N0 is the number of viable lactobacilli cells before gastric fluid treatment (CFU/Tablet). Ng is the number of viable lactobacilli cells after gastric fluid treatment (CFU/Tablet). Ne is the number of viable lactobacilli cells after enteric fluid treatment (CFU/Tablet).

After enteric juice treatment, there were no viable lactobacilli cells at 0.1 mL initial inoculum for three kinds of tablets, while viable cells could be examined in all the kinds of tablets at 1.0 mL initial inoculum (Table 5). This result also confirmed that more intake of the product would significantly increase the levels of probiotics in the intestine. However, compared with the control, the survival rate of lactobacilli cells after intestinal juice treatment only increased 2.95-, 0.13- and 2.77-fold for L-group, M-group, and H-group, respectively. Meanwhile, there was no dose-dependent protective effect of the herbs on the viability of lactobacilli cells after enteric juice treatment when compared to the gastric fluid treatment. Nagashima, Pansiera, Baracat and Gómez [12] had also found that the number of *L. acidophilus* decreased significantly after effervescent probiotics products contact with artificial enteric juice; they ascribed this decline to the sharp change in the pH from 2.0 in the gastric juice to 6.8 in the enteric juice, and the presence of bile salts. Therefore, the presence of bile salts in enteric juice and the pH variation from 1.7 to 6.8 might greatly destroy the viability of lactobacilli and thus weaken the protective effect of the herbs in this study. Additionally, an increase of antimicrobial activity and/or a decrease of probiotic activity of components in the herbs in the enteric juice environment may be another reason for the reduced protective effect of the herbs.

Generally, after gastrointestinal fluids treatment, the survival rates of lactobacilli for the L-group (0.0076%) and the H-group (0.0294%) were both higher than that for the control (0.0009%), while it was similar between the M-group (0.0006%) and the control. It is confirmed that the herbs supplemented in the lactobacilli effervescent tablets have a certain protective effect on the viability of lactobacilli during gastrointestinal fluids treatment, but for the better protective effect the optimal concentration of the herbs in probiotic products should be further investigated.

#### *3.4. Antibacterial Activity of the Tablets*

The goal in administering probiotic products is to induce a balanced enteric microbiota, which will have a favorable impact on consumer health. Enteric microbiota include both beneficial and harmful microflora. As mentioned above, the tablets supplemented with Chinese ginseng and *P. sibiricum* have a certain favorable effect on the activity of some lactobacilli. In order to investigate the antibacterial activity of the lactobacilli effervescent tablets, an in vitro bacteriostatic experiment against *S. aureus* and *E. coli* O157:H7, two common pathogens in clinical infection and food contamination, was conducted. After incubation with the control tablet for one hour, about 95.11% and 97.86% of *S. aureus* and *E. coli* O157:H7 were killed, respectively (Table 6). While the antibacterial ratio against both bacteria was decreased significantly with an increase in the content of the two herbs in the tablets (*p* < 0.05), there was still a high antibacterial ratio for these tablets, which was more than 78% for the H-group. Because the treatment time of the antibacterial experiment was relatively short, which was 1 h, we considered that the disintegration progress of tablets may be an important factor affecting the antibacterial activity. As shown in Table 6, the dissolution time of the tablets was prolonged with increasing content of the two herbs (*p* < 0.05), which is opposite to the change trend of antibacterial activity. The effervescence mediated by the citric acid and sodium bicarbonate ensures a rapid and complete distribution of the active ingredients in products to solutions [14], and can rapidly create an anaerobic microenvironment by released CO2 [30], which can also decrease pH of the solution (Table 6). These features are harmful to bacteria which cannot withstand the environment. As the dissolution time of the tablets was prolonged with increased content of the two herbs (Table 6), this would decrease release rates of active ingredients and CO2 with an increase in the content of the herbs, which may lower the antibacterial activity of the tablets with increased content of the herbs. Additionally, under the condition of the antibacterial experiment in this study, antagonist effects between antimicrobial and probiotic components of the herbs may lead to weak antibacterial activity of the tablets containing the herbs in a dose-dependent manner. Therefore, the antibacterial activity was highest for the control, which decreased significantly with an increase in the content of the two herbs in tablets under the condition of the antibacterial experiment in this study (Table 6). According to the previous results on the change of viability of lactobacilli after tablet disintegration for 48 h (Table 4), we deduced that the antibacterial activities of the four kinds of tablets against *S. aureus* and *E. coli* O157:H7 would be different when prolonged the treatment time.


**Table 6.** Antibacterial activity of different kinds of lactobacilli effervescent tablets against *Staphylococcus aureus* and *Escherichia coli* O157:H7 for one-hour incubation.

All data show the means ± SD. Different lowercase English letters in the same column indicate a significant difference; different uppercase English letters or lowercase Greek letters in the same row indicate a significant difference, respectively.

Meanwhile, the antibacterial activity of each kind of tablet was significantly higher against *E. coli* O157:H7 than against *S. aureus* (*p* < 0.05) (Table 6). Xue, Yang, Zhao, Hou, Zhang, Zhang and Ren [28] had found that ginsenosides cause bacterial death by directly destroying the membrane system. As we know, the cell wall of *S. aureus* is thicker than that of *E. coli* O157:H7, which will reduce the speed and content of CO2 and active ingredients (such as saponins) reaching the cell membrane, causing higher antibacterial activity against *E. coli* O157:H7 than against *S. aureus* for each kind of tablet.

