reclamation of chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection

G Model JIPH-677; No of Pages ARTICLE IN PRESS Journal of Infection and Public Health xxx (2017) xxx–xxx Contents lists available at ScienceDirect Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection Fanjing Meng a , Shaoguo Yang a , Xin Wang a , Tingtao Chen a,∗ , Xiaolei Wang a , Xianyao Tang a , Rongji Zhang a , Liang Shen b a b Institute of Translational Medicine, Nanchang University, Nanchang, Jiangxi 330031, PR China Department of Obstetrics and Gynecology, Shandong Provincial Hospital Affiliated with Shandong University, PR China a r t i c l e i n f o Article history: Received 21 July 2016 Received in revised form October 2016 Accepted 18 November 2016 Keywords: Herb residue Probiotics DGGE Lactobacilli ELISA a b s t r a c t Environmental pollution caused by herb residues and the huge waste of medicinal ingredients contained in herb residues hinder the development of traditional Chinese medicine enterprises To solve this problem, several probiotics were tested, and Lactobacillus plantarum (HM218749) was finally selected for the reuse of herb residues of Jianweixiaoshi tablets A mouse model of Helicobacter pylori infection was developed to evaluate the anti-H pylori infection activity of the herb residue fermentation supernatant using a urease activity test, histological imaging, an enzyme-linked immunosorbent assay (ELISA) and denaturing gel gradient electrophoresis (DGGE) The results demonstrated that the herb residue fermentation supernatant successfully inhibited urease activity, slowed cell infiltration in the gastric area and significantly reduced the production of interleukin-6 (IL-6), IL-8 and TNF-␣ in the treatment group (p < 0.01) In addition, the DGGE results indicated that the herb residue fermentation supernatant was beneficial for the recovery of the disturbed microbiota in the infected model to the normal condition, in which L gasseri (GU417842.1) and L johnsonii (HQ828141.1) were dominant in all groups Therefore, the probiotics exhibited strong potential for the development of herb residues in this study, and the products showed strong potential in curing H pylori infections © 2017 The Authors Published by Elsevier Limited This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Chinese herbal medicine (TCHM) is among the important traditional industries in China and is a treasure of the Chinese nation In recent years, the poor results of single-target drugs in the treatment of chronic diseases (e.g., diabetes and cardiovascular disease) and the urgent need for green and safe formulas have made TCHM a hot topic worldwide [1–3] However, the huge amounts of herb residues produced by the continuous development of the Chinese herbal medicine industry have become a serious problem for large pharmaceutical companies In China, the annual yield of herb residues is approximately 30 million tons, mostly disposed of through stacking in the open, sanitary burial or burning, causing serious environmental pollution, especially in water quality Although some studies have reused herb residues for feed additives, the preparation of activated carbon, raw material for papermaking, cultivation of edible fungi or preparation of ethanol, these methods consider only the nutritional benefits and texture of medical plants, ignoring their precious medicinal ingredients [4,5] As is well known, herb residues are the by-products of traditional Chinese medicinal materials extracted by water or ethanol, and approximately 30%–50% of the medicinally active substances are still contained in them The microorganism fermentation theory in TCHM suggests that the digestive enzymes (e.g., cellulase, protease, pectinase and lignin enzymes and lipase) produced by microorganisms could effectively degrade plant cell walls, expand the intercellular region and improve the extraction yield of active ingredients [6] In addition, microorganisms could degrade macromolecular material to small molecules for direct absorption by the human body, reducing the side effects of drugs by degrading toxic substances and introducing new medicinal effects by biological modification [7] Probiotics are now accepted as useful in the prevention and/or treatment of certain pathological conditions [8] At present, the most studied probiotics are lactic acid-producing bacteria, particularly Lactobacillus species [9], which have proven to be useful in the treatment of several gastrointestinal diseases such as acute infectious diar- ∗ Corresponding author E-mail address: chentingtao1984@163.com (T Chen) http://dx.doi.org/10.1016/j.jiph.2016.11.013 