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 cerevisiahaveas 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 Mn2+ 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).
$$\text{s}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l} \text{r}\text{a}\text{t}\text{e}= \frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{r}\text{e}\text{m}\text{a}\text{i}\text{n}\text{i}\text{n}\text{g} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{o}\text{r}\text{i}\text{g}\text{i}\text{n}\text{a}\text{l} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{b}\text{a}\text{c}\text{t}\text{e}\text{r}\text{i}\text{a}}\times 100\%C=\frac{2.57\times 46.07}{6300\times 1.0\times 0.10\times 2}\times 0.1266\times ({A}_{2}-{A}_{1})$$