Production of α-glucosidase Inhibitor in the Intestines by Bacillus Licheniformis

Alpha-glucosidase (EC.3.2.1.20) is involved in the absorption of monosaccharides in the small intestine of animals. We aimed to nd a microorganism capable of proliferating in the intestine and producing α-glucosidase inhibitor. We developed a strain capable of forming spores from dry grass and growing in an anaerobic environment was selected as Bacillus lichenformis. Mixing spores of this strain with a high-fat diet and high-carbohydrate diet, it was conrmed that the weight gain was signicantly reduced than the high-calorie diet group without spores. Furthermore it was conrmed that Bacillus lichenformis administered as spores eciently proliferated in the intestine and consistently produced α-glucosidase inhibitor by securing a constant amount of the strain and α-glucosidase inhibitor in feces after a certain period. This study shows an ecient process in which microorganisms capable of proliferating in the intestine directly produce and supply specic secondary metabolites in the intestine.

The AGI originating from aerobic microorganisms such as Streptomyces and Bacillus can be obtained through culture. It has the advantage of producing AGI in large quantities in a short period through the breeding of high-producing strains and optimization of the culture process (Zhang et al. 2019;Lee et al. 2018). However, the AGI of microbial origin is di cult to use into originals cause various factors such as fermentation odor and concentration of effective substances, there is a need to undergo puri cation (Zhu et al. 2013;Zhu et al. 2008).
The intestinal microbiota such as lactic acid microorganism proliferate in the intestine and exhibit various physiological effects that control the in ammatory response of the host or produce useful substances such as secondary metabolites and also generate energy for use by the host through metabolic processes (Wang et al. 2019;Riedl et al. 2017;Nieuwdorp et al. 2014;Sweeney & Morton 2013). This means that if microorganisms that are capable of growing in the anaerobic part of intestine produce large amounts of secondary metabolites, the possibility of using the intestine as a production site and using space for speci c secondary metabolites is high.
Several speci c microorganisms, such as Bacillus and Streptomyces are known to produce large amounts of AGI in an aerobic environment (Onose et al. 2013;Paek et al. 1997;Hardick et al. 1992), however they are di cult to proliferate in the intestine which is an anaerobic environment, they are di cult to supply AGI in the intestine.
In this study, a new strain that is capable to proliferate in the intestine for intestinal production of useful substances and capable of producing large amounts of AGI in an anaerobic environment such as the intestine was explored, a new strain was examined that is capable of intestinal proliferation, production of AGI in the intestine and weight loss effect accordingly.

Results And Discussion
Screening strain About 400 pieces of rice straw and hay collected from all over the world such as Korea, China, Japan and the United States were used as samples to isolate microorganisms. A small amount of sterile saline is added to each sample and suspended, and the spore solution obtained by heat treatment in an 80℃ water bath for 20 minutes is used in an LB plate medium containing 2% agar (1% tryptone, 0.2% sucrose, 0.5% yeast extract and 0.5% NaCl, pH 7.0) and anaerobically cultured in an incubator at 55℃. for two days to isolate colony-forming microorganisms. Single colonies obtained by separating from hay were inoculated in 5 mL of 5% soy our suspension medium and cultured with shaking at 37℃ for 24 hours, and then 60 species with high AGI activity in the supernatant were selected. The selected 60 strains were capable of growing for anaerobic growth at 50℃ and three strains with high AGI activity were selected as strains that were capable of using propionic acid. The selected three strains were identi ed as Bacillus licheniformis as a result of classi cation by taxonomic characteristics ( The microorganism with the highest AGI production capacity was nally selected and to increase the AGI production capacity of the strain after inducing mutation by treatment with NTG (100μg/mL) to reach 99.9% kill rate, spread to 200-300 per sheet on L-broth plate medium and incubate anaerobically for two days at 37℃. The resulting colonies were randomly inoculated in 5 percent soybean our medium and cultured with shaking at 40℃ for two days, and then the centrifuged supernatant was measured for the AGI activity. By repeating the mutation twice in the same way, a strain having high AGI activity was selected and named B. licheniformis NY1505. As a result of analyzing the 16s RNA nucleotide sequence of B. licheniformis NY1505 strain, it showed high homology with B. licheniformis. The dendrogram shows that B. licheniformis NY1505 (the accession number KCTC13021BP) is allied species with the B. licheniformis type strain ( Fig. 1).
Analysis of AGI 5 x 10 5 spores of NY 1505 were inoculated into 500g of steaming soybeans and covered the lm, incubated at 37℃ for 24 hours, added 2.5L of 70% (v/v) ethanol, extract twice, and evaporated under reduced pressure. To 150 mL of the concentrated extract, 300 mL of hexane, dichloromethane, and Ethyl acetate were sequentially added twice, stirred for 2 hours, allowed to stand for 2 hours, and fractionated to wash the aqueous layer. After drying the aqueous layer, 100 mL of 90% ethanol was added to dissolve it, followed by silica gel column (100 mL) chromatography. The mobile phase was stepwise gradient from 1:1 solution of acetonitrile and methanol to 3 : 2 solution (Total 1000 mL). The AGI activity of each fraction was measured to obtain two fractions, AGI 1 and AGI 2. AGI 1 and AGI 2 each appeared as a single spot in thin layer chromatography, and the structure was determined through NMR analysis.
The chemical shift of AGI 1 is as follows. AGI 1 is a triterpene-type substance in which ve rings are connected and has a structure similar to betulinic acid. The chemical structure was found to be 3-oxo-11α-hydroxy-lup-20(29)-en-28-oic acid.
The inhibition pattern of AGI 1 through the Lineweaver-Burk plot was con rmed as non-competitive inhibition (Fig. 2).
AGI 2 was presumed to be 1-deoxynojirimycin (DNJ), and as a result of running NMR, DNJ and NMR results were con rmed to be consistent. The chemical shift of AGI 2 is as follows. AGI 2 showed a tendency to inhibit competitive inhibition (Fig. 3).