1876-0341/© 2017 The Authors Published by Elsevier Limited This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/) Please cite this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013 G Model ARTICLE IN PRESS JIPH-677; No of Pages F Meng et al / Journal of Infection and Public Health xxx (2017) xxx–xxx Table Concentrations of IL-6, IL-8 and TNF-␣ in the gastric mucosa (mean ± SD, pg/mg) Group N IL-6 IL-8 TNF-␣ Control group Model group Treatment group 6 63.52 ± 11.12 123.43 ± 20.52 85.87 ± 16.64b 68.42 ± 16.12 110.83 ± 18.23 76.34 ± 16.31b 87.39 ± 20.23 132.44 ± 25.65 97.45 ± 16.89a a b Preparation of herb residue extract and fermentation supernatant p < 0.05 p < 0.01 (Compared with the Model group) Table Sequencing results of typical bands of DGGE patterns from Fig Strain No Closest relatives Similarity (%) GenBank No Bacterial DGGE a b c d L gasseri L johnsonii L plantarum L casei 99 100 100 100 GU417842.1 HQ828141.1 EF439684.1 JN974254.1 99 100 100 GU417842.1 HQ828141.1 FJ171327.1 Lactobacilli DGGE L gasseri a L johnsonii b L lactis c ter: 6.0 ± 0.1 mm; height: 10.0 ± 0.1 mm), which was placed on the surface of the agar The size of the inhibition zone was measured until the formation of a clear zone around the Oxford cup The experiment was conducted in duplicate [14] rhea or pouchitis The intake of probiotics can also be beneficial in Helicobacter pylori-infected subjects [8] H pylori, first recognized in 1982, is now regarded as a major cause of gastritis and peptic ulcers and is a risk factor for peptic ulcers, chronic gastritis, and gastric malignancy This organism can be found in 70%–90% of the population in developing countries and in 25%–50% of the population in developed countries [8,10–12] At present, combination therapy consisting of antibiotics and a proton pump inhibitor (PPI)6 is regarded as the treatment of choice to eradicate H pylori infection, and this regimen is 90% effective However, it is expensive and subject to side effects and antibiotic resistance [13] In this study, the herb residues of Jianweixiaoshi tablets, which consist of hawthorn, malt, pseudostellaria root, Chinese yam, and orange peel and have been approved by the Ministry of Public Health as both a medicine and a food, were chosen for reuse by probiotics, and the anti-H pylori infection effect was evaluated based on the invigoration of the stomach and promotion of digestion by the Jianweixiaoshi tablets in addition to the anti-H pylori effects of the probiotics (Tables and 2) Materials and methods Acid, salt and antibacterial testing of the probiotics The Lactobacillus plantarum (HM218749), L reuteri (EU547310), L rhamnosus (RS05630), L johnsoni (RS03965) and L paracasei (ATCC 334) were grown in de Man Rogosa Sharpe (MRS) media at 37 ◦ C overnight and were sub-cultured times The cultures were then centrifuged at 4500 × g for 10 to obtain a pure culture To test acid tolerance, each isolate was diluted 1:100 (v/v) in phosphate-buffered saline (PBS) at pH 1.5, 3.5, 4.5, 5.5 and 7.0 for h To test salt tolerance, freshly prepared cultures were inoculated into corresponding media containing 0.1–0.5% (w/w) bile salts and were incubated at 37 ◦ C for another h All bacterial colonies were enumerated using the plate count method For antimicrobial activity, overnight (12–16 h) cultures of pathogenic micro-organisms, including Shigella dysenteriae 301, Sh dysenteriae 2457, Staphylococcus aureus COWAN1, S aureus CMC, Enterobacter sakazakii 45402, Candida albicans SC5314 and H pylori SS1, were spread on the surfaces of their corresponding plates, and the culture supernatant (200 ␮L) of the probiotics was loaded into an Oxford cup (outer diameter: 7.8 ± 0.1 mm; inner diame- The herb residue of Jianweixiaoshi tablets was obtained from River Pharmaceutical Co., Ltd and was mashed using a pulper within h The probiotics L plantarum (HM218749), L reuteri (EU547310), L rhamnosus (RS05630), L Johnsoni (RS03965) and L paracasei (ATCC 334)(108 cfu/mL) were used as an inoculum to prepare the herb residue fermentation supernatant, which was incubated for 24–36 h at 37 ◦ C, and the bacterial number was evaluated using the plate count method H pylori infection model and treatment The study was approved by the Ethical Committee of the Second Affiliated Hospital of Nanchang University, and all methods were conducted in accordance with the approved guidelines The H pylori SS1 strain was routinely cultured under microaerophilic conditions (85% N2 , 5% H2 , 10% CO2 ) at 37 ◦ C on Wilkins-Chalgren agar enriched with 7% (vol/vol) horse blood and 1% (vol/vol) VITOX (Oxoid, Basingstoke, United Kingdom) Specificpathogen-free 