Effects of inulin on the growth performance and tolerance in adverse environments of several probiotics

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The results showed that inulin could significantly promote the growth of B. subtilis , S. cerevisiae , and L. fermentum (p < 0.05). When inulin replaced glucose, the ethanol concentration in S. cerevisiae fermentation broth could be increased by 15%. Inulin could significantly improve the acid tolerance of B. subtilis under acidic conditions. It could significantly improve the bile salt tolerance of L. fermentum and S. cerevisiae and significantly increase the ethanol tolerance of L. fermentum and B. subtilis . It could also significantly increase the survival rate of these three probiotics under low-temperature conditions. Our findings prove that inulin positively affects the growth ability and poor environmental tolerance of probiotics, and can be used as a prebiotic for several probiotics. Prebiotics Bacillus subtilis Saccharomyces cerevisiae Lactobacillus fermentum Tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Probiotics can enter the animal’s gastrointestinal tract and multiply rapidly and colonize the surface of intestinal epithelial cells to form a microbial barrier in the intestine to prevent the invasion of pathogenic microorganisms. Since probiotics colonize the digestive tract of the host body, the probability of infection by harmful pathogenic microorganisms is reduced, thereby reducing the risk of infection of the body (Brownawell et al. 2012 ). Probiotics can also produce various hydrolytic enzymes to decompose materials that are not easily decomposed in the intestinal tract of animals to promote the digestion and absorption of nutrients in the intestinal tract. Lactobacillus is the most commonly used type in probiotic preparations, colonized in the human digestive tract. Some nutrients are more favorable for human absorption after fermentation and metabolism by Lactobacillus itself or promote the host’s health by antagonistic biological effects and reducing intestinal pH. Lactobacillus can grow at pH 3.0 ~ 4.5 and have a certain tolerance to gastric acid (Jorjao et al. 2015 ). Bacillus is a common probiotic because it can form spores and has good stability, acid, alkali, high temperature, and pressure resistance. It can exist in the form of spores during storage. Bacillus can consume oxygen in the intestinal tract, maintain an anaerobic environment, inhibit harmful bacteria growth, and maintain intestinal flora balance. Wang et al. found that feeding 15% Bacillus subtilis fermented feed to lactating sows can significantly improve the apparent digestibility of its dry matter, total phosphorus, and total energy (Wang et al. 2018 ). Yeast is a facultative anaerobic microorganism primarily used in the fermentation industry for alcohol fermentation and can also be used as a microecological agent in feed additives. Given that the host's endogenous digestive enzymes cannot decompose the mannan of the yeast cell wall, the mannan of the yeast itself can be used as a prebiotic to promote the proliferation of other probiotics, lower the pH in the intestine, and inhibit the growth of harmful bacteria such as E. coli. In aquaculture, given the rich mannan content of yeast cell walls, it can significantly promote microvilli growth in aquatic animals' intestinal epithelial cells, change their morphology, increase the surface area of intestinal folds, and better absorb nutrients (Machová and Bystrický 2012 ). Prebiotics selectively stimulate the growth of probiotics resident in the gut, especially Bifidobacteria, through the production of β-fructosidase, changing the colonic microflora to a healthier composition. The common prebiotics is fructooligosaccharides, isomalt oligosaccharides, dextran, inulin, Hieracium, and lentinan. Probiotics are degraded, digested and absorbed after reaching the posterior intestine. Their products can provide energy and nutrients for the growth and proliferation of intestinal epithelial cells to promote or repair the intestinal mucosa. Prebiotics also significantly promote the growth of probiotics in vitro (Ogueke et al. 2010 ). Inulin is a natural reserve polysaccharide that is widely distributed in nature. It mainly exists in composite plants. The primary raw materials of inulin are chicory ( Cichorium intybus L.) and Jerusalem artichoke . Inulin can promote the proliferation of probiotics and improve the community of probiotics. No enzyme in the human intestine can digest inulin. Thus inulin can be used by probiotics through the small intestine of the human body and promote the growth and proliferation of probiotics, reduce the pH in the intestine, and then inhibit the proliferation of many harmful bacteria, reduce the production of harmful toxins in the intestine and improve the health level of the intestine (Sasajima et al. 2010 ). Inulin can reduce the number of E. coli in the intestine, increase the number of probiotics such as Lactobacillus and Bifidobacterium , and improve the community structure of intestinal microorganisms (Samanta et al. 2013 ). It is reported that adding inulin to the diet of obese mice can reduce the abundance of Bacteroides in the intestine of mice and increase the proportion of Firmicum (Vandeputte et al. 2017 ). Valcheva et al. found that after taking 15 g of inulin daily, the intestinal microbial community structure changed (Valcheva et al. 2019 ). In vitro experiments have found that using Jerusalem artichoke root extract can significantly increase the number of Lactobacillus and Enterococcus faecium . Inulin can increase butyric acid production in the intestinal tract by increasing the number of Bifidobacteria or lactic acid bacteria in the intestinal tract and indirectly reducing inflammation and harmful bacteria (Hoentjen et al. 2005 ). Guo et al. found that adding inulin to the diet can improve gastrointestinal discomfort and intestinal inflammation and reduce obesity-related diseases caused by high-fat food intake (Guo et al. 2018 ). Inulin has a significant role in promoting the growth and proliferation of various probiotics in the intestine and the colonization of probiotics in the intestine. At present, the research on the probiotic function of inulin is mostly in vivo to explore the effect of inulin on the growth performance of intestinal probiotics. However, only some studies reported on the probiotic effect of inulin in vitro . This study focused on adding different inulin to the fermentation broth and using inulin as an alternative carbon source. The colony count was measured to investigate the effect of inulin on the proliferation ability of three probiotics Bacillus subtilis , Lactobacillus fermentum , and Saccharomyces cerevisiae . A study on the effect of inulin improving the tolerance of these three probiotics in an adverse environment was also conducted. Materials and methods Materials and reagents Inulin comes from the Jerusalem Artichoke Industry Engineering Technology Research Center, Henan Academy of Sciences. Beef meal and peptone were purchased from Beijing Obosing Biotechnology Co., Ltd (Beijing, China). Yeast extract powder was purchased from Qingdao Science and Technology Industrial Park Haibo Biological Technology Co., Ltd (Qingdao, China). Glucose, sodium acetate, and disodium hydrogen citrate are of analytical purity, purchased from the Tianjin Damao chemical reagent factory. Manganese sulfate and magnesium sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and Tianjin Zhiyuan Chemical Reagent Co., Ltd (Tianjin, China). YPD medium: peptone 20.0 g, glucose 20.0 g, yeast extract 10.0 g, distilled water 1000 mL, pH 6.5 ± 0.2. MRS medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dimethyl hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, sulfuric acid Manganese 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH 5.7 ± 0.2. Glucose peptone medium: glucose 5g, peptone 6g, yeast extract powder 6 g, beef extract powder 1.5 g, Tween 80 1 g, distilled water 1000 mL, pH 7.4 ± 0.2. Preparation of seed solution Preparation of Lactobacillus fermentum seed liquid: Selected Lactobacillus fermentum strain into 20 mL MRS liquid medium and shook it at 37°C and 150 r/min for 24 h the seed liquid of Lactobacillus fermentum . Preparation of Bacillus subtilis seed solution: The Bacillus subtilis slant strain was picked into 20 mL of glucose peptone medium, cultured at 37°C, and 150 r/min for 12 h Bacillus subtilis seed solution. Preparation of Saccharomyces cerevisiae seed liquid: The yeast (Angel Yeast Co., Ltd., Yichang, China) was activated and then gradually diluted using the dilution-coated plate method to purify the strain. After 24 h of culture at 25°C, a single colony was picked and cultured on the slope of the test tube. Then it was placed in the 20 mL YPD medium and cultured at 28°C and 150 r/min for 24 h, which was the Saccharomyces cerevisiae seed liquid. Effect of inulin on the growth performance of the probiotics Different concentrations of 1%, 2%, 3%, 4%, and 5% inulin were added to MRS liquid medium, YPD liquid medium, and glucose peptone medium. Moreover, the sample in which no inulin was added was set as a control group (CON). The other group is the alternative inulin group (IAG). Inulin was used as a substitute for carbon sources and the same quantity of inulin was used as an alternative to glucose in the basal medium. The inoculation volume of all groups was 5%. Bacillus subtilis was inoculated in a glucose peptone medium, cultured at 37°C, and shaken at 150 r/min for 12 h. Saccharomyces cerevisiae was inoculated into YPD liquid medium, cultured at 25°C, and shaken at 150 r/min for 24 h. Lactobacillus fermentum was inoculated in MRS liquid medium, cultured at 37°C, and shaken at 150 r/min for 24 h. Effect of inulin on the improvement of the tolerance of several probiotics in an adverse environment Effect of inulin on acid and bile salt tolerance of the probiotics The method used in this study was described as Kusada (Kusada et al. 2021 ). The pH of the culture medium was adjusted to 3.0 with 0.1 M HCl to simulate the gastric acid experiment. The 0.3% bovine bile salt was added to the culture broth of Saccharomyces cerevisiae and Bacillus subtilis , and 0.05% bovine bile salt was added to the culture broth Lactobacillus fermentum to perform the simulated bile test. The 5% seed solution of each probiotic strain was inoculated into the simulated gastric acid and bile culture solution, respectively. Bacillus subtilis and Lactobacillus fermentum were then cultured at 37°C and 150 r/min for 24 h, and Saccharomyces cerevisiae was cultured at 28°C and 150 r/min for 24 h. Then the total plate count was determined. Effect of inulin on ethanol tolerance of the probiotics Added 10% absolute ethanol to the medium. Next, inoculated each probiotic strain's 5% seed solution into the medium. Bacillus subtilis and Lactobacillus fermentum were then cultured at 37°C and 150 r/min for 24 h, and Saccharomyces cerevisiae was cultured at 28°C and 150 r/min for 24 h. Then the total plate count was determined. Effect of inulin on pepsin resistance of the probiotics Prepared 10% pepsin solution and then added it to the culture medium to reach a final concentration of 1%. Then inoculated 5% seed solution of each probiotic strain was put into the medium. Bacillus subtilis and Lactobacillus fermentum were then cultured at 37°C and 150 r/min for 24 h, and Saccharomyces cerevisiae was cultured at 28°C and 150 r/min for 24 h. Then the total plate count was determined. Effect of inulin on salt tolerance of the probiotics Added 10% sodium chloride solution to the culture medium. Next, inoculated each probiotic strain's 5% seed solution into the medium. Bacillus subtilis and Lactobacillus fermentum were then cultured at 37°C and 150 r/min for 24 h, and Saccharomyces cerevisiae was cultured at 28°C and 150 r/min for 24 h. Then the total plate count was determined. Effect of inulin on the survival rate of the probiotics at low temperature Inoculated each probiotic strain's 5% seed solution into the medium. Bacillus subtilis and Lactobacillus fermentum were then cultured at 37°C and 150 r/min for 24 h, and Saccharomyces cerevisiae was cultured at 28°C and 150 r/min for 24 h. Then they were placed at 4°C for 7 d. Moreover, the total plate count was determined. The survival rate of the strains was obtained as follows. $$\text{s}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l} \text{r}\text{a}\text{t}\text{e}= \frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{r}\text{e}\text{m}\text{a}\text{i}\text{n}\text{i}\text{n}\text{g} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{o}\text{r}\text{i}\text{g}\text{i}\text{n}\text{a}\text{l} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}}\times 100\%$$ Determination of the probiotics content The dilution coating plate method was used to determine the probiotics content. Appropriate gradient dilutions of the cultured bacterial liquid in sterile water were prepared in a sterile environment. Totally 0.1 mL of the three dilutions of the culture was inoculated on the surface of the corresponding solid medium for coating. After coating, the plates of Saccharomyces cerevisiae and Bacillus subtilis were inverted and cultured in a constant temperature incubator for 12 h. The plate of Lactobacillus fermentum was inverted and cultured for 24 h. Then the number of colonies on each plate was recorded. Determination of the physical and chemical indicators Determination of pH of Lactobacillus fermentum fermentation broth The acidity of the fermentation broth of Lactobacillus fermentum was directly measured using a pH meter (Mettler-Toledo Measurement Instrument Shanghai Co Ltd., Shanghai, China). Determination of ethanol production capacity of Saccharomyces cerevisiae Totally 1.5 mL of the inoculated Saccharomyces cerevisiae fermentation broth was centrifuged at 8 000 r/min and filtered, and the ethanol content in the fermentation broth was determined by the enzyme labeling method. The 0.1 mL of filtered bacterial solution