselves against insects, pathogens, and herbivores
[4]. Recently, researchers have found that SFN possesses antioxidant, anti-inflammatory, and antibacterial functions
[5,6][5][6]. In addition, SFN could prevent and cure cancer
[7[7][8][9],
8,9], improve the nervous system
[10[10][11][12],
11,12], and prevent coronavirus disease 2019
[13]. Therefore, SFN has attracted extensive attention from researchers around the world.
Although SFN has shown great positive effects on human health, it is an unstable compound. Therefore, it is difficult to obtain SFN directly from cruciferous plant tissues owing to its structural instability
[14,15,16,17][14][15][16][17]. It has always been a research topic of interest to maintain the function of SFN stably in the human body. Exogenous hydrolysis and transformation research of glucoraphanin have suggested that SFN could be obtained outside plant tissues with high biological activity. Meanwhile, the key controlling factors for the exogenous transformation are glucoraphanin concentration and the activity of myrosinase
[18,19][18][19]. Triska et al. found that SFN formation was controlled by a temperature-specific epithiospecifier protein (a myrosinase cofactor). The most suitable exogenous transformation condition is to ensure sufficient radish sprout content to continuously add myrosinase and to maintain a stable transformation temperature during the process
[20].
However, external factors can also affect myrosinase synthesis during the cultivation of cruciferous plants. Polyethylene glycol can enhance the expression of the myrosinase gene, which can increase myrosinase synthesis
[21]. Lower planting temperatures can also result in higher activity of the myrosinase obtained from cruciferous plants and increase the ability of myrosinase to convert glucoraphanin into SFN
[22]. Although the production of plant-derived SFN is increased through exogenous environmental regulation, it is difficult for the human body to take up enough SFN from plant tissues due to the instability of the SFN structure
[23]. Therefore, determining how to enhance the absorption rate of SFN in intestinal tissues has attracted the attention of researchers.
Studies show that the gut microbiome not only helps digest food ingested into the gastrointestinal tract, but also converts dietary pairs into more active products
[24,25,26][24][25][26]. Lai et al. showed that after feeding F344 rats with cooked
broccoli (without myrosinase), the microbes in the gut of the rats converted glucoraphanin into SFN, and SFN was detected in the blood
[27]. However, studies on humans show that there are individual differences in the ability of intestinal flora to produce SFN
[28,29][28][29].
Meanwhile, studies have reported that in the microbial conversion of glucoraphanin, probiotics, which are a class of active microorganisms that colonize the human intestinal environment, could improve the composition of human intestinal flora and enhance intestinal digestion
[30,31][30][31]. Probiotics have also shown preventive and therapeutic effects on colorectal cancer, mental disorders, diabetes, and other diseases
[32,33][32][33]. Therefore, probiotics are widely used in health products, dietary supplements, and prebiotics
[34,35,36][34][35][36]. In contrast, studies have shown that lactic acid bacteria (
LAB) can convert glucoraphanin from broccoli into SFN under suitable conditions, and high SFN concentrations were found in fermented broccoli puree
[37,38][37][38]. The
LAB fermentation results also indicated that, in addition to the mammalian gut flora, microorganisms from other sources could also degrade glucoraphanin and produce SFN.
Although microorganisms can convert glucoraphanin into SFN in the gut, the production of SFN is limited due to the insufficient number of microorganisms that can synthesize myrosinase in the intestine
[4]. With the discovery of exogenous myrosinase, and the ability of
LAB to ferment
broccoli puree to produce high concentrations of SFN, researchers attempted to replicate this process in vivo
[37,38][37][38].
2. Intestinal Microorganisms Can Enhance the Utilization of SFN
2.1. Structure and Biochemical Characteristics of SFN, Glucoraphanin, and Myrosinase
SFN (1-isothiocyanate-4-(methylsulfonyl) is an isothiocyanates (a general formula R-N=C=S). Due to the presence of an active electrophilic carbon atom in the SFN group (-N=C=S), SFN is easily reversible with thiols under physiological conditions, resulting in pH-sensitive dithiocarbamates reacting with amines and forming thiourea
[1,39,40][1][39][40]. This is the reason why SFN is sensitive to temperature and other conditions. SFN is converted by its precursor glucoraphanin under the enzymatic hydrolysis reaction of myrosinase. Meanwhile, glucoraphanin (4-methylsulfinyl butyl glucosinolate) is a methionine-derived aliphatic glucosinolate. Glucoraphanin is widely found in cruciferous plants, especially
broccoli. Glucoraphanin is a water-soluble sulfur-containing anionic secondary metabolite consisting of a β-glucosinolate N-hydroxysulfate with a side chain center and a β-D-glucopyranose residue
[41,42,43][41][42][43]. Glucoraphanin demonstrates no physiological activity. Therefore, the best form of utilization for SFN is to preserve.
Myrosinase, also known as β-glucosidase, is a ubiquitous enzyme in cruciferous plants that can efficiently degrade glucosinolates. The essence of myrosinase is a glycoprotein, and currently found in plants, aphids, and other myrosinase of the glycoside hydrolase family 1
[44,45][44][45]. The differences in myrosinase from different sources are mainly reflected in molecular weight, subunit number, and side chain sugar content, which leads to the different ability of myrosinase to degrade glucoraphanin.