Animal experiment
High carbohydrate diet After starting the diet and measuring the weight of each group every week, the lowest and highest values were excluded and statistically processed. In addition, more than 3g of fresh feces were collected for each cage to examine the number of microorganism.
The high carbohydrate diet group showed a weight gain rate about 130% compared to the standard diet group in week 4, but the high carbohydrate diet group with spores of the B. licheniformis NY1505 strain showed the weight gain rate of about 90% compared to the standard diet group ( Fig 4).

High fat diet
The high fat diet group showed a weight gain rate about 150% compared to the standard diet group in week 6, but when the high fat diet group with spores of the B. licheniformis NY1505 strain showed the weight gain rate about 120% compared to the standard diet group ( Fig. 5).
Comparing the results in Fig. 4 and 5 the high fat diet group was more sensitive than the high carbohydrate group to the administration of B. licheniformis NY1505 spores at the week 3 or 4. In particular, it has been reported that when DNJ, a component of AGI 2, is administered for a long period of 12 weeks or longer, it activates β-oxidation, which decomposes fatty acids in mitochondria, and inhibits liver fat formation (Tsuduki et al. 2013;Tsuduki et al. 2009). Therefore, this is expected to be because B. licheniformis NY1505 proliferates in the intestine and produces AGI that activates β-oxidation, which is the catabolic action of fatty acids. It is known that betulinic acid, which has a similar structure to AGI 1, also inhibits adipogenesis by inhibiting differentiation in adipocyte growth ( (Fig. 6). Spores were administered for seven weeks and the number of microorganism rapidly decreased at 8th week when not administered for one week. It can be judged that it inhabits temporarily without adhering to the intestine.

The number of α-glucosidase inhibitor NY1505 microorganism excreted in feces
The B. licheniformis NY1505 strain proliferates vigorously in the intestine and produces AGI. From the 2nd week when the B. licheniformis NY1505 strain which had proliferated in the intestine was being detected in the feces, AGI that produced in the intestine was excreted into the feces equally (Fig. 7). As shown in Fig. 6, the amount of B. licheniformis NY1505 excreted is stabilized, and the amount of AGI excreted is also constant from 4thweek to 7th week. In other words, AGI is always continuously produced in the intestine, its concentration is maintained and a constant amount of the produced AGI is excreted. Considering that the AGI activity of natto produced from the B. licheniformis NY1505 strain is 90-95 units/g (data not shown), it can be seen that a signi cant amount of AGI is produced in the intestine and some of it is excreted.