was transferred to the test tube. Then 2 mL distilled water, 0.20 mL solution 1 (buffer), 0.20 mL solution 2 (NAD + ), and 0.05 mL solution 3 (aldehyde dehydrogenase) were added successively. It was stood for 30 min and diluted to 4 times. Then 1 mL sample was mixed with 3 mL distilled water. The absorbance A 1 at 340 nm was then measured. The 0.02 mL suspension 4 (ethanol dehydrogenase) was added to the sample. The absorbance A 2 at 340 nm was measured. The ethanol (C) content in the fermentation broth was calculated as follows. $$C=\frac{2.57\times 46.07}{6300\times 1.0\times 0.10\times 2}\times 0.1266\times ({A}_{2}-{A}_{1})$$ Statistical analyses The statistical significance of the experimental data was determined by one-way analysis of variance (ANOVA) using the SPSS 22.0 for Windows statistical package (SPSS, Inc., Chicago, IL). The mean and standard deviation of the data were determined using Excel 2003 (Microsoft, Inc., Seattle, WA). Results The effect of inulin on the growth performance of Bacillus subtilis The effect of inulin as an added and alternative carbon source on the growth performance of Bacillus subtilis is shown in Fig. 1 . When inulin replaced the glucose in the basal medium, the number of Bacillus subtilis in the fermentation broth was 1×10 8 CFU/mL, which was 5.7 times that of the control group. The results indicated that the utilization ability of Bacillus subtilis to inulin was significantly better than that of glucose. In addition, adding different proportions of inulin could significantly increase the content of Bacillus subtilis in the fermentation broth compared with the control group. When 1% inulin was added, the content of Bacillus subtilis in the fermentation broth was the highest, reaching 2×10 8 CFU/mL, which was 10.6 times that of the control group. However, with the increase of inulin, the content of viable Bacillus subtilis showed a downward trend. The results showed that inulin could significantly increase the content of viable bacteria in the fermentation broth of Bacillus subtilis ( P < 0.05). The effect of inulin on the growth performance of Saccharomyces cerevisiae It can be seen from Fig. 2 that when inulin replaced the glucose in the basal medium, the number of Saccharomyces cerevisiae in the fermentation broth was 4.2×10 9 CFU/mL, which is 2.6 times that of the control group. The results indicated that the utilization ability of inulin by Saccharomyces cerevisiae was significantly better than that of glucose. In addition, adding different proportions of inulin could significantly increase the content of Saccharomyces cerevisiae in the fermentation broth compared with the control group. The content of Saccharomyces cerevisiae in the fermentation broth increased with the increase of the inulin amount. When 5% inulin was added, the content of Saccharomyces cerevisiae in the fermentation broth was the highest, reaching 5.5×10 9 CFU/mL, 3.3 times that of the control group. The results showed that inulin could significantly increase the viable bacteria content of Saccharomyces cerevisiae ( P < 0.05). The effect of inulin on ethanol production of Saccharomyces cerevisiae is shown in Fig. 3 . It can be seen that when inulin replaced glucose in the basic medium, the volume concentration of ethanol in the fermentation broth was 6.9%, which was 1.15 times that in the control group. This result indicated that the ability of Saccharomyces cerevisiae to use inulin to metabolize ethanol is significantly better than that of glucose. In addition, adding different proportions of inulin could significantly increase ethanol content compared with the control group. The volume concentration of ethanol increased with the increase of inulin. When added 5% inulin was, the volume of ethanol in the solution was the highest, reaching 7.85%, 1.3 times that of the control group. The results showed that inulin could significantly improve the ethanol production capacity of Saccharomyces cerevisiae as a substitute or as a carbon source. The effect of inulin on the growth performance of Lactobacillus fermentum The effect of inulin on the growth performance of Lactobacillus fermentum is shown in Fig. 4 . It can be seen that when inulin replaced the glucose in the basal medium, the number of Lactobacillus fermentum in the fermentation broth was 4.4×10 8 CFU/mL, which was 0.6 times that of the control group. This result showed that the use of Lactobacillus fermentum was significantly better than that of inulin. In addition, adding different proportions of inulin could significantly increase the content of Lactobacillus fermentum in the fermentation broth compared with the control group. When 3% inulin was added, the Lactobacillus fermentum content in the fermentation broth was the highest, reaching 9.9×10 8 CFU/mL, which was 1.35 times that of the control group. When the inulin was added at 4% and 5%, the content of viable bacteria tended to be stable. The results showed that inulin could significantly increase the content of viable bacteria in the fermentation broth of Lactobacillus fermentum ( P < 0.05); however, it could inhibit the proliferation of Lactobacillus fermentum when inulin was added as an alternative carbon source to the basic medium. As shown in Fig. 5 , when inulin replaced glucose in the basic medium, the pH value of the fermentation liquid was 4.39, which was significantly higher than that of the control group. The results showed that the ability of Lactobacillus fermentation to produce acid by inulin was significantly lower than that of glucose. In addition, adding different proportions of inulin could significantly reduce the pH of the fermentation broth compared with the control group. The pH of the fermentation broth decreased with the increase of inulin. When the amount of inulin reached about 3%, the pH value was the lowest and did not decrease. The results showed that inulin could significantly improve the acid production capacity of Lactobacillus fermentans ( P < 0.05). However, inulin could not promote the acid production capacity of Lactobacillus ferments when it was used to replace glucose in the basic medium. This result was consistent with the result of viable bacteria obtained above. The effect of inulin on the acid tolerance, bile salt tolerance, ethanol tolerance, pepsin tolerance, salt tolerance, and low-temperature tolerance of the three probiotics The acid tolerance of inulin to the three probiotics is shown in Table 1 . It can be seen that under pH 3, the viable content of Bacillus subtilis was significantly higher than that of the control group ( P < 0.05). However, no significant difference was observed in the viable content of Saccharomyces cerevisiae and Lactobacillus fermentum between the experimental and control groups. Table 1 Effect of inulin on the acid tolerance of the probiotics Viable content of Saccharomyces cerevisiae ×10 7 (CFU/mL) Viable Lactobacillus content of fermentans ×10 7 (CFU/mL) Viable content of Bacillus subtilis ×10 8 (CFU/mL) Experimental group 6.3 ± 1.48 a 4.5 ± 0.70 a 1.19 ± 0.11 a Control group 5.2 ± 1.35 a 3.6 ± 0.59 a 0.96 ± 0.06 b Different letters in the same column indicate significant differences ( P < 0.05). As shown in Table 2 , the viable contents of Saccharomyces cerevisiae and Lactobacillus fermentum were significantly higher ( P < 0.05) than that of the control group. The results indicated that inulin significantly improved the bile tolerance of Saccharomyces cerevisiae and Lactobacillus fermentum . However, no significant difference was observed in the viable content of Bacillus subtilis between the experimental and control groups. Table 2 Effect of inulin on bile salt tolerance of probiotics Viable content of Saccharomyces cerevisiae ×10 7 (CFU/mL) Viable content of Lactobacillus fermentans ×10 7 (CFU/mL) Viable content of Bacillus subtilis ×10 6 (CFU/mL) Experimental group 8.1 ± 1.18 a 7.4 ± 0.47 a 5.6 ± 1.48 a Control group 0.1 ± 0.06 b 4.9 ± 1.03 b 4.0 ± 0.62 a Different letters in the same column indicate significant differences ( P < 0.05). Table 3 showed that when 10% absolute ethanol was added, the viable contents of Lactobacillus fermentum and Bacillus subtilis in the experimental group were significantly higher ( P < 0.05) than that of the control group. However, no significant difference was observed in the viable content of Saccharomyces cerevisiae between the experimental group and the control group. Table 3 Effect of inulin on ethanol tolerance of probiotics Viable content of Saccharomyces cerevisiae ×10 7 (CFU/mL) Viable content of Lactobacillus fermentans ×10 8 (CFU/mL) Viable content of Bacillus subtilis ×10 7 (CFU/mL) Experimental group 8.3 ± 0.66 a 2.7 ± 0.15 a 9.8 ± 1.5 a Control group 7.5 ± 0.70 a 2.0 ± 0.28 b 0.2 ± 0.05 b Different letters in the same column indicate significant differences ( P < 0.05). It can be seen from Table 4 that under the condition of adding 1% pepsin to the culture medium when the carbon source in the original culture medium was replaced by inulin, the viable content of the Saccharomyces cerevisiae was significantly higher ( P < 0.05) than that of a control group. However, no significant difference was observed in the viable content of Lactobacillus fermentum and Bacillus subtilis between the experimental and control groups. Table 4 Effects of inulin on the pepsin tolerance of probiotics Viable content of Saccharomyces cerevisiae ×10 5 (CFU/mL) Viable content of Lactobacillus fermentans ×10 5 (CFU/mL) Viable content of Bacillus subtilis ×10 2 (CFU/mL) Experimental group 2.6 ± 0.09 a 1.9 ± 0.12 a 7.4 ± 1.94 a Control group 2.4 ± 0.16 b 1.8 ± 0.13 a 7.3 ± 1.12 a Different letters in the same column indicate significant differences ( P < 0.05). The effect of inulin on the salt tolerance of the probiotics is shown in Table 5 . It can be seen that when 10% NaCl was added, the viable content of Bacillus subtilis was significantly higher ( P < 0.05) than that of the control group. However, no significant difference was observed in the viable content of Saccharomyces cerevisiae and Lactobacillus fermentum between the experimental and control groups. Table 5 Effect of inulin on the salt tolerance of probiotics Viable content of Saccharomyces cerevisiae ×10 5 (CFU/mL) Viable content of Lactobacillus fermentans ×10 7 (CFU/mL) Viable content of Bacillus subtilis ×10 5 (CFU/mL) Experimental group 7.1 ± 1.68 a 1.3 ± 0.05 a 7.3 ± 0.79 a Control group 4.8 ± 0.90 a 1.2 ± 0.15 a 5.2 ± 0.48 b Different letters in the same column indicate significant differences ( P < 0.05). The inulin results on low-temperature tolerance of three probiotics are shown in Table 6 . The results showed that the survival rate of Lactobacillus fermentum and Bacillus subtilis was significantly higher than that of the control group. However, the survival rate of Saccharomyces cerevisiae was significantly lower than that of the control group. Table 6 Effect of inulin on low-temperature tolerance of the probiotics Survival rate Saccharomyces cerevisiae Lactobacillus fermentans Bacillus subtilis Experimental group 82.10% 88.70% 31.7% Control group 102.10% 73.10% 23.2% Discussion As a prebiotic, inulin has been widely used for decades. Although the human diet is generally rich in complex carbohydrates, the human body contains fewer glycosidases, so it can only hydrolyze sucrose, lactose, and some starches, but not inulin (Goh and Klaenhammer 2015 ). Therefore, most of these indigestible complex carbohydrates reach the gut and are transported and metabolized by some gut microbiota members. Inulin can improve intestinal probiotics, reduce intestinal pH and promote the production of short-chain fatty acids to improve intestinal health (Liu et al. 2020 ). Inulin intake can significantly increase the relative abundance of probiotics such as Bifidobacteria and Lactobacillus and inhibit the abundance of pathogenic bacteria. Maintaining the activity of probiotics and their ability is essential in the processing and applying of probiotics (Kim et al. 2019 ). This research studied the effects of inulin as a carbon source and alternative carbon source on three probiotics' growth and production performance. The results showed that inulin had the most apparent promoting effect on the growth performance of Bacillus subtilis . The addition of inulin had a promoting effect on the acid production capacity of Lactobacillus fermentum and the ethanol production capacity of Saccharomyces cerevisiae . In this paper, when inulin was used as a carbon source, it could significantly enhance the growth performance of Bacillus subtilis . Adding a carbon source to the medium could also promote the growth of Bacillus to a certain extent. Bacillus is a bacterium that can produce inulinase, decompose, and utilize inulin (Seydlova et al. 2012 ). Early research found that Bacillus subtilis could produce inulinase with good heat stability (Vullo et al. 1991 ). Zherebtsov et al. found that when garlic, onion extract, inulin, and soluble starch were used as carbon sources, the productivity of inulinase was the highest when microorganisms were inoculated on soluble starch (Zherebtsov et al. 2002 ). However, the result is different from this study. Therefore, it can be seen that inulin is not inducible to inulinase production by Bacillus subtilis. In this study, when 1% inulin was added as a supplementary energy source, the content of Bacillus subtilis was the highest, about 10.6 times that of the control group. Qian et al. found that supplementing inulin could increase biomass and improve metabolic pathways (Qian et al. 2015 ). At the same time, the combined use of Bacillus subtilis and inulin can improve the immune performance of animals and improve intestinal flora (Cerezuela et al. 2012 ). Therefore, inulin has a good role in promoting Bacillus subtilis . In previous studies, it has been found that inulin can be directly used as a carbon source for Saccharomyces cerevisiae without being pretreated by acid and enzymes, and the yeast can ferment it to produce ethanol (Lim et al. 2011 ). The degree of polymerization of inulin has a more significant