2.2. Increasing the Intestinal SFN Production Rate Is a Scientific Approach to Enhance SFN Utilization
Although SFN has strong anticancer properties, it is very unstable and loses its biological activity under certain conditions, such as the presence of oxygen, which could reduce the utilization of SFN
[46,47,48][46][47][48]. To improve SFN utilization, researchers considered the possibility of directly producing SFN in vivo and conducted in vitro simulation studies
[49]. Xu et al. found that glucoraphanin could be converted into SFN under a simulated gastrointestinal environment in vitro, and the maximum conversion rate could reach 46.2%. Moreover, when glucoraphanin was directly fed to germ-free and human-microbiota-associated mice, SFN degradation products were found in the urine of the mice, which indicated that SFN could be produced in the intestinal environment and that SFN could be utilized
[50].
Lai et al. used male F344 rats as an animal model and demonstrated that the cecum can also degrade glucoraphanin and produce SFN
[27]. Once glucoraphanin was directly gavaged into male F344 rats, SFN was detected in the plasma of the rats after 120 min, and the level of SFN in the plasma remained constant for 1 h. In addition, a study indicated that the intestinal environment not only is a limiting factor for SFN utilization, but also has a promoting effect on SFN utilization
[29]. However, humans can consume glucoraphanin-rich
Brassica vegetables directly instead of consuming some glucoraphanin. Raw
broccoli is used for studies on humans, and the results indicate that glucosinolates in
broccoli are degraded in the human body and the degradation products of glucosinolates are detected in the blood and urine of volunteers
[51]. Therefore, the studies on humans suggest that SFN can be produced in the human intestinal environment.
Meanwhile, the bioavailability of SFN in raw
broccoli could reach 37%, which is significantly higher than that in cooked
broccoli, and the consumption of cooked
broccoli would delay the absorption of SFN
[52]. Egner et al. found that the bioavailability of SFN was far superior to glucoraphanin in the human body
[53]. Another study showed that the main reason for the higher bioavailability of SFN than sulforaphane is that glucoraphanin must be hydrolyzed to be absorbed
[54].
However, because humans consume more cooked than raw vegetables, researchers have used cooked white
cabbage to simulate the in vivo degradation of glucoraphanin in a rat duodenal model
[55]. The results show that 82% of glucoraphanin is released from white
cabbage seeds after 10 min, but no degradation of glucoraphanin is detected. However, the in vitro simulation results for the rat duodenum are different from the results from the study on male F344 rats. Therefore, Wu et al. explored the possibility of SFN production by the gut microbiota
[28,56][28][56]. Research using a male C57BL/6 mouse shows that glucoraphanin could be degraded to SFN in the intestine, and the production of SFN is related to the intestinal flora. Hwang et al. show that 13 pmol/g fresh weight of SFN is produced in the gut after 120 min, and approximately 29% of the SFN is taken up and utilized by cells, indicating that the intestinal environment has a promoting effect on the production and utilization of SFN
[57].
Meanwhile, Sangkret et al. found that the main elements affecting SFN production were myrosinase activity, temperature, pH, and reaction time
[14]. Recently, researchers obtained a new myrosinase-producing bacterium from marine sediment (Marine Bacterium
Shewanella baltica Myr-37)
[58]. Once the reaction temperature is 40 °C and pH = 7.0, myrosinase can efficiently degrade sulforaphane to SFN in 25 min, the yield is 0.57 mg/mL, and the corresponding SFN conversion efficiency is 89%. However, intestinal myrosinase is also affected by epithiospecifier protein (EP) and sulfur–selenium interaction(S–Se) in the process of SFN formation
[48,59,60][48][59][60]. EP interferes with the production of SFN, while S–Se induces the expression of myrosinase gene to produce more myrosinase.
The intestinal environment can degrade glucoraphanin to produce SFN without the action of plant-derived myrosinase, and the main factor for SFN production is the effect of the intestinal flora. However, the mechanisms by which the gut microbiota degrades glucoraphanin to produce SFN, and the gut microbes involved, are not clear. Therefore, researchers investigated the mechanisms of the microbial transformation of glucoraphanin.
2.3. Microorganisms Converted Glucoraphanin into SFN Using Myrosinase Synthesis
Myrosinase is a beta-thioglucosidase glucohydrolase that was originally discovered in cruciferous plants; it can resist in vitro damage and degrade glucoraphanin
[61,62,63][61][62][63]. Studies have found that some microorganisms can also synthesize myrosinase (
Table 1)
[64,65,66][64][65][66]. Naoki Tani et al. first isolated a species of
Enterobacter cloacae, which could synthesize myrosinase, but its molecular weight was smaller than the endogenous myrosinase of the plant. Meanwhile,
Bacteroides thetaiotaomicron (another dominant species derived from the human colon) could convert glucosinolates into allyl isothiocya-nate
[67]. With the further development of research on SFN production by intestinal flora, it was discovered that a variety of intestinal strains can degrade glucoraphanin to produce SFN
[68].
Table 1.
Microorganisms with myrosinase synthesis function.