Conclusions
Gut microbiota inhabiting in the intestine proliferate in the intestine and produce secondary metabolites (Kopp-Hoolihan 2001). Secondary metabolites produced by the gut microbiota may affect the host depending on the amount. In other words, various physiological activities are capable to expect by intentionally administering gut microbiota that can produce a large amount of useful secondary metabolites (Parvez et al. 2006;Kopp-Hoolihan 2001). In this study, we investigated the possibility of intestinal production of physiologically active substances as AGI that involved in the absorption of sugars in the digestive tract by microbiota capable of proliferating in the intestine.
The conditions for the strains to produce physiologically active substances in the intestine should be safe, they should be able to proliferate in the anaerobic environment in the intestines, they should form spores to reach the intestines e ciently, the high productivity of physiologically active substances in the anaerobic environment (Parvez et al. 2006;Kopp-Hoolihan 2001). Therefore, strains were screened from the nature. As a result B. licheniformis NY1505 was obtained. B. licheniformis NY1505 e ciently reaches the intestine in the form of spores, then proliferates, produces a large amount of AGI, and exhibits the physiological activity of the host that slows the rate of weight gain (Figs. 4, 5).
On the other hand, how the administered microbiota adheres and inhabits in the intestine depends on the need for supply of the secondary metabolite. In other words, it is preferable to inhabit and produce the secondary metabolite in the intestine only for a necessary period. As shown in Fig. 6, following the intestinal habitat of B. licheniformis NY1505, the numbers of strain excreted out of the body decreases after the administration of the strain is nished. B. licheniformis NY1505 inhabits and is excreted in a relatively short time rather than adhering and inhabiting in the intestine, so it is capable to use only for a necessary period.
It takes a lot of time and cost to process such as extract, purify, and drying physiologically active substances from fermented products of animals and plants or microorganisms (Zhu et  In this study, it is shown an e cient process of producing and supplying secondary metabolites directly in the intestine by administering strain capable of proliferating in the intestine. AGI is a compound that works inside the intestine, but it is expected that this process will work. This study suggests that by oral administration of microbiota that is capable of intestinal proliferation, the intestinal environment is used as a factory to produce secondary metabolites. It can be a new supply and intake method for physiologically active substances. Methods/experimental Materials Reagents such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ρ-nitrophenyl α-D-glucopyranoside(pNPG), sodium propionate, potassium phosphate, and sodium carbonate were used with guaranteed reagents.
The reaction was stopped by adding 100 µL of 0.1M sodium carbonate, and the inhibition rate was measured by substituting the absorbance at 405 nm into the following equation (Kim et al. 2005).

The inhibition rate = (A-B)/A × 100
In the above equation, A is the absorbance of the control and B is the absorbance of the sample containing the inhibitor. Inhibition rate 1 unit was de ned as the amount of inhibitor when 100% inhibition of α-glucosidase used in the assay.

Animals
Experimental animals were bred in research facilities. Three-week-old ICR mice were used as experimental animals after the acclimation period before the experiment were classi ed by weight equally and classi ed into ten animals per group and 5 per cage. The illuminance was controlled by turning it on every 12 hours and the temperature of the breeding room was adjusted to 21℃. The feed composition of each group is as shown in Table 1, and the high carbohydrate group and the fat group was reared by dividing the spores administered group and the non-administered group, respectively. The spore administration diet was prepared by directly mixing 10 8 cells of spores per 1 kg of feed. During the breeding period, body weight was measured, feces were collected and the amount of microorganism contained in the feces and excreted and the activity of AGI were measured at regular intervals. This study was approved by the Animal Care and Use Committee of Kangwon National University (permit no. KW-190103-11) Statistical analysis The results of each experiment were expressed as the mean with standard deviations (± SD). A one-way analysis of variance (ANOVA) test (Bonferroni, SPSS, v.32, for Windows) was performed to determine the group means. Values were considered to be signi cant when P was less than 0.05(P ≤ 0.05). Chemical structure of AGI 1 and the enzyme inhibition type of AGI 1 a Chemical Structure of AGI 1 b Inhibition type of AGI 1 showed non-competitive inhibition by Lineweaver -Burk plot. Each symbol showed amounts of AGI 1. is 1140 μg, □ is 570 μg, △ is 228 μg, ○ is no inhibitor Figure 3 Chemical structure of AGI 2 and the enzyme inhibition type of AGI 2 a Chemical Structure of AGI 2 b Inhibition type of AGI 2 showed competitive inhibition by Lineweaver -Burk plot. Each symbol showed amounts of AGI 2. is 48.9μg, □ is 32.6μg, △ is 16.3μg, ○ is no inhibitor Figure 4 Comparison of weight alteration in High carbohydrate diet clade with a standard diet Each symbol □ showed weight alteration of standard diet group, ○ showed weight alteration of high carbohydrate diet group, • showed weight alteration of high carbohydrate diet with NY1505 spore group. Values were considered to be signi cant ( ) when P was less than 0.05(P ≤ 0.05). meant that high carbohydrate diet group with and without NY1505 showed to be accompanied reliability meaning signi cant.

Figure 5
Comparison of weight alteration in fat diet clade with a standard diet Each symbol □ showed weight alteration of standard diet group, ○ showed weight alteration of fat diet group, • showed weight alteration of fat diet with NY1505 spore group Values were considered to be signi cant ( ) when P was less than 0.05(P ≤ 0.05). meant that high fat diet group with and without NY1505 showed to be accompanied reliability meaning signi cant.

Figure 6
A secreting amount of NY 1505 CFU per 1g from feces Each symbol showed secreting amount of NY 1505 CFU per 1g from feces. ○ meant high carbohydrate diet with NY1505 spore group, • meant high fat diet with NY1505 spore group.