impact on its processing properties, and studies have shown that inulin mixtures with different degrees of polymerization can increase the effect of prebiotics in the colon (Lopes et al. 2015 ). Nowadays, synthetic methods can generally be used to control the degree of polymerization of synthetic inulin. Different Saccharomyces cerevisiae has different ability to utilize inulin. Studies have shown that the yield of two different Saccharomyces cerevisiae using inulin to produce ethanol can differ by 1.6 times (Lim et al. 2011 ). Invertase Suc2 is a key hydrolase for the degradation of inulin by Saccharomyces cerevisiae . Previous research found that when using inulin as a carbon source for Saccharomyces cerevisiae , the activity of extracellular enzymes is 4.3 times higher than when using sucrose (Wang and Li 2013 ). In the study of the activity of the invertase Suc2, it was found that among the 13 N-glycosylation sites of Suc2, the sugar chains of N4, N45, N78, and N146 play an essential role in the host strain's inulin transformation. The desugar effect of N4, N7,8, and N146 sequences increased Suc2 activity and ethanol production (Yang et al. 2020 ). This may be why different Saccharomyces cerevisiahave as have different ethanol producticapacitiesity. In this study, inulin used as an alternative carbon source can improve the growth performance of Saccharomyces cerevisiae . Saccharomycbollarddii and inulin are added to the yogurt together to ensure the activity of the yeast during the 28-day storage period, and the number of viable cells in the yogurt can exceed 6.0 log CFU/g (Sarwar et al. 2019 ). Moreover, inulin as an added carbon source can increase ethanol output and is dose-dependent. Because Saccharomyces cerevisiae has a solid ability to produce inulinase, the utilization of inulin by Saccharomyces cerevisiae increases ethanol production (Chi et al. 2009 ). Li et al. found that Saccharomyces cerevisiae has the highest endo-inulinase activity in the microbial decontamination test of the crude Jerusalem artichoke inulin extract compared with 9 other yeasts (Li et al. 2019b ). It suggests that the addition of inulin positively affects the growth and fermentation of Saccharomyces cerevisiae . Lactobacillus and Bifidobacterium are both important acid-producing microorganisms in the human intestinal flora. In previous studies, it has been found that Bifidobacteria can use inulin-type fructans as the only energy source, and inulin can also increase the number of Bifidobacteria in the intestine (Rossi et al. 2005 ). Lactobacillus is highly nutritious and mainly grows in an environment rich in carbohydrates. When Lactobacillus uses inulin as the only energy source and the final product, lactic acid, it also produces acetic acid, formic acid, and ethanol (Makras et al. 2005 ). The results showed that inulin used as an added carbon source could significantly increase the content of viable bacteria in the fermentation broth of Lactobacillus fermentum . In contrast, inulin as an alternative carbon source added to the basic medium can inhibit the proliferation of Lactobacillus fermentum . It is speculated that the inulinase produced by Lactobacillus fermentum may have a singularity or tend to be short-chain or long-chain inulinase, weakening the Lactobacillus fermentum 's ability to utilize inulin. This may be related to the strain of Lactobacillus fermentum . A recent study screened the ability of Lactobacillus to ferment fructooligosaccharides, and the results showed that 12 of the 16 strains of Lactobacillus fermented fructooligosaccharides (Kaplan and Hutkins 2003 ). It suggests that not all Lactobacillus can ferment inulin. In lactobacilli capable of metabolizing fructooligosaccharides, using fructooligosaccharides appears to be through two catabolic pathways. The first is that the oligofructose is transported into the cytoplasm intact and is hydrolyzed by GH32 β-fructosidase, and different lactobacilli have different ways of transporting and ingesting oligofructose (Goh and Klaenhammer 2015 ; Velikova et al. 2017 ). The second transformation method combines fructooligosaccharides with GH32 β-fructosidase on the cell surface.The oligofructose is hydrolyzed outside the cell, and then the decomposition products, fructose, glucose, etc., are transported and taken up (Velikova et al. 2017 ). Lactobacillus paracasei , commonly used in research, is the second approach. Lactobacillus paracasei can produce large amounts of lactic acid through simultaneous saccharification and fermentation of inulin. Studies have shown that high concentrations of Mn 2+ accelerate the hydrolysis of inulin by increasing the activity of β-fructosidase, and increasing the conversion of sugar to lactic acid by increasing the overall glycolytic flux (Petrov et al. 2017 ). Extracellular β-fructosidase plays an important role in the decomposition of long-chain inulin. In addition, β-fructosidase decomposes and metabolizes long-chain inulin and releases monomer fructose through the extracellular matrix, which can be cross-fed to other consumers in the gut (Zhu et al. 2020 ). Previous studies have shown that Lactobacillus para case W20 can stimulate the growth of Lactobacillus salivarius w57 by accumulating fructooligosaccharides (2–3 degrees of polymerization) using a family of GH32 enzymes. Therefore, when inulin is used by probiotics such as Lactobacillus , it will also promote the growth of other microorganisms. In addition, the inhibition of Lactobacillus fermentum may also be related to the different inulin actions of Lactobacillus fermentum . The previous report pointed out that Lactobacillus paracasei can use long-chain inulin as the sole carbon source and ferment fructooligosaccharide and oligofructose-enriched inulin. In contrast, Lactobacillus acidophilus can only ferment oligofructose-enriched inulin (Makras et al. 2005 ). In this paper, it is found that inulin is used as a carbon source, which can significantly improve the acid production capacity of Lactobacillus fermentum . It showed that glucose is the carbon source that Lactobacillus fermentum preferentially selects during fermentation. Makras et al. found that when inulin rich in fructooligosaccharides was used as the sole energy source of Lactobacillus paracasei , the free fructose in the fermentation medium was first fermented, then fructooligosaccharides and inulin were degraded, resulting in the accumulation of fructose, glucose and sucrose, and then consumed. The degradation of long-chain inulin generally occurs in the later decomposition stage and conforms to the above decomposition rules (Makras et al. 2005 ). By observing the growth curve of Lactobacillus agilis YZ050 in the mixture of glucose and inulin, it is found that Lactobacillus agilis YZ050 has a secondary growth phenomenon, which may be due to the initial consumption of glucose. Then inulin is a carbon source, and the latter's speed is significantly higher than the former's (Wang et al. 2021 ). Petrova et al. (Petrova et al. 2015 ) used Lactobacillus paracasei to saccharify and ferment inulin, the productivity of lactic acid was 1.08 g/L/h, and the raw material conversion rate of inulin was 91%. This result indicates that inulin can promote the fermentation of Lactobacillus , which is consistent with the results when inulin is used as a carbon source in this experiment. Therefore, the fermentation of inulin to Lactobacillus is affected by factors such as the species of Lactobacillus and the type of inulin. However, in general, it promotes the growth and fermentation of Lactobacillus . Since inulin has a good effect on the growth and fermentation of probiotics, many studies have used probiotics to produce inulinase, thereby expanding the application of inulin (Jiang et al. 2019 ). This paper studied the survival rate of Bacillus subtilis , Saccharomyces cerevisiae , and Lactobacillus fermentum under different environmental conditions when inulin was used as the carbon source influence of inulin on the growth of probiotics was explored. Due to probiotics' excellent effect on intestinal health, many studies have processed probiotics into oral liquid and tablet preparations to treat and prevent intestinal diseases. Inulin has been used in industry as a prebiotic. Gut microbes can ferment inulin to produce short-chain fatty acids, which can induce the growth of beneficial microorganisms, thereby changing the composition of the organisms in the gut microbiota and enhancing the host’s immune system (Seifert and Watzl 2007 ). Probiotics need to reach the intestine smoothly and maintain activity because it is a particular environment in the intestine. Safely delivering probiotics to the intestines requires the harsh low-acid environment of the stomach and the influence of gastric protein. The main component of gastric acid in human gastric juice is hydrochloric acid, its pH is usually around 3.0, and it can reach 2.5 under fasting conditions (Mulaw et al. 2019 ). A recent study conjugated phthalate groups with inulin and combined them with probiotics into compressed tablets. In a simulated intestinal environment, the activity of probiotics is also significantly more robust than compressed tablets without inulin (Kim et al. 2019 ).In this study, it was found that under the condition of pH 3, the use of inulin as a carbon source significantly increased the content of live Bacillus subtilis . The viable bacteria content of Saccharomyces cerevisiae and Lactobacillus fermentum was also increased by a certain amount after using inulin as a carbon source. Lactobacillus itself can produce acid and has good acid resistance. The study of the symbiotic cheese of inulin and Lactobacillus delbrueckii found that the number of viable bacteria under pH 3.5 can be the same as that under the neutral condition (Araújo et al. 2010 ). Under lower pH conditions, on the one hand, it will affect the transmembrane transport of nutrients by microorganisms. On the other hand, it will affect the activity of various enzymes in microbial cells, thereby affecting the growth of microorganisms (Mulaw et al. 2019 ). The optimum pH of inulinase produced by most microorganisms is 4.0–6.0, and the inulinase can remain relatively stable near the optimum pH (Atia et al. 2016 ). Therefore, adding inulin as a carbon source can increase the acid tolerance of probiotics and improve the body's intestinal health. Some enzymes and membrane proteins present on the cell surface of microorganisms will be hydrolyzed by pepsin because pepsin has a strong proteolytic ability, thereby inhibiting the growth of microorganisms, resulting in poor tolerance of microorganisms to pepsin (Castañeda-Valbuena et al. 2022 ). The presence of inulin significantly increased the resistance to gastrointestinal conditions. The survival after simulated gastrointestinal conditions in 245 control conditions for L. plantarum CIDCA8727 (39.91 ± 9.02%) resulted significantly higher than the 246 survival for L. paracasei BGP1 (21.97 ± 1.98%) (Mahboubi and Kazempour 2016 ). Similarly, using calcium alginate gelatinized starch, chitosan coating, and inulin are encapsulated by emulsion technology to microencapsulate Lactobacillus casei and Bifidobacterium survival rate of probiotics is significantly increased under the condition of simulated gastric juice. However, the results of this study showed that using inulin as a carbon source could improve the stomach protein tolerance of yeast. At the same time, the effect on the other two microorganisms is not obvious. Under the same conditions of gastric protein solution, Bacillus subtilis had the tiniest viable bacteria in the study. It has the lowest tolerance to gastric protein fluid, which may be because the membrane protein on its surface is greatly affected by pepsin, resulting in its low activity. Pepsin is a highly specific protease with a certain amino acid sequence selection specificity, preferentially breaking the peptide bond formed by aromatic amino acids (phenylalanine, tyrosine, and tryptophan) or leucine (Kageyama 2002 ). Animal intestines usually contain a certain concentration of bile salts, which can change the permeability of cell membranes and cause cell death by destroying the integrity of cell membranes (Taranto et al. 2006 ). Bile salts will undergo an uncoupling reaction in the intestine, and this reaction is mainly catalyzed by bile salt hydrolyzing enzymes secreted by Bifidobacterium and Lactobacillus in the intestine (Pereira et al. 2004 ). The study found that adding 1% inulin and Lactobacillus acidophilus can promote the release of bile acid in vitro, which can affect serum cholesterol (Adebola et al. 2020 ). Adding of inulin in this article can improve the bile tolerance of Lactobacillus fermentum , which is similar to the experiment results. Lactobacillus casei and Lactobacillus rhamnosus isolated from human milk can also use inulin and oligofructose as carbon sources and have good acid and bile resistance (Tulumoglu et al. 2018 ). The inulin and Lactobacillus delbrueckii UFV h2b20 were processed into symbiotic cheese together, and it was found that inulin has protective effects on Lactobacillus delbrueckii UFV h2b20 under different bile salt concentrations and can enhance its resistance (Araújo et al. 2010 ). In addition, by studying the effect of adding inulin to alginate beads and observing its ability to protect the three probiotic strains, it turns out that beads containing 5% w/v inulin are the most effective at resisting bile salts (Atia et al. 2016 ). In this study, inulin as a carbon source can improve the bile salt tolerance of Saccharomyces cerevisiae , which can be increased about 81 times. It is speculated that yeast has a solid ability to use inulin. Saccharomyces cerevisiae has good tolerance to ethanol because of the characteristics of ethanol produced by Saccharomyces cerevisiae . However, the study of this article found that when inulin is used as a carbon source for probiotics, the tolerance of Bacillus subtilis to ethanol is most significantly improved. The research on the ethanol tolerance