Evaluation of tolerance to artificial gastroenteric juice and fermentation characteristics of Lactobacillus strains isolated from human

Abstract Fifty‐seven strains of Lactobacillus were isolated from fecal samples of healthy young people in Tibet, Xinjiang, and Inner Mongolia using pure culture methods. Lactobacillus ruminis and Lactobacillus gasseri were the dominant Lactobacillus species isolated from the intestinal microflora, accounting for 54.4% and 14.0% of the total isolates, respectively. Isolated strains were identified by 16S rRNA sequencing, and their tolerance to gastric acid and bile salt, and fermentation characteristics were evaluated. The results of experiments in vitro showed that nine of the isolated strains of Lactobacillus grew well at pH 3.0. After 11 h of incubation in artificial digestive juices, the isolated L. plantarum and the control strain L. plantarum P8 still had high survival rates. Most of the isolates and control isolates have strong tolerance to bile salts. The evaluation of fermentation characteristics indicated that the ability of the intestinal Lactobacillus to ferment skimmed milk was lower than that of the reference L. plantarum P8. In the process of storage, the viable count of screened isolates of human origin in fermented milk decreased to some extent, but remained above 7.01 ± 0.22 log CFU/ml, showing good storage characteristics.

as a hidden organ in the human body (Sekirov et al., 2010;Zhao & Shen, 2010). Human intestinal microflora was affected by many factors, such as geographical environment, eating habits, lifestyle, and age. Xinjiang, Tibet, and Inner Mongolia are the gathering places of nomads, following the ancient and traditional way of living. At the same time, these areas are located in the frontier and have a unique climate. With the growth and development of human body, the structure of human intestinal microflora is relatively stable, so we studied the intestinal microflora of young people in this area.
The term "probiotic" refers to microorganisms that benefit the host when administered in appropriate quantities (Hotel, 2001;Hill et al., 2014;Roberfroid, 2000). The best probiotics are of human origin that is safe and does not harbor genes associated with antibiotic resistance, pathogenicity, or virulence factors (Yasmin et al., 2020).
At the same time, they must be able to survive under intestinal conditions (e.g., acid pH, enzymes, and bile salts) . It must also be demonstrated that the activity, viability, growth, and efficacy of probiotics are maintained in technical treatments (Plaza-Díaz et al. 2017, 2018. Probiotics include microorganisms, most of which are similar to the beneficial bacteria naturally produced in the human intestine. To date, probiotics have been used in the treatment of a variety of gastrointestinal diseases and have been widely studied; the most studied species are from the genera Lactobacillus and Bifidobacterium, and also yeast (Wilkins & Sequoia, 2017). As one of the main probiotics, Lactobacillus species are also extremely important microflora in the intestine and their ability to survive in the gut environment is of great significance to host's health.
Lactobacillus species are important in intestinal homeostasis throughout the host's life. They can help correct imbalances in the intestinal microflora, thereby restoring and improving host's health. Clinical studies have confirmed that probiotics are effective against gastrointestinal disorders, for example, irritable bowel syndrome (Zhang et al., 2016), Helicobacter pylori (Zheng et al., 2013), inflammatory bowel disease (Saez-Lara et al., 2015), and diarrhea (McFarland, 2007); allergic diseases for example atopic dermatitis (Rather et al., 2016); in treating obesity (Sharafedtinov et al., 2013), insulin resistance syndrome (Rajkumar et al., 2014), type 2 diabetes and non-alcoholic fatty liver disease (Sáez-Lara et al. 2016;Buss et al., 2014), and the side effects of cancer (Redman et al., 2014). Positive effects of probiotics on human health can also be achieved through immune regulation (Hevia et al., 2015).
Lactobacillus is also commonly used in the production of fermented dairy products. In the food industry and dairy processing industry, it is used as a starter in the production of yogurt and cheese. In recent years, the use of lactic acid bacteria, particularly Lactobacillus species, has extended from food production into microecological preparations for use as health products. Lactobacillus has a very important role and economic value in our daily life. For this reason, there has been much research on the isolation, screening, metabolism, physiology, and genomics of intestinal Lactobacillus species.
In this study, the pure culture method was used to isolate and identify the Lactobacillus from the intestinal tracts of healthy young people in Tibet, Inner Mongolia, and Xinjiang. Preliminary evaluations were made on the acid and bile salt resistance and fermentation characteristics of the intestinal isolates of Lactobacillus. The experimental results may provide basic data for the screening and the evaluation of probiotic Lactobacillus and provide theoretical guidance for the industrial production of Lactobacillus.