6- to 8-week-old male C57BL/6 mice were housed and fed a commercial diet, with water ad libitum H pylori infections by the SS1 strain were conducted as previously described [15] Briefly, freshly prepared aliquots (500 ␮l, 109 CFU) of the H pylori SS1 strain in brain heart infusion broth (Oxoid) were administered to mice via orogastric inoculation five times (days 1, 3, 5, and 9) All noninfected control animals were inoculated with the same volume of plain brain heart infusion broth Eight weeks after the last gavage of H pylori, urease activity and histological imaging were examined to confirm successful modeling Next, the mice were divided into groups: control group (N = 12): normal mice only given PBS; model group (N = 12): the infectious model, only given PBS; treatment group (N = 12): the infectious model, given herb residue fermentation supernatant Ten weeks after the last gavage of H pylori SS1, 0.5 ml of PBS (control group and model group) or herb residue fermentation supernatant (treatment group) was administered to mice for weeks, and then the stomachs of mice in each group were sterilely obtained and divided into parts for the urease activity test, histological imaging, ELISA testing (IL-6, IL-8 and TNF-␣), and DGGE analysis Two weeks later, the stomachs of mice in each group were obtained for DGGE analysis in the recovery stage Determination of urease activity and ELISA Urease activity was determined by a method based on the commercial rapid urease test (Sanqiang Biochemical Industry Corporation in Fujian, China) with a sensitivity of 102 bacteria [16] Following the manufacturer’s instructions, each strip of the stomach antrum and body was homogenized and placed in mL of reaction solution (1 g of urea/mL (wt/vol) containing 850 ␮g of phenol red/mL (wt/vol) as a pH indicator) The solution became pink red or dark red within as a positive result and remained yellow as a negative results The products of IL-1␤, TNF-␣ and IL-6 in cell supernatants were determined using the ELISA kit for IL-1␤ (eBioscience), TNF-␣ (eBioscience) and IL-6 (eBioscience) Please cite this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013 G Model JIPH-677; No of Pages ARTICLE IN PRESS F Meng et al / Journal of Infection and Public Health xxx (2017) xxx–xxx Fig Evaluation of the probiotic characteristics of selected Lactobacillus plantarum (HM218749), L reuteri (EU547310), L rhamnosus (RS05630), L Johnsoni (RS03965) and L paracasei (ATCC 334) A, pH tolerance; B, bile salt tolerance; C, pathogen inhibition capability; D, growth in herb residues Histopathological analysis of gastric tissue samples Excised stomachs were opened along the lesser curvature, and the longitudinal half was fixed in 10% neutral buffered formalin solution, embedded in paraffin, and processed for histopathological analysis [17] All samples were stained with hematoxylin-eosin (H&E) for the evaluation of gastric inflammation and by the MayGrünwald Giemsa stain method for the assessment of H pylori SS1 colonization Histopathological evaluation was performed by a histopathologist with no prior knowledge of the identity of the samples DGGE analysis DNA was isolated by a bead-beating method, and the bacterial primers and Lactobacillus primers were used for the DGGE analysis [18] The bands of interest in DGGE gels were excised using a sterile blade and were incubated overnight at ◦ C in TE buffer (pH 8.0) to allow DNA diffusion for further amplifications The PCR products for sequencing were purified using the QIA quick PCR purification kit and were sub-cloned using the pMD18-T vector system I (Takara) according to the manufacturer’s instructions, and the transformants were randomly picked and sequenced in Invitrogen (Shanghai, China) Results Acid, salt and antibacterial test of the probiotics The populations of L plantarum (HM218749), L reuteri (EU547310), L rhamnosus (RS05630), L Johnsoni (RS03965) and L paracasei (ATCC 334) after exposure to acid and bile salt are shown in Fig 1A and B As shown in the figures, the most suitable condition for all strains was pH 7.0 without bile salt supplementation in the medium When the pH was lowered and the bile salt concentration was increased, an obvious reduction in microbial biomass was observed for all strains In addition, the high population of 6.0 log CFU/mL at pH 1.5 and 8.5 log CFU/mL at pH 3.5 (the actual pH in the stomach is 2.0–4.0) for L plantarum (HM218749) and that of 7.5 log CFU/mL at a bile salt concentration of 0.3% suggested strong survival during passage through the gastrointestinal tract (GI) Moreover, the strong inhibition of food-borne pathogens (especially H pylori SS1, 24 