of Bacillus subtilis is because it can promote ethanol conversion into aromatic compounds through esterification and Maillard reaction during the flavor formation stage of liquor processing. It is a crucial probiotic in liquor processing (Mukherjee et al. 2009 ). Studies have found that the tolerance of Bacillus subtilis to ethanol is related to the phospholipids on its cell membrane. Placing Bacillus subtilis in a volume fraction of 7.6–30%, ethanol will cause the membrane to swell, accompanied by a decrease in membrane thickness (Gurtovenko and Anwar 2009 ). The high salt environment will cause the osmotic pressure to rise, resulting in the loss of microbial activity. Bacillus subtilis E221 was selected from the gut of Nile tilapia fed with 0.8% inulin at a salinity of 16 PSU for 8 weeks. Studies have found that Bacillus subtilisE221 has good salt tolerance, improving the growth stress of high salt to Nile tilapia (Tang et al. 2020 ). This result is the same as in this experiment. Inulin as a carbon source significantly improved the salt tolerance of Bacillus subtilis . The experimental results suggest that inulin significantly affects the salt tolerance of Bacillus subtilis . The growth of probiotics is usually inhibited at low temperatures. Low temperature may lead to the destruction of cell structure and also affect the activity of enzymes and the expression of related genes (Abadias et al. 2001 ). It can be seen from the experimental results that using inulin as a carbon source can improve the survival rate of Lactobacillus fermentum and Bacillus subtilis at 4°C. Nevertheless, it can reduce the survival rate of Saccharomyces cerevisiae , indicating that in a low-temperature environment, Saccharomyces cerevisiae's ability to utilize glucose could be stronger than that of inulin. It is speculated that the reason may be that inulin is a soluble dietary fiber, which is mainly a linear polysaccharide connected by D-furan fructose molecules (Li et al. 2019a ). Therefore, when bacteria use it, it produces less heat and energy. However, in another experiment, adding inulin can increase the viable bacteria of ice cream during the storage period of 120 days and keep the viability of yeast above 6 log cfu/g (Sarwar et al. 2021 ). Currently, inulin and other fructans are also used as freeze-drying protective agents for probiotics, because vacuum freeze-drying is the most successful and convenient way to preserve yeast, bacteria, and spore-forming fungi (Abadias et al. 2001 ). $$\text{s}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l} \text{r}\text{a}\text{t}\text{e}= \frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{r}\text{e}\text{m}\text{a}\text{i}\text{n}\text{i}\text{n}\text{g} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{o}\text{r}\text{i}\text{g}\text{i}\text{n}\text{a}\text{l} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}}\times 100\%C=\frac{2.57\times 46.07}{6300\times 1.0\times 0.10\times 2}\times 0.1266\times ({A}_{2}-{A}_{1})$$ Conclusion The addition of inulin can promote the growth performance of the three probiotics in different degrees, in which the effect of inulin on the growth performance of Bacillus subtilis the most obvious, followed by Saccharomyces cerevisiae , and finally, Lactobacillus fermentum . Adding inulin can promote the acid production capacity of Lactobacillus fermentum and the ethanol production capacity of Saccharomyces cerevisiae . Bacillus subtilis and Saccharomyces cerevisiae preferred inulin in the experiment of inulin replacing glucose in the medium, and the utilization effect of inulin was higher than that of glucose. In contrast, Lactobacillus fermentans preferred glucose, and glucose's utilization effect was higher than inulin's. Generally, inulin can improve the tolerance of probiotics in adverse environments to varying degrees. However, in different adverse environments, the effects of inulin on the tolerance of different probiotics are significantly different. Specifically, yeast's bile salt tolerance and pepsin tolerance have significantly improved. The ethanol tolerance, bile salt tolerance, and low-temperature tolerance of Lactobacillus fermentum are significantly improved. The ethanol, acid, and salt tolerance of Bacillus subtilis are significantly improved. This article proves that inulin positively affects the poor environmental tolerance of probiotics. However, its mechanism of action is still unclear, and follow-up research is still needed. Declarations Funding This research was funded by Education Department of Henan Province (No. 2019GGJS259) and Henan Academy of Sciences (No. 230503005, No.231003029, No. 240603015). Author contributions YGF: Conceptualization, Investigation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. YCN: Investigation, Data curation, Formal analysis, Funding acquisition. JC: Investigation, Validation, Data curation, Writing editing. CCQ: Methodology, Validation, Formal analysis. HFW: Investigation, Methodology, Formal analysis. NFH: Resources. Conflict of interest The authors declare that they have no conflict of interest in the publication. 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J Anim Sci 96 (1):206-214. https://doi.org/10.1093/jas/skx019 Wang H, Simpson JH, Kotra ME, Zhu Y, Wickramasinghe S, Mills DA, Chiu NHL (2021) Epitranscriptomic profile of Lactobacillus agilis and its adaptation to growth on inulin. BMC Res Notes 14 (1):154. https://doi.org/10.1186/s13104-021-05563-2 Wang SA, Li FL (2013) Invertase SUC2 Is the key hydrolase for inulin degradation in Saccharomyces cerevisiae. Appl Environ Microbiol 79 (1):403-406. https://doi.org/10.1128/AEM.02658-12 Yang F, Zhang X, Lu Y, Wang B, Chen X, Sun Z, Li X (2020) Inulin catabolism in Saccharomyces cerevisiae is affected by some key glycosylation sequons of invertase Suc2. Biotechnol Lett 42 (3):471-479. https://doi.org/10.1007/s10529-020-02791-7 Zherebtsov NA, Shelamova SA, Abramova IN (2002) [Biosynthesis of inulinases by Bacillus bacteria]. Applied Biochemistry & Microbiology 38 (6):544-548. https://doi.org/10.1023/A:1020722510374 Zhu Y, Liu J, Lopez JM, Mills DA (2020) Inulin Fermentation by Lactobacilli and Bifidobacteria from Dairy Calves. Appl Environ Microbiol 87 (1). https://doi.org/10.1128/AEM.01738-20 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4167997","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284809181,"identity":"8a08dcb9-f7e4-43c7-a956-8d3753c080b2","order_by":0,"name":"Yan-Ge Fan","email":"","orcid":"","institution":"Henan Institute of Chemistry Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yan-Ge","middleName":"","lastName":"Fan","suffix":""},{"id":284809182,"identity":"d6c3df09-3835-4f96-840f-03c2271428a6","order_by":1,"name":"Yu-Chang Ning","email":"","orcid":"","institution":"Henan University of Animal Husbandry and Economy","correspondingAuthor":false,"prefix":"","firstName":"Yu-Chang","middleName":"","lastName":"Ning","suffix":""},{"id":284809183,"identity":"7d633191-d6c1-4dae-9c04-184e13bab1c1","order_by":2,"name":"Jin Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDACCRA2YKhvkz984MCHHyRoYeyTYEs8OLOHWC1AwDhPgsf4MAcbETr4Zzcfe2BRcIeZTbrnw2EGHgZ5frEDBCy5cyzdQMLgGRubzNkNhwssGAxnzk7Ar8VAIsdMQsLgMA8bQ+6GwzN4GBIMbhPUkv8NpEWCjSHnAUgjMVpy2EBaDNgkchiI0yJxIw3ssAQ2nmMGwECWIOwX/hnJz6Ql/hxOkG9vfvzhww8beX5pAlpAgFkCyVbCykGA8QNx6kbBKBgFo2CkAgAgSkCJgEXymAAAAABJRU5ErkJggg==","orcid":"","institution":"Henan Institute of Chemistry Henan Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jin","middleName":"","lastName":"Chen","suffix":""},{"id":284809186,"identity":"2b5bf70e-0b63-41ec-b378-54cda7a89a9b","order_by":3,"name":"Chang-Qing Cao","email":"","orcid":"","institution":"Henan Institute of Chemistry Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chang-Qing","middleName":"","lastName":"Cao","suffix":""},{"id":284809188,"identity":"0ee13096-6757-47de-9574-d20d48caf762","order_by":4,"name":"Hui-Feng Wang","email":"","orcid":"","institution":"Henan Institute of Chemistry Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hui-Feng","middleName":"","lastName":"Wang","suffix":""},{"id":284809190,"identity":"da2eccb7-bb3f-4395-b20e-58546f266c62","order_by":5,"name":"Nan-Feng Han","email":"","orcid":"","institution":"Henan Institute of Chemistry Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nan-Feng","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2024-03-26 07:54:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4167997/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4167997/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53847941,"identity":"1a79d282-cd37-4385-9714-cf16f5bca40e","added_by":"auto","created_at":"2024-04-01 09:02:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114597,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inulin on the content of viable bacteria in the fermentation broth of \u003cem\u003eBacillus subtilis. \u003c/em\u003eIAG; inulin alternative group, CON; control group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4167997/v1/6ced13958640e7ec39d4909c.png"},{"id":53847940,"identity":"c31d2fb8-90f0-4df7-ba05-0414499675e2","added_by":"auto","created_at":"2024-04-01 09:02:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60886,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inulin on the content of viable bacteria in the fermentation broth of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4167997/v1/0e6a6414e1df74697b105207.png"},{"id":53847939,"identity":"bf52e844-9a9f-47ea-9875-f4de13d97c16","added_by":"auto","created_at":"2024-04-01 09:02:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79999,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inulin on the ethanol production capacity of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4167997/v1/afdb82e3317b34644508a355.png"},{"id":53847942,"identity":"8a1261a5-c584-483b-9376-eae027fa0f7a","added_by":"auto","created_at":"2024-04-01 09:02:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58723,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inulin on the content of viable bacteria in the fermentation broth of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4167997/v1/bfed18f652527f0d4f9c944b.png"},{"id":53847943,"identity":"e19a963a-ffe0-4475-90f6-5c37b9c095f7","added_by":"auto","created_at":"2024-04-01 09:02:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43423,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inulin on the acid production capacity of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4167997/v1/ae640261de768832cd887c9f.png"},{"id":55610283,"identity":"c52801ea-41e6-466b-9b4b-c7ca06d8693a","added_by":"auto","created_at":"2024-04-30 14:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1451757,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4167997/v1/f1ddb8e1-5c8a-4b23-b774-1765c9056b78.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of inulin on the growth performance and tolerance in adverse environments of several probiotics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProbiotics can enter the animal\u0026rsquo;s gastrointestinal tract and multiply rapidly and colonize the surface of intestinal epithelial cells to form a microbial barrier in the intestine to prevent the invasion of pathogenic microorganisms. Since probiotics colonize the digestive tract of the host body, the probability of infection by harmful pathogenic microorganisms is reduced, thereby reducing the risk of infection of the body (Brownawell et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Probiotics can also produce various hydrolytic enzymes to decompose materials that are not easily decomposed in the intestinal tract of animals to promote the digestion and absorption of nutrients in the intestinal tract. \u003cem\u003eLactobacillus\u003c/em\u003e is the most commonly used type in probiotic preparations, colonized in the human digestive tract. Some nutrients are more favorable for human absorption after fermentation and metabolism by \u003cem\u003eLactobacillus\u003c/em\u003e itself or promote the host\u0026rsquo;s health by antagonistic biological effects and reducing intestinal pH. \u003cem\u003eLactobacillus\u003c/em\u003e can grow at pH 3.0\u0026thinsp;~\u0026thinsp;4.5 and have a certain tolerance to gastric acid (Jorjao et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus\u003c/em\u003e is a common probiotic because it can form spores and has good stability, acid, alkali, high temperature, and pressure resistance. It can exist in the form of spores during storage. \u003cem\u003eBacillus\u003c/em\u003e can consume oxygen in the intestinal tract, maintain an anaerobic environment, inhibit harmful bacteria growth, and maintain intestinal flora balance. Wang et al. found that feeding 15% \u003cem\u003eBacillus subtilis\u003c/em\u003e fermented feed to lactating sows can significantly improve the apparent digestibility of its dry matter, total phosphorus, and total energy (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYeast is a facultative anaerobic microorganism primarily used in the fermentation industry for alcohol fermentation and can also be used as a microecological agent in feed additives. Given that the host's endogenous digestive enzymes cannot decompose the mannan of the yeast cell wall, the mannan of the yeast itself can be used as a prebiotic to promote the proliferation of other probiotics, lower the pH in the intestine, and inhibit the growth of harmful bacteria such as E. coli. In aquaculture, given the rich mannan content of yeast cell walls, it can significantly promote microvilli growth in aquatic animals' intestinal epithelial cells, change their morphology, increase the surface area of intestinal folds, and better absorb nutrients (Machov\u0026aacute; and Bystrick\u0026yacute; \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrebiotics selectively stimulate the growth of probiotics resident in the gut, especially Bifidobacteria, through the production of β-fructosidase, changing the colonic microflora to a healthier composition. The common prebiotics is fructooligosaccharides, isomalt