| Sample collection
Fecal samples were collected from healthy young people (average age 24 years) in Inner Mongolia, Xinjiang, and Tibet (n = 38) (

| Isolation, purification, and preservation of Lactobacillus
Lactic acid bacteria were counted using the pouring culture method (Liu Hongxin et al., 2017). The stool sample (5 g) was diluted with normal saline (45 ml), and the appropriate concentration gradients (10 -5 , 10 -6 , and 10 -7 ) were selected by the 10-fold gradient dilution method (Yu et al., .2015). The diluent of 1 ml sample was absorbed and shaken in the unsolidified MRS solid medium, and 0.5% l-cysteine and 0.05 mg/ml mupirocin (Qingdao Hope Biotechnology Co., Ltd., China) (Ferraris et al., 2010) were added to modify MRS Agar (Oxoid). After the culture medium was thoroughly dried, the dishes were incubated at 37℃ in an anaerobic environment for 48 h (Jousimies-Sommer & Summanen, 2002). At the same time, 100 μL of sample dilution with the same dilution multiple as living bacteria count was spread on MRS solid medium. After colony formation, single colonies with different morphological characteristics were randomly selected on the corresponding solid medium for 2-3 times of lineation purification (Gooch Jan, 2011), in order to screen out colonies with different morphology.
The single colony was stained with Gram, and the cell morphology was observed under a microscope. The pure isolate identified as grampositive was cultured in modified MRS solid medium. After catalase test, the catalase-positive bacteria were discarded. Finally, the grampositive and catalase-negative isolates were tentatively designated as lactic acid bacteria (Daiwen., 1999). Those tentatively designated as Lactobacillus were cultured at 37℃ in an anaerobic environment for 18-24 h and prepared for DNA extraction (see below) and preservation. After centrifugation at 3500 × g for 10 min, the supernatant was discarded and replaced with about 5 ml normal saline into which the bacteria in the precipitate were resuspended. This was repeated three times in more saline to wash the bacterial cells which were finally resuspended using a Pap needle in a sterile protective solution containing: 10 g skimmed milk powder, 0.1 g sodium glutamate, 90 ml distilled water, all sterilized at 121℃ for 7 min. Some of the isolates were stored in a cryopreservation tube at −80℃, and the other part was freeze-dried in an ampoule tube and cryopreserved in a refrigerator at −80℃.

| Sequence analysis of 16S rRNA gene
For extraction of DNA from each tentatively identified Lactobacillus strain, 2 ml of cells in liquid culture medium was centrifuged for 1 min at 12000 rpm. The supernatant was discarded, and the bacterial precipitate was collected. The genomic DNA extracted using DNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd., China) needs to be determined by a ND-1000 micro-ultraviolet spectrophotometer. Fifty microliters of purified DNA was diluted to 100 ng/ μL A BLAST search (http://BLAST.ncbi.nlm.nih.gov/BLAST.cgi) of the NCBI database was used to analyze the nucleic acid sequences of bacteria in relation to sequences in GenBank. When sequence similarity to GenBank sequences achieved 97%, they were considered to be the same genus; when sequence similarity reached 99%, they were determined to be the same species (Drancourt et al., 2000).
Finally, MEGA software version 6.0 was used build the system evolution tree (Tamura et al., 2007;Yu et al., 2011).

| In vitro growth curve
Cryopreserved isolate was inoculated into modified MRS liquid medium, incubated at 37℃ for 24 h, and then continuously cultured for

| Tolerance to artificial digestive juices
Cryopreserved Isolated Lactobacillus strains were activated as described previously. Activated isolate was centrifuged (3800 × g) for 10 min and the supernatant removed. The bacterial pellets were collected and washed three times in PBS (8.0 g/L NaCl, 0.2 g/L KH 2 PO 4, and 1.15 g/L Na 2 HPO 4 , pH 7.2). The bacteria were then resuspended in 5 ml of sterilized PBS. For each strain, 0.5 ml of bacterial suspension was added to 4.5 ml (pH 2.5) of artificial gastric fluid and then incubated at 37℃ under anaerobic conditions.
Samples were taken at 0 h and 3 h, respectively, and plate viable bacteria were counted (Fernández et al., 2003). The artificial gastric juice cultured with pH 2.5 for 3 h was transferred into artificial intestinal juice (4.5 ml) by absorbing 0.5 ml, and then anaerobic culture was performed. The number of living bacteria was determined after 4 h and 8 h (Hosono, 1998). Finally, the survival rate was calculated using the plate counting method (Bao et al., 2010).   (Pochart et al., 1992).

| Bile salt tolerance
The tolerance of isolate to bile salt was judged according to the delay time of each strain.

| Statistical analysis
Three parallel samples were made for the determination of each index of the test sample. The experimental data were statistically analyzed by the CORR program of SAS9.0 software, and the drawing was made by Origin7.0 software.

| Preliminary identification of Lactobacillus
Continuous culture of isolates in modified MRS solid medium for 48 h resulted in colonies that were white, transparent, with neat edges, smooth or dull surfaces, flattened, or protruding in the center.
By Gram staining and other phenotypic tests, a total of 57 isolates were identified as Lactobacillus species. All isolates showed grampositive and catalase-negative properties.