mm) and high growth number in herb residues without any other additives (8.8 log CFU/g) suggested that L plantarum (HM218749) could be used as a potential strain for the development of herb residues (Fig 1C and D) Evaluation of the H pylori SS1 infection model The urease experiments showed that the mice administered H pylori SS1 were urease positive (S-1), and the histopathological evaluation revealed a significant increase in mucosal inflammation in the gastric body (H&E).The Giemsa stain also indicated a high colonization number of H pylori SS1 (S-1B and S-1C) All the data showed that we had successfully established the H pylori SS1 infection model Treatment of H pylori SS1-infected mice with herb residue fermentation supernatant Before the formal experiment, 0.1 ml, 0.3 ml, 0.5 ml and 0.7 ml of herb residue fermentation supernatants were used to evaluate the suitable dose for SS1-infected mice, and the dose of 0.5 ml was chosen for its sound treating effect Fig shows that the herb residue fermentation supernatant possessed high urease inhibitory activity, and all the H pylori SS1-infected mice in the treatment Please cite this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013 G Model JIPH-677; No of Pages ARTICLE IN PRESS F Meng et al / Journal of Infection and Public Health xxx (2017) xxx–xxx Fig Evaluation of the treatment of Helicobacter pylori infection with herb residue fermentation supernatant by urease activity test (1, positive control; 2, negative control; 3–8, samples) and histological image of gastric area in mice (B, HE staining, original magnification ×100; C, Giemsa stain, original magnification ×100) group showed negative results (Fig 2A) In addition, the histological image of the gastric area indicated that the gavage of the herb residue fermentation supernatant had clearly lowered the chronic inflammation of the gastric mucosa and reduced the colonization of H pylori SS1 (Fig 2B and C), and the ELISA results confirmed that the herb residue fermentation supernatant had significantly reduced the production of IL-6 (123.43 ± 20.52–85.87 ± 16.64), (110.83 ± 18.23–76.34 ± 16.31) and TNF-␣ IL-8 (132.44 ± 25.65–97.45 ± 16.89) in the gastricmucosa Effects of the herb residue fermentation supernatant on the microbial diversity in the stomach As shown in Fig 4, the dominant bacteria in the stomach were relatively low in number, and only several lactobacilli species, mostly L gasseri (GU417842.1) and L johnsonii (HQ828141.1), maintained dominance regardless of the administration of H pylori SS1 or herb residue fermentation supernatant (Fig 4) The lactobacilli DGGE results also confirmed the same trends (Fig 4) Discussion In China, Jianweixiaoshi tablets bring more than 1.2 billion RMB income for the enterprise each year, but they also pro- duce approximately 100 000 t of herb residue The hawthorn, malt tangerine peel, radix pseudostellariae and yam contained in the Jianweixiaoshi tablets are useful for digestion, anorexia, abdominal distension, invigorating the stomach and tonifying the spleen, and the Jianweixiaoshi tablets are claimed to promote gastrointestinal peristalsis, promote the gastric secretion of digestive juices, enhance the activity of pepsin, enhance physique and enhance immune function Therefore, the medicinal components remaining in the herb residue should be useful for stomach diseases In our previous studies, L plantarum (HM218749) was isolated from the human intestine and was shown to tolerate low pH and high concentrations of salt bile, as well as to possess high adherence capability and anti-adherence properties against food-borne pathogens both in vivo and in vitro [19] In this study, its high survival rate in extreme acid (pH = 1.5–3.5) and bile salt conditions (0.3%) ensure its survival in the stomach to exert its probiotic effect and strong anti-H pylori SS1effect (Fig 1) Moreover, the high number of L plantarum (HM218749) in herb residue (8.5 LG CFU/g) without any additives offers us more options for the development of drugs or healthcare products To verify the anti-H pylori effect of the herb residue fermentation supernatant, we employed the SS1 H pylori mouse model and verified its reliability using urease activity testing and histological imaging (S-1) H pylori is a unique type of bacteria that can sur- Please cite this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013 G Model JIPH-677; No of Pages ARTICLE IN PRESS F Meng et al / Journal of Infection and Public Health xxx (2017) xxx–xxx Fig DGGE profiles of bacterial V3 16S rRNA gene fragments amplified from the mouse stomach A, negative group; B, positive group; C, treatment group 