oligosaccharides, dextran, inulin, Hieracium, and lentinan. Probiotics are degraded, digested and absorbed after reaching the posterior intestine. Their products can provide energy and nutrients for the growth and proliferation of intestinal epithelial cells to promote or repair the intestinal mucosa. Prebiotics also significantly promote the growth of probiotics in vitro (Ogueke et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInulin is a natural reserve polysaccharide that is widely distributed in nature. It mainly exists in composite plants. The primary raw materials of inulin are chicory (\u003cem\u003eCichorium intybus\u003c/em\u003e L.) and \u003cem\u003eJerusalem artichoke\u003c/em\u003e. Inulin can promote the proliferation of probiotics and improve the community of probiotics. No enzyme in the human intestine can digest inulin. Thus inulin can be used by probiotics through the small intestine of the human body and promote the growth and proliferation of probiotics, reduce the pH in the intestine, and then inhibit the proliferation of many harmful bacteria, reduce the production of harmful toxins in the intestine and improve the health level of the intestine (Sasajima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Inulin can reduce the number of \u003cem\u003eE. coli\u003c/em\u003e in the intestine, increase the number of probiotics such as \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, and improve the community structure of intestinal microorganisms (Samanta et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It is reported that adding inulin to the diet of obese mice can reduce the abundance of \u003cem\u003eBacteroides\u003c/em\u003e in the intestine of mice and increase the proportion of \u003cem\u003eFirmicum\u003c/em\u003e (Vandeputte et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Valcheva et al. found that after taking 15 g of inulin daily, the intestinal microbial community structure changed (Valcheva et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In vitro experiments have found that using \u003cem\u003eJerusalem artichoke\u003c/em\u003e root extract can significantly increase the number of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eEnterococcus faecium\u003c/em\u003e. Inulin can increase butyric acid production in the intestinal tract by increasing the number of \u003cem\u003eBifidobacteria\u003c/em\u003e or \u003cem\u003elactic acid bacteria\u003c/em\u003e in the intestinal tract and indirectly reducing inflammation and harmful bacteria (Hoentjen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Guo et al. found that adding inulin to the diet can improve gastrointestinal discomfort and intestinal inflammation and reduce obesity-related diseases caused by high-fat food intake (Guo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInulin has a significant role in promoting the growth and proliferation of various probiotics in the intestine and the colonization of probiotics in the intestine. At present, the research on the probiotic function of inulin is mostly \u003cem\u003ein vivo\u003c/em\u003e to explore the effect of inulin on the growth performance of intestinal probiotics. However, only some studies reported on the probiotic effect of inulin \u003cem\u003ein vitro\u003c/em\u003e. This study focused on adding different inulin to the fermentation broth and using inulin as an alternative carbon source. The colony count was measured to investigate the effect of inulin on the proliferation ability of three probiotics \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. A study on the effect of inulin improving the tolerance of these three probiotics in an adverse environment was also conducted.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and reagents\u003c/h2\u003e \u003cp\u003eInulin comes from the Jerusalem Artichoke Industry Engineering Technology Research Center, Henan Academy of Sciences. Beef meal and peptone were purchased from Beijing Obosing Biotechnology Co., Ltd (Beijing, China). Yeast extract powder was purchased from Qingdao Science and Technology Industrial Park Haibo Biological Technology Co., Ltd (Qingdao, China). Glucose, sodium acetate, and disodium hydrogen citrate are of analytical purity, purchased from the Tianjin Damao chemical reagent factory. Manganese sulfate and magnesium sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and Tianjin Zhiyuan Chemical Reagent Co., Ltd (Tianjin, China).\u003c/p\u003e \u003cp\u003eYPD medium: peptone 20.0 g, glucose 20.0 g, yeast extract 10.0 g, distilled water 1000 mL, pH 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2.\u003c/p\u003e \u003cp\u003eMRS medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dimethyl hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, sulfuric acid Manganese 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH 5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2.\u003c/p\u003e \u003cp\u003eGlucose peptone medium: glucose 5g, peptone 6g, yeast extract powder 6 g, beef extract powder 1.5 g, Tween 80 1 g, distilled water 1000 mL, pH 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of seed solution\u003c/h2\u003e \u003cp\u003ePreparation of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e seed liquid: Selected \u003cem\u003eLactobacillus fermentum\u003c/em\u003e strain into 20 mL MRS liquid medium and shook it at 37\u0026deg;C and 150 r/min for 24 h the seed liquid of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePreparation of \u003cem\u003eBacillus subtilis\u003c/em\u003e seed solution: The \u003cem\u003eBacillus subtilis\u003c/em\u003e slant strain was picked into 20 mL of glucose peptone medium, cultured at 37\u0026deg;C, and 150 r/min for 12 h \u003cem\u003eBacillus subtilis\u003c/em\u003e seed solution.\u003c/p\u003e \u003cp\u003ePreparation of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e seed liquid: The yeast (Angel Yeast Co., Ltd., Yichang, China) was activated and then gradually diluted using the dilution-coated plate method to purify the strain. After 24 h of culture at 25\u0026deg;C, a single colony was picked and cultured on the slope of the test tube. Then it was placed in the 20 mL YPD medium and cultured at 28\u0026deg;C and 150 r/min for 24 h, which was the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e seed liquid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEffect of inulin on the growth performance of the probiotics\u003c/h2\u003e \u003cp\u003eDifferent concentrations of 1%, 2%, 3%, 4%, and 5% inulin were added to MRS liquid medium, YPD liquid medium, and glucose peptone medium. Moreover, the sample in which no inulin was added was set as a control group (CON). The other group is the alternative inulin group (IAG). Inulin was used as a substitute for carbon sources and the same quantity of inulin was used as an alternative to glucose in the basal medium.\u003c/p\u003e \u003cp\u003eThe inoculation volume of all groups was 5%. \u003cem\u003eBacillus subtilis\u003c/em\u003e was inoculated in a glucose peptone medium, cultured at 37\u0026deg;C, and shaken at 150 r/min for 12 h. \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was inoculated into YPD liquid medium, cultured at 25\u0026deg;C, and shaken at 150 r/min for 24 h. \u003cem\u003eLactobacillus fermentum\u003c/em\u003ewas inoculated in MRS liquid medium, cultured at 37\u0026deg;C, and shaken at 150 r/min for 24 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of inulin on the improvement of the tolerance of several probiotics in an adverse environment\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEffect of inulin on acid and bile salt tolerance of the probiotics\u003c/h2\u003e \u003cp\u003eThe method used in this study was described as Kusada (Kusada et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The pH of the culture medium was adjusted to 3.0 with 0.1 M HCl to simulate the gastric acid experiment. The 0.3% bovine bile salt was added to the culture broth of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e, and 0.05% bovine bile salt was added to the culture broth \u003cem\u003eLactobacillus fermentum\u003c/em\u003e to perform the simulated bile test. The 5% seed solution of each probiotic strain was inoculated into the simulated gastric acid and bile culture solution, respectively. \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e were then cultured at 37\u0026deg;C and 150 r/min for 24 h, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was cultured at 28\u0026deg;C and 150 r/min for 24 h. Then the total plate count was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffect of inulin on ethanol tolerance of the probiotics\u003c/h2\u003e \u003cp\u003eAdded 10% absolute ethanol to the medium. Next, inoculated each probiotic strain's 5% seed solution into the medium. \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e were then cultured at 37\u0026deg;C and 150 r/min for 24 h, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was cultured at 28\u0026deg;C and 150 r/min for 24 h. Then the total plate count was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of inulin on pepsin resistance of the probiotics\u003c/h2\u003e \u003cp\u003ePrepared 10% pepsin solution and then added it to the culture medium to reach a final concentration of 1%. Then inoculated 5% seed solution of each probiotic strain was put into the medium. \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e were then cultured at 37\u0026deg;C and 150 r/min for 24 h, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was cultured at 28\u0026deg;C and 150 r/min for 24 h. Then the total plate count was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect of inulin on salt tolerance of the probiotics\u003c/h2\u003e \u003cp\u003eAdded 10% sodium chloride solution to the culture medium. Next, inoculated each probiotic strain's 5% seed solution into the medium. \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e were then cultured at 37\u0026deg;C and 150 r/min for 24 h, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was cultured at 28\u0026deg;C and 150 r/min for 24 h. Then the total plate count was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of inulin on the survival rate of the probiotics at low temperature\u003c/h2\u003e \u003cp\u003eInoculated each probiotic strain's 5% seed solution into the medium. \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e were then cultured at 37\u0026deg;C and 150 r/min for 24 h, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was cultured at 28\u0026deg;C and 150 r/min for 24 h. Then they were placed at 4\u0026deg;C for 7 d. Moreover, the total plate count was determined. The survival rate of the strains was obtained as follows.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{s}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l} \\text{r}\\text{a}\\text{t}\\text{e}= \\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{r}\\text{e}\\text{m}\\text{a}\\text{i}\\text{n}\\text{i}\\text{n}\\text{g} \\text{v}\\text{i}\\text{a}\\text{b}\\text{l}\\text{e} \\text{b}\\text{a}\\text{c}\\text{t}\\text{e}\\text{r}\\text{i}\\text{a}}{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{o}\\text{r}\\text{i}\\text{g}\\text{i}\\text{n}\\text{a}\\text{l} \\text{v}\\text{i}\\text{a}\\text{b}\\text{l}\\text{e} \\text{b}\\text{a}\\text{c}\\text{t}\\text{e}\\text{r}\\text{i}\\text{a}}\\times 100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the probiotics content\u003c/h2\u003e \u003cp\u003eThe dilution coating plate method was used to determine the probiotics content. Appropriate gradient dilutions of the cultured bacterial liquid in sterile water were prepared in a sterile environment. Totally 0.1 mL of the three dilutions of the culture was inoculated on the surface of the corresponding solid medium for coating. After coating, the plates of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e were inverted and cultured in a constant temperature incubator for 12 h. The plate of \u003cem\u003eLactobacillus fermentum\u003c/em\u003ewas inverted and cultured for 24 h. Then the number of colonies on each plate was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the physical and chemical indicators\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of pH of Lactobacillus fermentum fermentation broth\u003c/h2\u003e \u003cp\u003eThe acidity of the fermentation broth of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e was directly measured using a pH meter (Mettler-Toledo Measurement Instrument Shanghai Co Ltd., Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of ethanol production capacity of Saccharomyces cerevisiae\u003c/h2\u003e \u003cp\u003eTotally 1.5 mL of the inoculated \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e fermentation broth was centrifuged at 8 000 r/min and filtered, and the ethanol content in the fermentation broth was determined by the enzyme labeling method. The 0.1 mL of filtered bacterial solution was transferred to the test tube. Then 2 mL distilled water, 0.20 mL solution 1 (buffer), 0.20 mL solution 2 (NAD\u003csup\u003e+\u003c/sup\u003e), and 0.05 mL solution 3 (aldehyde dehydrogenase) were added successively. It was stood for 30 min and diluted to 4 times.\u003c/p\u003e \u003cp\u003eThen 1 mL sample was mixed with 3 mL distilled water. The absorbance A\u003csub\u003e1\u003c/sub\u003e at 340 nm was then measured. The 0.02 mL suspension 4 (ethanol dehydrogenase) was added to the sample. The absorbance A\u003csub\u003e2\u003c/sub\u003e at 340 nm was measured. The ethanol (C) content in the fermentation broth was calculated as follows.