| Molecular identification by 16S rRNA sequencing
The 57 isolates of Lactobacillus from human feces could be divided into 13 species by 16S rRNA gene sequencing (Figure 1 Lactobacillus among individual and between regions (Liu Hongxin et al., 2017). The fact that the dominant Lactobacillus species from young people in Tibet, Xinjiang, and Inner Mongolia detected in this experiment was similar to that of Europeans may be because the diet of these three regions is similar to that of Europeans, that is, a lot of dairy products.

| In vitro growth curve
We evaluated the growth of nine of the Lactobacillus (from seven species) isolated from fecal samples and two reference strains: L.
plantarum P8 and L. casei Zhang. It can be seen from the Figure 2 that the growth curves of nine isolates of Lactobacillus isolated in MRS liquid medium were basically very similar to each other. They all grew slowly between 0 and 4 h, entered a stable growth period after 14 h which lasted until 24 h. In this experiment, growth characteristics were evaluated under aerobic conditions and so differences in growth were also affected by oxygen. Thus, in subsequent experiments we adjusted the culture time according to the growth curve.

| Acid and bile tolerance
Preliminary analysis of acid tolerance (pH 2.0) and artificial gastric juice tolerance (pH 3.0) of 11 Lactobacillus can be seen in Table 2. All the tested Lactobacillus could grow well in the pH 3.0 environment, but were unable to survive in the pH 2.0 environment for very long.
This is consistent with the results of Marteau (Marteau et al., 1997).
After 3 h at pH 3.0 in artificial gastric juice, survival rates of the different isolates of Lactobacillus varied significantly from that of the two reference strains (p < .05) ( Table 3). From the experimental results, it can be seen that the survival rate of the isolated L. plantarum IMAUFB042, IMAUFB063, and the control strain L. plantarum P8 in pH 3.0 gastric juice environment reached more than 90%, showing a strong tolerance to artificial gastric juice, which is consistent with the conclusion that part of Lactobacillus isolated from human body has good resistance to gastrointestinal fluid, which was found by Fernández, Boris, and Barbés (2003). In other studies, it was also found that L. plantarum had strong tolerance to artificial gastric juice with low pH value (Peng et al. 2010;Wang et al. 2004).
The 11 isolates of Lactobacillus that were digested at pH 3. Bile concentration in the human intestinal tract generally fluctuates between 0.3% and 1.8%. Of the 11 Lactobacillus evaluated, 10 were tolerant to bile salts in the range of 0.3% -2.0% (Table 4).  and L. plantarum P8 to 1.8% bile salts was consistent with previous studies on these isolates (Wu et al., 2009;Ying et al., 2007). Another strain of the same species, L. plantarum MB3182, also has good tolerance to 2.0% bile salt (Missotten et al., 2008), confirming the conclusion that L. plantarum has good bile salt tolerance.
The delay time of each strain in response to bile salts was significantly different among isolates (p < .05), indicating significant differences in bile salt tolerance among isolate ( The pH value of each strain decreased with fermentation time; the decrease was slow in the first 3 h of fermentation, and then accelerated ( Figure 4). Fermentation by most of the Lactobacillus was strong, reducing the pH to 4.5 within 22 h, while the ability of L.
casei IMAUFB059 and IMAUFB013 to ferment skimmed milk was weak and the pH achieved during fermentation was not as low as for the other Lactobacillus. The pH of each strain changed slowly during storage because of growth, and therefore, acid production was suppressed at 4℃. While the pH of L. casei IMAUFB059 and IMAUFB013 did continue to decrease during storage, it was still higher than 4.5 after 21 days. Overall, acid production during fermentation was lower for Lactobacillus than for L. plantarum P8 and L. casei Zhang.
During the fermentation of skimmed milk, the titration acidity of each strain increased significantly with the fermentation time. The changing trend of titration acidity between 0 and 3 h is slow, the titration acidity is lower, and the titration acidity increases rapidly between  After 21 days of storage, the number of living bacteria in the fermented milk had decreased by about 3 logarithmic units (Table 6). This is consistent with other studies that found the number of living bacteria of some probiotic decreased to a certain extent during low-temperature storage of fermented milk (Kailasapathy, 2006;Shah, 2000). While the number of living bacteria in fermented milk produced by some isolates remained above 1.0 × 10 7 CFU/ml after 21 days of storage, the number of living bacteria of the reference strain, L. casei Zhang, after 21 days of storage was slightly higher than for the Lactobacillus isolated from human feces and also the other reference strain, L. plantarum P8. This indicates that the storage characteristics of L. casei Zhang-fermented milk were better than for some of the Lactobacillus isolated from the intestinal tract.

FU N D PROJ EC T S U PP O RT
The study was funded by National Key R&D Program (Project Code:

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included in the article.