#### *3.5. Organoleptic Assessment*

An organoleptic assessment was performed to measure the acceptability of the four kinds of effervescent tablets. As shown in Table 7, except for appearance and overall liking attributes, there was no significant difference among different kinds of tablets. In respect of the appearance of the tablet solution, panelists evaluated the L-group as largely like (7.30 ± 1.17), while the H-group was considered slightly appreciated (5.90 ± 1.86). After disintegration, a small amount of light-yellow foam was floating on the solution of the H-group, resulting in a lower appearance score for it. The overall liking classified the H-group as slightly like (5.30 ± 1.56) and the L-group and the control as moderately like (6.45 ± 1.43 and 6.05 ± 1.39, respectively), while the evaluation for the M-group (5.70 ± 1.45) was between them. Besides the appearance attribute, the flavor was also a main reason for a lower overall liking score for the H-group solution, although there was no significant difference in the flavor attribute between the four kinds of tablets (*p* > 0.05). As can be seen from Table 7, the lowest score of flavor attribute was found for H-group solution (5.70 ± 1.34), as most panelists felt relatively high bitterness in the H-group solution. Panelists participated in the organoleptic assessment aged between 22 and 25 years, do not like consuming bitter foods. Perhaps these effervescent tablets may be evaluated differently if older people participate in the organoleptic assessment. According to the preference ranking test results (Table 8), the L-group received the highest value in the sum of the orders (59), but its preference did not present statistical difference (*p* > 0.05) for the control and the M-group. The H-group was the less preferred along with the M-group, reinforcing the results obtained in the organoleptic assessment.

**Table 7.** Organoleptic assessment of the lactobacilli effervescent tablets supplemented with different contents of Chinese ginseng and *Polygonatum sibiricum.*


All data show the means ± SD. Different letters in the same column indicate a significant difference.


**Table 8.** Results of the preference ranking test of the lactobacilli effervescent tablets supplemented with different contents of Chinese ginseng and *Polygonatum sibiricum.*

\* Significant critical difference (5%) minimum of 21, for four samples and 20 panelists, according to the Friedman test. ns non-significant.

#### *3.6. Stability of the Tablets during Storage*

Figure 1 shows the survival of lactobacilli in different kinds of lactobacilli effervescent tablets after storage at 25 ◦C for 60 days. After storage for 15 days, the number of lactobacilli cells was reduced by 1.20, 1.79, 2.08, and 2.35 logarithmic cycles for the control, L-group, M-group, and H-group, respectively. This result demonstrated that the viability of lactobacilli decreased significantly with the increase of the content of the herbs in tablets in the early stage of storage (0–15 days). Nevertheless, in the later stage of storage (15–60 days), the viability of lactobacilli in the tablets supplemented with the herbs was similar to that in the control (Figure 1). Considering the diversity of drug-resistance ability of microorganism cells to antimicrobial substances, we speculated that some sensitive cells in the tablets may be inactivated by the herbs in a dose-dependent manner in the early stage of storage, while the cells that can tolerate the herbs could maintain their viability throughout the storage period. Nagashima, Pansiera, Baracat and Gómez [12] had also reported that the number of *L. acidophilus* in effervescent probiotics tablets decreased from 5.18 to 2.91 logarithmic cycles after storage for 60 days at 25 ◦C. In this study, the number of viable lactobacilli cells was about 5.80 logarithmic cycles in all kinds of tablets at the end of storage (Figure 1). In order to obtain more probiotic properties, it should be considered to add protectants for lactobacilli viability to the formula.

**Figure 1.** Viability of lactobacilli in different kinds of lactobacilli effervescent tablets stored for 0, 15, 30, 45 and 60 days at 25 ◦C. All data show the means ± SD. At the same storage period, different letters indicate a significant difference.

#### **4. Conclusions**

Our findings demonstrate that it is possible to develop an optimal formulation of lactobacilli effervescent tablets supplemented with Chinese ginseng and *P. sibiricum*, combining functional benefits of lactobacilli and both herbs. In terms of form, the effervescent tablet product exceeds the stability of the liquid form, thereby prolonging the shelf life. Compared with the capsule product, the effervescent tablet has the advantages of simple processing, rapid disintegration, dissolving in water, and easy to take. The effervescent tablet is more suitable for children, the elderly, and people who have difficulty in swallowing pills. According to the results in this study, the formulation of the L-group (60 mg herbs/tablet) would be the best choice for industrial production, which can effectively protect the viability of lactobacilli after tabletting and gastrointestinal juices treatment, together with the high antibacterial ability and the preferred organoleptic characteristics. Meanwhile, the L-group was relatively stable during storage. For a better probiotic effect of the L-group, consumers should drink it in time after disintegration, which would be conducive to retaining higher viability of lactobacilli reaching the intestine and help to create better antibacterial activity in the intestine, leading to a more beneficial enteric microbiota. The main purpose of this study was to investigate the effect of Chinese ginseng and *P. sibiricum* on the properties of effervescent tablets, thus no sweetener was added to the formulations of the lactobacilli effervescent tablets. Considering the younger consumer perspective, manufacturers can add some beneficial sweeteners, such as the prebiotic fiber inulin, to the formula. Additionally, an in vivo experiment should be studied for understanding more health care functions of the lactobacilli effervescent tablets supplemented with Chinese ginseng and *P. sibiricum*.

**Author Contributions:** Conceptualization, F.Z. and T.Z.; methodology, F.Z., M.L., L.M. and J.Y.; validation, M.L. and L.M.; formal analysis, F.Z.; investigation, M.L., L.M. and J.Y.; resources, F.Z.; data curation, F.Z.; writing—original draft preparation, F.Z.; writing—review and editing, M.L. and L.M.; visualization, F.Z.; supervision, F.Z.; project administration, F.Z.; funding acquisition, F.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by No. 2018YFC1602202 of the National Key Research and Development Program of China, and the Fundamental Research Funds for the Central Universities.

**Acknowledgments:** We would like to acknowledge technician Yan Chen for equipment maintenance, and thank Shuning Zhong for assistance in provision of some necessary literature.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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