\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$C=\\frac{2.57\\times 46.07}{6300\\times 1.0\\times 0.10\\times 2}\\times 0.1266\\times ({A}_{2}-{A}_{1})$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe statistical significance of the experimental data was determined by one-way analysis of variance (ANOVA) using the SPSS 22.0 for Windows statistical package (SPSS, Inc., Chicago, IL). The mean and standard deviation of the data were determined using Excel 2003 (Microsoft, Inc., Seattle, WA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of inulin on the growth performance of Bacillus subtilis\u003c/h2\u003e \u003cp\u003eThe effect of inulin as an added and alternative carbon source on the growth performance of \u003cem\u003eBacillus subtilis\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. When inulin replaced the glucose in the basal medium, the number of \u003cem\u003eBacillus subtilis\u003c/em\u003e in the fermentation broth was 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL, which was 5.7 times that of the control group. The results indicated that the utilization ability of \u003cem\u003eBacillus subtilis\u003c/em\u003e to inulin was significantly better than that of glucose. In addition, adding different proportions of inulin could significantly increase the content of \u003cem\u003eBacillus subtilis\u003c/em\u003e in the fermentation broth compared with the control group. When 1% inulin was added, the content of \u003cem\u003eBacillus subtilis\u003c/em\u003e in the fermentation broth was the highest, reaching 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL, which was 10.6 times that of the control group. However, with the increase of inulin, the content of viable \u003cem\u003eBacillus subtilis\u003c/em\u003e showed a downward trend. The results showed that inulin could significantly increase the content of viable bacteria in the fermentation broth of \u003cem\u003eBacillus subtilis\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of inulin on the growth performance of Saccharomyces cerevisiae\u003c/h2\u003e \u003cp\u003eIt can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e that when inulin replaced the glucose in the basal medium, the number of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in the fermentation broth was 4.2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL, which is 2.6 times that of the control group. The results indicated that the utilization ability of inulin by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was significantly better than that of glucose. In addition, adding different proportions of inulin could significantly increase the content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in the fermentation broth compared with the control group. The content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in the fermentation broth increased with the increase of the inulin amount. When 5% inulin was added, the content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in the fermentation broth was the highest, reaching 5.5\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL, 3.3 times that of the control group. The results showed that inulin could significantly increase the viable bacteria content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of inulin on ethanol production of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It can be seen that when inulin replaced glucose in the basic medium, the volume concentration of ethanol in the fermentation broth was 6.9%, which was 1.15 times that in the control group. This result indicated that the ability of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e to use inulin to metabolize ethanol is significantly better than that of glucose. In addition, adding different proportions of inulin could significantly increase ethanol content compared with the control group. The volume concentration of ethanol increased with the increase of inulin. When added 5% inulin was, the volume of ethanol in the solution was the highest, reaching 7.85%, 1.3 times that of the control group. The results showed that inulin could significantly improve the ethanol production capacity of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e as a substitute or as a carbon source.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe effect of inulin on the growth performance of Lactobacillus fermentum\u003c/h2\u003e \u003cp\u003eThe effect of inulin on the growth performance of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. It can be seen that when inulin replaced the glucose in the basal medium, the number of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e in the fermentation broth was 4.4\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL, which was 0.6 times that of the control group. This result showed that the use of \u003cem\u003eLactobacillus fermentum\u003c/em\u003ewas significantly better than that of inulin. In addition, adding different proportions of inulin could significantly increase the content of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e in the fermentation broth compared with the control group. When 3% inulin was added, the \u003cem\u003eLactobacillus fermentum\u003c/em\u003e content in the fermentation broth was the highest, reaching 9.9\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL, which was 1.35 times that of the control group. When the inulin was added at 4% and 5%, the content of viable bacteria tended to be stable. The results showed that inulin could significantly increase the content of viable bacteria in the fermentation broth of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); however, it could inhibit the proliferation of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e when inulin was added as an alternative carbon source to the basic medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, when inulin replaced glucose in the basic medium, the pH value of the fermentation liquid was 4.39, which was significantly higher than that of the control group. The results showed that the ability of \u003cem\u003eLactobacillus\u003c/em\u003e fermentation to produce acid by inulin was significantly lower than that of glucose. In addition, adding different proportions of inulin could significantly reduce the pH of the fermentation broth compared with the control group. The pH of the fermentation broth decreased with the increase of inulin. When the amount of inulin reached about 3%, the pH value was the lowest and did not decrease. The results showed that inulin could significantly improve the acid production capacity of \u003cem\u003eLactobacillus fermentans\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, inulin could not promote the acid production capacity of \u003cem\u003eLactobacillus ferments\u003c/em\u003e when it was used to replace glucose in the basic medium. This result was consistent with the result of viable bacteria obtained above.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of inulin on the acid tolerance, bile salt tolerance, ethanol tolerance, pepsin tolerance, salt tolerance, and low-temperature tolerance of the three probiotics\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe acid tolerance of inulin to the three probiotics is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It can be seen that under pH 3, the viable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e was significantly higher than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant difference was observed in the viable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e between the experimental and control groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of inulin on the acid tolerance of the probiotics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViable \u003cem\u003eLactobacillus\u003c/em\u003e content of \u003cem\u003efermentans\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e8\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferent letters in the same column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the viable contents of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of the control group. The results indicated that inulin significantly improved the bile tolerance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. However, no significant difference was observed in the viable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e between the experimental and control groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of inulin on bile salt tolerance of probiotics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViable content of \u003cem\u003eLactobacillus fermentans\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e6\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferent letters in the same column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e showed that when 10% absolute ethanol was added, the viable contents of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eBacillus subtilis in\u003c/em\u003e the experimental group were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of the control group. However, no significant difference was observed in the viable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e between the experimental group and the control group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of inulin on ethanol tolerance of probiotics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViable content of \u003cem\u003eLactobacillus fermentans\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e8\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferent letters in the same column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eIt can be seen from Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e that under the condition of adding 1% pepsin to the culture medium when the carbon source in the original culture medium was replaced by inulin, the viable content of the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of a control group. However, no significant difference was observed in the viable content of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e between the experimental and control groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of inulin on the pepsin tolerance of probiotics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e5\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViable content of \u003cem\u003eLactobacillus fermentans\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e5\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e2\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferent letters in the same column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe effect of inulin on the salt tolerance of the probiotics is shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It can be seen that when 10% NaCl was added, the viable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of the control group. However, no significant difference was observed in the viable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e between the experimental and control groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of inulin on the salt tolerance of probiotics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e5\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViable content of \u003cem\u003eLactobacillus fermentans\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e7\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViable content of \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u0026times;10\u003csup\u003e5\u003c/sup\u003e(CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDifferent letters in the same column indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe inulin results on low-temperature tolerance of three probiotics are shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The results showed that the survival rate of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e was significantly higher than that of the control group. However, the survival rate of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was significantly lower than that of the control group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of inulin on low-temperature tolerance of the probiotics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eSurvival rate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus fermentans\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e88.70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e102.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a prebiotic, inulin has been widely used for decades. Although the human diet is generally rich in complex carbohydrates, the human body contains fewer glycosidases, so it can only hydrolyze sucrose, lactose, and some starches, but not inulin (Goh and Klaenhammer \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, most of these indigestible complex carbohydrates reach the gut and are transported and metabolized by some gut microbiota members. Inulin can improve intestinal probiotics, reduce intestinal pH and promote the production of short-chain fatty acids to improve intestinal health (Liu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Inulin intake can significantly increase the relative abundance of probiotics such as \u003cem\u003eBifidobacteria\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e and inhibit the abundance of pathogenic bacteria. Maintaining the activity of probiotics and their ability is essential in the processing and applying of probiotics (Kim et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This research studied the effects of inulin as a carbon source and alternative carbon source on three probiotics' growth and production performance. The results showed that inulin had the most apparent promoting effect on the growth performance of \u003cem\u003eBacillus subtilis\u003c/em\u003e. The addition of inulin had a promoting effect on the acid production capacity of Lactobacillus fermentum and the ethanol production capacity of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. In this paper, when inulin was used as a carbon source, it could significantly enhance the growth performance of \u003cem\u003eBacillus subtilis\u003c/em\u003e. Adding a carbon source to the medium could also promote the growth of \u003cem\u003eBacillus\u003c/em\u003e to a certain extent. \u003cem\u003eBacillus\u003c/em\u003e is a bacterium that can produce inulinase, decompose, and utilize inulin (Seydlova et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEarly research found that \u003cem\u003eBacillus subtilis\u003c/em\u003e could produce inulinase with good heat stability (Vullo et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Zherebtsov et al. found that when garlic, onion extract, inulin, and soluble starch were used as carbon sources, the productivity of inulinase was the highest when microorganisms were inoculated on soluble starch (Zherebtsov et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, the result is different from this study. Therefore, it can be seen that inulin is not inducible to inulinase production by \u003cem\u003eBacillus subtilis.