1–5, treatment period; 7–10, recovery period gel electrophoresis (PCR-DGGE) method was used to investigate the effect of herb residue fermentation supernatant on microbial diversity DGGE is a powerful tool based on the direct analysis of the extract of DNA from the microbial environment and does not require cell cultivation [14] This method has been used successfully to evaluate the bacterial composition of probiotic products and to identify or profile bacteria in dairy products [19,21–23] As DGGE could only identify bacteria representing more than 1–3% of total bacteria, this method only identified three dominant bacteria, namely L gasseri, L johnsonii and L plantarum, of which L gasseri and L johnsonii could tolerate the extremely low pH in the stomach and were dominant in all groups with or without the administration of H pylori SS1 or herb residue fermentation supernatant, both in bacterial DGGE and lactobacilli DGGE (Figs and 4) L gasseri has long been used as a probiotic and in the treatment of H pylori SS1 infection [9] Moreover, although outside interference exhibited little influence on the dominant lactobacilli species, it seemed that the addition of H pylori SS1 had disturbed the microbial diversity in H pylori SS1 infection model, and the administration of herb residue fermentation supernatant helped to restore the former microbial balance in the mouse stomachs (Fig 3) In conclusion, we have shown in the H pylori SS1 mouse model that herb residue fermentation supernatant can inhibit urease activity, reduce the level of the gastric inflammatory cytokines IL-6, IL-8 and TNF-␣, alleviate histological lesions and help to recover the disturbed microbiota to a normal level Therefore, the combination of Jianweixiaoshi herb residue and L plantarum (HM218749) provides a novel method for the use of herb residue to better address the environmental pollution problem and waste of medicinal ingredients contained in herb residues Funding No funding sources Competing interests None declared Ethical approval Fig DGGE profiles of LAB species-specific fingerprints amplified from the mouse stomach A, negative group; B, positive group; C, treatment group 1–5, treatment period; 7–10, recovery period vive in the acidic environment of the animal stomach because of its ability to neutralize gastric acids with the ammonia produced by urease The urease activity test is an important indicator of H pylori infection, and our results showed that the herb residue fermentation supernatant successfully inhibited urease activity in the treatment group (Fig 2) Moreover, H pylori achieved the substantial infiltration of lymphocytes and neutrophils in the infected group, while the herb residue fermentation supernatant clearly slowed the inflammatory response of the gastric mucosa and reduced the colonization of H pylori SS1 (Fig 2), which was confirmed by the IL-6, IL-8 and TNF-␣ protein levels evaluated by ELISA Excess levels of IL-6, IL-8 and TNF-␣ induced by activated inflammatory cells (e.g., eosinophils, macrophages, mononuclear phagocytes, neutrophils) will lead to the damage of cells and tissues, eventually causing the inflammation-associated diseases Therefore, the overexpression of IL-6, IL-8 and TNF-␣ is strongly connected to the gastric mucosal inflammation caused by H pylori SS1 [20] As the microbial diversity in the stomach is crucial for stomach disease, the polymerase chain reaction denaturing gradient Not required Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No 81503364, 31560264) and Jiangxi Province (20151BAB205001) Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jiph.2016.11.013 References [1] Wang X, Sun H, Zhang A, Sun W, Wang P, Wang Z Potential role of metabolomics apporoaches in the area of traditional Chinese medicine: as pillars of the bridge between Chinese and Western medicine J Pharm Biomed Anal 2011;55:859–68 [2] Zhang H-P, Pan J-B, Zhang C, Ji N, Wang H, Ji Z-L Network understanding of herb medicine via rapid identification of ingredient-target interactions Sci Rep 2014;4 [3] Lee SJ, Bose S, Seo J-G, Chung W-S, Lim C-Y, Kim H The effects of coadministration of probiotics with herbal medicine on obesity, metabolic endotoxemia and 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beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013 ... this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013... this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.013... type of bacteria that can sur- Please cite this article in press as: Meng F, et al Reclamation of Chinese herb residues using probiotics and evaluation of their beneficial effect on pathogen infection