\u003c/em\u003e In this study, when 1% inulin was added as a supplementary energy source, the content of \u003cem\u003eBacillus subtilis\u003c/em\u003e was the highest, about 10.6 times that of the control group. Qian et al. found that supplementing inulin could increase biomass and improve metabolic pathways (Qian et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). At the same time, the combined use of \u003cem\u003eBacillus subtilis\u003c/em\u003e and inulin can improve the immune performance of animals and improve intestinal flora (Cerezuela et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Therefore, inulin has a good role in promoting \u003cem\u003eBacillus subtilis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn previous studies, it has been found that inulin can be directly used as a carbon source for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e without being pretreated by acid and enzymes, and the yeast can ferment it to produce ethanol (Lim et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The degree of polymerization of inulin has a more significant impact on its processing properties, and studies have shown that inulin mixtures with different degrees of polymerization can increase the effect of prebiotics in the colon (Lopes et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nowadays, synthetic methods can generally be used to control the degree of polymerization of synthetic inulin. Different \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e has different ability to utilize inulin. Studies have shown that the yield of two different \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e using inulin to produce ethanol can differ by 1.6 times (Lim et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInvertase Suc2 is a key hydrolase for the degradation of inulin by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Previous research found that when using inulin as a carbon source for \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, the activity of extracellular enzymes is 4.3 times higher than when using sucrose (Wang and Li \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the study of the activity of the invertase Suc2, it was found that among the 13 N-glycosylation sites of Suc2, the sugar chains of N4, N45, N78, and N146 play an essential role in the host strain's inulin transformation. The desugar effect of N4, N7,8, and N146 sequences increased Suc2 activity and ethanol production (Yang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This may be why different \u003cem\u003eSaccharomyces cerevisiahave\u003c/em\u003eas have different ethanol producticapacitiesity. In this study, inulin used as an alternative carbon source can improve the growth performance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. \u003cem\u003eSaccharomycbollarddii\u003c/em\u003e and inulin are added to the yogurt together to ensure the activity of the yeast during the 28-day storage period, and the number of viable cells in the yogurt can exceed 6.0 log CFU/g (Sarwar et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, inulin as an added carbon source can increase ethanol output and is dose-dependent. Because \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e has a solid ability to produce inulinase, the utilization of inulin by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e increases ethanol production (Chi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Li et al. found that \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e has the highest endo-inulinase activity in the microbial decontamination test of the crude Jerusalem artichoke inulin extract compared with 9 other yeasts (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). It suggests that the addition of inulin positively affects the growth and fermentation of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e are both important acid-producing microorganisms in the human intestinal flora. In previous studies, it has been found that \u003cem\u003eBifidobacteria\u003c/em\u003e can use inulin-type fructans as the only energy source, and inulin can also increase the number of \u003cem\u003eBifidobacteria\u003c/em\u003e in the intestine (Rossi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eLactobacillus\u003c/em\u003e is highly nutritious and mainly grows in an environment rich in carbohydrates. When \u003cem\u003eLactobacillus\u003c/em\u003e uses inulin as the only energy source and the final product, lactic acid, it also produces acetic acid, formic acid, and ethanol (Makras et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The results showed that inulin used as an added carbon source could significantly increase the content of viable bacteria in the fermentation broth of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, inulin as an alternative carbon source added to the basic medium can inhibit the proliferation of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. It is speculated that the inulinase produced by \u003cem\u003eLactobacillus fermentum\u003c/em\u003e may have a singularity or tend to be short-chain or long-chain inulinase, weakening the \u003cem\u003eLactobacillus fermentum\u003c/em\u003e's ability to utilize inulin. This may be related to the strain of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. A recent study screened the ability of \u003cem\u003eLactobacillus\u003c/em\u003e to ferment fructooligosaccharides, and the results showed that 12 of the 16 strains of \u003cem\u003eLactobacillus\u003c/em\u003e fermented fructooligosaccharides (Kaplan and Hutkins \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). It suggests that not all \u003cem\u003eLactobacillus\u003c/em\u003e can ferment inulin.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003elactobacilli\u003c/em\u003e capable of metabolizing fructooligosaccharides, using fructooligosaccharides appears to be through two catabolic pathways. The first is that the oligofructose is transported into the cytoplasm intact and is hydrolyzed by GH32 β-fructosidase, and different \u003cem\u003elactobacilli\u003c/em\u003e have different ways of transporting and ingesting oligofructose (Goh and Klaenhammer \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Velikova et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The second transformation method combines fructooligosaccharides with GH32 β-fructosidase on the cell surface.The oligofructose is hydrolyzed outside the cell, and then the decomposition products, fructose, glucose, etc., are transported and taken up (Velikova et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eLactobacillus paracasei\u003c/em\u003e, commonly used in research, is the second approach. \u003cem\u003eLactobacillus paracasei\u003c/em\u003e can produce large amounts of lactic acid through simultaneous saccharification and fermentation of inulin. Studies have shown that high concentrations of Mn\u003csup\u003e2+\u003c/sup\u003e accelerate the hydrolysis of inulin by increasing the activity of β-fructosidase, and increasing the conversion of sugar to lactic acid by increasing the overall glycolytic flux (Petrov et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExtracellular β-fructosidase plays an important role in the decomposition of long-chain inulin. In addition, β-fructosidase decomposes and metabolizes long-chain inulin and releases monomer fructose through the extracellular matrix, which can be cross-fed to other consumers in the gut (Zhu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Previous studies have shown that \u003cem\u003eLactobacillus para case W20\u003c/em\u003e can stimulate the growth of \u003cem\u003eLactobacillus salivarius w57\u003c/em\u003e by accumulating fructooligosaccharides (2\u0026ndash;3 degrees of polymerization) using a family of GH32 enzymes. Therefore, when inulin is used by probiotics such as \u003cem\u003eLactobacillus\u003c/em\u003e, it will also promote the growth of other microorganisms.\u003c/p\u003e \u003cp\u003eIn addition, the inhibition of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e may also be related to the different inulin actions of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. The previous report pointed out that \u003cem\u003eLactobacillus paracasei\u003c/em\u003e can use long-chain inulin as the sole carbon source and ferment fructooligosaccharide and oligofructose-enriched inulin. In contrast, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e can only ferment oligofructose-enriched inulin (Makras et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In this paper, it is found that inulin is used as a carbon source, which can significantly improve the acid production capacity of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. It showed that glucose is the carbon source that \u003cem\u003eLactobacillus fermentum\u003c/em\u003e preferentially selects during fermentation. Makras et al. found that when inulin rich in fructooligosaccharides was used as the sole energy source of \u003cem\u003eLactobacillus paracasei\u003c/em\u003e, the free fructose in the fermentation medium was first fermented, then fructooligosaccharides and inulin were degraded, resulting in the accumulation of fructose, glucose and sucrose, and then consumed. The degradation of long-chain inulin generally occurs in the later decomposition stage and conforms to the above decomposition rules (Makras et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). By observing the growth curve of \u003cem\u003eLactobacillus agilis YZ050\u003c/em\u003e in the mixture of glucose and inulin, it is found that \u003cem\u003eLactobacillus agilis YZ050\u003c/em\u003e has a secondary growth phenomenon, which may be due to the initial consumption of glucose.\u003c/p\u003e \u003cp\u003eThen inulin is a carbon source, and the latter's speed is significantly higher than the former's (Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Petrova et al. (Petrova et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) used \u003cem\u003eLactobacillus paracasei\u003c/em\u003e to saccharify and ferment inulin, the productivity of lactic acid was 1.08 g/L/h, and the raw material conversion rate of inulin was 91%. This result indicates that inulin can promote the fermentation of \u003cem\u003eLactobacillus\u003c/em\u003e, which is consistent with the results when inulin is used as a carbon source in this experiment. Therefore, the fermentation of inulin to \u003cem\u003eLactobacillus\u003c/em\u003e is affected by factors such as the species of \u003cem\u003eLactobacillus\u003c/em\u003e and the type of inulin. However, in general, it promotes the growth and fermentation of \u003cem\u003eLactobacillus\u003c/em\u003e. Since inulin has a good effect on the growth and fermentation of probiotics, many studies have used probiotics to produce inulinase, thereby expanding the application of inulin (Jiang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis paper studied the survival rate of \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e under different environmental conditions when inulin was used as the carbon source influence of inulin on the growth of probiotics was explored. Due to probiotics' excellent effect on intestinal health, many studies have processed probiotics into oral liquid and tablet preparations to treat and prevent intestinal diseases. Inulin has been used in industry as a prebiotic. Gut microbes can ferment inulin to produce short-chain fatty acids, which can induce the growth of beneficial microorganisms, thereby changing the composition of the organisms in the gut microbiota and enhancing the host\u0026rsquo;s immune system (Seifert and Watzl \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProbiotics need to reach the intestine smoothly and maintain activity because it is a particular environment in the intestine. Safely delivering probiotics to the intestines requires the harsh low-acid environment of the stomach and the influence of gastric protein. The main component of gastric acid in human gastric juice is hydrochloric acid, its pH is usually around 3.0, and it can reach 2.5 under fasting conditions (Mulaw et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A recent study conjugated phthalate groups with inulin and combined them with probiotics into compressed tablets. In a simulated intestinal environment, the activity of probiotics is also significantly more robust than compressed tablets without inulin (Kim et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).In this study, it was found that under the condition of pH 3, the use of inulin as a carbon source significantly increased the content of live \u003cem\u003eBacillus subtilis\u003c/em\u003e. The viable bacteria content of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e was also increased by a certain amount after using inulin as a carbon source. \u003cem\u003eLactobacillus\u003c/em\u003e itself can produce acid and has good acid resistance. The study of the symbiotic cheese of inulin and \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e found that the number of viable bacteria under pH 3.5 can be the same as that under the neutral condition (Ara\u0026uacute;jo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Under lower pH conditions, on the one hand, it will affect the transmembrane transport of nutrients by microorganisms.\u003c/p\u003e \u003cp\u003eOn the other hand, it will affect the activity of various enzymes in microbial cells, thereby affecting the growth of microorganisms (Mulaw et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The optimum pH of inulinase produced by most microorganisms is 4.0\u0026ndash;6.0, and the inulinase can remain relatively stable near the optimum pH (Atia et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, adding inulin as a carbon source can increase the acid tolerance of probiotics and improve the body's intestinal health.\u003c/p\u003e \u003cp\u003eSome enzymes and membrane proteins present on the cell surface of microorganisms will be hydrolyzed by pepsin because pepsin has a strong proteolytic ability, thereby inhibiting the growth of microorganisms, resulting in poor tolerance of microorganisms to pepsin (Casta\u0026ntilde;eda-Valbuena et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The presence of inulin significantly increased the resistance to gastrointestinal conditions. The survival after simulated gastrointestinal conditions in 245 control conditions for L. plantarum CIDCA8727 (39.91\u0026thinsp;\u0026plusmn;\u0026thinsp;9.02%) resulted significantly higher than the 246 survival for L. paracasei BGP1 (21.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98%) (Mahboubi and Kazempour \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, using calcium alginate gelatinized starch, chitosan coating, and inulin are encapsulated by emulsion technology to microencapsulate \u003cem\u003eLactobacillus casei\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e survival rate of probiotics is significantly increased under the condition of simulated gastric juice. However, the results of this study showed that using inulin as a carbon source could improve the stomach protein tolerance of yeast. At the same time, the effect on the other two microorganisms is not obvious. Under the same conditions of gastric protein solution, \u003cem\u003eBacillus subtilis\u003c/em\u003e had the tiniest viable bacteria in the study. It has the lowest tolerance to gastric protein fluid, which may be because the membrane protein on its surface is greatly affected by pepsin, resulting in its low activity. Pepsin is a highly specific protease with a certain amino acid sequence selection specificity, preferentially breaking the peptide bond formed by aromatic amino acids (phenylalanine, tyrosine, and tryptophan) or leucine (Kageyama \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Animal intestines usually contain a certain concentration of bile salts, which can change the permeability of cell membranes and cause cell death by destroying the integrity of cell membranes (Taranto et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Bile salts will undergo an uncoupling reaction in the intestine, and this reaction is mainly catalyzed by bile salt hydrolyzing enzymes secreted by \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e in the intestine (Pereira et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The study found that adding 1% inulin and \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e can promote the release of bile acid in vitro, which can affect serum cholesterol (Adebola et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Adding of inulin in this article can improve the bile tolerance of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, which is similar to the experiment results. \u003cem\u003eLactobacillus casei\u003c/em\u003e and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e isolated from human milk can also use inulin and oligofructose as carbon sources and have good acid and bile resistance (Tulumoglu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The inulin and \u003cem\u003eLactobacillus delbrueckii UFV h2b20\u003c/em\u003e were processed into symbiotic cheese together, and it was found that inulin has protective effects on \u003cem\u003eLactobacillus delbrueckii UFV h2b20\u003c/em\u003e under different bile salt concentrations and can enhance its resistance (Ara\u0026uacute;jo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, by studying the effect of adding inulin to alginate beads and observing its ability to protect the three probiotic strains, it turns out that beads containing 5% w/v inulin are the most effective at resisting bile salts (Atia et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, inulin as a carbon source can improve the bile salt tolerance of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, which can be increased about 81 times. It is speculated that yeast has a solid ability to use inulin.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e has good tolerance to ethanol because of the characteristics of ethanol produced by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. However, the study of this article found that when inulin is used as a carbon source for probiotics, the tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e to ethanol is most significantly improved. The research on the ethanol tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e is because it can promote ethanol conversion into aromatic compounds through esterification and Maillard reaction during the flavor formation stage of liquor processing. It is a crucial probiotic in liquor processing (Mukherjee et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Studies have found that the tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e to ethanol is related to the phospholipids on its cell membrane. Placing \u003cem\u003eBacillus subtilis\u003c/em\u003e in a volume fraction of 7.6\u0026ndash;30%, ethanol will cause the membrane to swell, accompanied by a decrease in membrane thickness (Gurtovenko and Anwar \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The high salt environment will cause the osmotic pressure to rise, resulting in the loss of microbial activity. \u003cem\u003eBacillus subtilis E221\u003c/em\u003e was selected from the gut of Nile tilapia fed with 0.8% inulin at a salinity of 16 PSU for 8 weeks. Studies have found that \u003cem\u003eBacillus subtilisE221\u003c/em\u003e has good salt tolerance, improving the growth stress of high salt to Nile tilapia (Tang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This result is the same as in this experiment. Inulin as a carbon source significantly improved the salt tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e. The experimental results suggest that inulin significantly affects the salt tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe growth of probiotics is usually inhibited at low temperatures. Low temperature may lead to the destruction of cell structure and also affect the activity of enzymes and the expression of related genes (Abadias et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It can be seen from the experimental results that using inulin as a carbon source can improve the survival rate of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e at 4\u0026deg;C. Nevertheless, it can reduce the survival rate of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, indicating that in a low-temperature environment, \u003cem\u003eSaccharomyces cerevisiae's\u003c/em\u003e ability to utilize glucose could be stronger than that of inulin. It is speculated that the reason may be that inulin is a soluble dietary fiber, which is mainly a linear polysaccharide connected by D-furan fructose molecules (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Therefore, when bacteria use it, it produces less heat and energy. However, in another experiment, adding inulin can increase the viable bacteria of ice cream during the storage period of 120 days and keep the viability of yeast above 6 log cfu/g (Sarwar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Currently, inulin and other fructans are also used as freeze-drying protective agents for probiotics, because vacuum freeze-drying is the most successful and convenient way to preserve yeast, bacteria, and spore-forming fungi (Abadias et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\text{s}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l} \\text{r}\\text{a}\\text{t}\\text{e}= \\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{r}\\text{e}\\text{m}\\text{a}\\text{i}\\text{n}\\text{i}\\text{n}\\text{g} \\text{v}\\text{i}\\text{a}\\text{b}\\text{l}\\text{e} \\text{b}\\text{a}\\text{c}\\text{t}\\text{e}\\text{r}\\text{i}\\text{a}}{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{o}\\text{r}\\text{i}\\text{g}\\text{i}\\text{n}\\text{a}\\text{l} \\text{v}\\text{i}\\text{a}\\text{b}\\text{l}\\text{e} \\text{b}\\text{a}\\text{c}\\text{t}\\text{e}\\text{r}\\text{i}\\text{a}}\\times 100\\%C=\\frac{2.57\\times 46.07}{6300\\times 1.0\\times 0.10\\times 2}\\times 0.1266\\times ({A}_{2}-{A}_{1})$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe addition of inulin can promote the growth performance of the three probiotics in different degrees, in which the effect of inulin on the growth performance of \u003cem\u003eBacillus subtilis\u003c/em\u003e the most obvious, followed by \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and finally, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. Adding inulin can promote the acid production capacity of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and the ethanol production capacity of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e preferred inulin in the experiment of inulin replacing glucose in the medium, and the utilization effect of inulin was higher than that of glucose. In contrast, \u003cem\u003eLactobacillus fermentans\u003c/em\u003e preferred glucose, and glucose's utilization effect was higher than inulin's. Generally, inulin can improve the tolerance of probiotics in adverse environments to varying degrees. However, in different adverse environments, the effects of inulin on the tolerance of different probiotics are significantly different. Specifically, yeast's bile salt tolerance and pepsin tolerance have significantly improved. The ethanol tolerance, bile salt tolerance, and low-temperature tolerance of \u003cem\u003eLactobacillus fermentum\u003c/em\u003e are significantly improved. The ethanol, acid, and salt tolerance of \u003cem\u003eBacillus subtilis\u003c/em\u003e are significantly improved. This article proves that inulin positively affects the poor environmental tolerance of probiotics. However, its mechanism of action is still unclear, and follow-up research is still needed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Education Department of Henan Province (No. 2019GGJS259) and Henan Academy of Sciences (No. 230503005, No.231003029, No. 240603015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYGF:\u0026nbsp;\u003c/strong\u003eConceptualization, Investigation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration, Funding acquisition. \u003cstrong\u003eYCN:\u003c/strong\u003e Investigation, Data curation, Formal analysis, Funding acquisition. \u003cstrong\u003eJC:\u003c/strong\u003e Investigation, Validation, Data curation, Writing editing. \u003cstrong\u003eCCQ:\u003c/strong\u003e Methodology, Validation, Formal analysis.\u003cstrong\u003e\u0026nbsp;HFW:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology, Formal analysis. \u003cstrong\u003eNFH:\u003c/strong\u003e Resources.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest in the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical standards\u003c/strong\u003e This research does not involve any human participants and animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbadias M, Benabarre A, Teixid\u0026oacute; N, Usall J, Vi\u0026ntilde;as I (2001) Effect of freeze drying and protectants on the viability of the biocontrol yeast Candida sake. 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Appl Environ Microbiol 87 (1). https://doi.org/10.1128/AEM.01738-20\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Prebiotics, Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus fermentum, Tolerance","lastPublishedDoi":"10.21203/rs.3.rs-4167997/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4167997/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study focused on the effects of inulin on growth performance and tolerance in an adverse environment of several probiotics \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and \u003cem\u003eLactobacillus fermentum\u003c/em\u003e. The results showed that inulin could significantly promote the growth of \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eS. cerevisiae\u003c/em\u003e, and \u003cem\u003eL. fermentum\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When inulin replaced glucose, the ethanol concentration in \u003cem\u003eS. cerevisiae\u003c/em\u003e fermentation broth could be increased by 15%. Inulin could significantly improve the acid tolerance of \u003cem\u003eB. subtilis\u003c/em\u003e under acidic conditions. It could significantly improve the bile salt tolerance of \u003cem\u003eL. fermentum\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e and significantly increase the ethanol tolerance of \u003cem\u003eL. fermentum\u003c/em\u003e and \u003cem\u003eB. subtilis\u003c/em\u003e. It could also significantly increase the survival rate of these three probiotics under low-temperature conditions. Our findings prove that inulin positively affects the growth ability and poor environmental tolerance of probiotics, and can be used as a prebiotic for several probiotics.\u003c/p\u003e","manuscriptTitle":"Effects of inulin on the growth performance and tolerance in adverse environments of several probiotics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 09:02:49","doi":"10.21203/rs.3.rs-4167997/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3710c94d-afe0-4095-82f7-ebf62c9efe41","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-30T14:08:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-01 09:02:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4167997","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4167997","identity":"rs-4167997","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}