RSC Advances View Article Online PAPER View Journal | View Issue Metabonomic strategy for the detection of metabolic effects of probiotics combined with Cite this: RSC Adv.,2018,8,5042 prebiotic supplementation in weaned rats† Mengxia Wang,a Xiaojun Zhang,a Yuanyuan Wang,c Yuan Li,a Yongxiong Chen,a Haihui Zheng,b Fangli Ma,c Chung Wah Ma,c Biyu Lu,a Zhiyong Xieb and Qiongfeng Liao *a The purpose of this study is to investigate the effects of probiotics combined with prebiotics (PP) supplementation on weaned rat metabolism. A metabonomic strategy employing 1H-NMR spectroscopy and multivariate data analysis was used to examine weaned rat biological responses to PP supplementation. Male Sprague-Dawley rats (post-natal day 21, PD 21) received probiotics (Lactobacillus acidophilus NCFM (L-NCFM) and Bifidobacterium lactis Bi-07 (B-LBi07), 1 : 1, 1.0 Â 1011 cfu kgÀ1) and prebiotics (Lycium barbarum polysaccharides (LBP), Poria cocos polysaccharides (PCPs) and Lentinan, À1 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 1 : 1 : 1, 24 g kg ) via intragastric administration for 28 consecutive days. Urine and feces were collected for analysis. Significant topographical metabolic variations were present in urine and feces. Urinary metabolites upregulated by PP treatment included alanine, N-acetylglycine, glutamine, dimethylamine, phosphorylcholine, ethylene glycol, mannitol, phenylacetylglycine and glycoate, which were related to alanine, aspartate and glutamate metabolism, and choline metabolism. Feces-derived metabolites, including caproate, valerate, butyrate, propionate, lactate, acetate, succinate, methanol, threonine and methionine, were significantly increased, which were related to short-chain fatty acid (SCFA) metabolism Received 2nd November 2017 and TCA cycle metabolism. These results indicate that dietary PP supplementation can regulate common Accepted 15th January 2018 systemic metabolic processes, including energy metabolism, amino acid metabolism, lipid metabolism, This article is licensed under a DOI: 10.1039/c7ra12067b nucleic acid metabolism, and gut microbiota-related metabolism. This study also illuminates the vital role rsc.li/rsc-advances of PP supplementation in regulating the metabolism of weaned rats. Open Access Article. Published on 30 January 2018. Downloaded 9/27/2021 5:54:34 AM. Introduction increasing susceptibility to disease.4 In addition, weaned animals are also affected by the separation from their mother A multiplicity of factors and forces, such as genetic, environ- and littermates, communication with unfamiliar compatriots, mental and stochastic ones, affect how organisms grow and and establishment of a new social hierarchy.5 The above develop. Weaning, which is an essential and critical event for combined stresses could result in metabolic disorder, gastro- human and rodent animals, may inuence nutritional metab- intestinal dysfunction, impaired mucosal barrier function, and olism, and growth and immunity during adulthood.1,2 The imbalance of intestinal microbiota, which are responsible for – period that an infant grows into a child is also a crucial phase the impaired growth and health of an animal.6 8 Therefore, illustrated by raised nutritional stress and mortality in nding methods to solve the problems associated with weaned humans.3 Weaned animals are subjected to their diet changing stress is of vital importance. Previous studies have shown that from breastfeeding to solid foods. Changes in nutritional status problems related to weaned stress in animals can be alleviated during early periods of life may lead to a decrease in food intake by nutritional modulation.9,10 and impair immune function, both of which contribute to an Probiotics are living microorganisms that is benecial to the health of the host organism when administered in appropriate amounts.11 Probiotics can improve the digestibility of starch 12 aSchool of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, and protein in vitro. Additionally, several studies propose that Guangzhou, 510006, P. R. China. E-mail: [email protected]; Fax: +86 20 probiotics can stimulate the immune system, to prevent or even 39358081 treat bowel infection.13 Moreover, other studies have shown that b School of Pharmaceutical Sciences (Shen Zhen), Sun Yat-sen University, Guangzhou, probiotics might contribute to the integrity of the intestinal 510006, P. R. China barrier and that they are effective in treating inammatory cInnitus (China) Company Ltd, Guangzhou, 510623, China 14 † Electronic supplementary information (ESI) available. See DOI: bowel diseases. Lactobacilli and Bi dobacteria, the most 10.1039/c7ra12067b commonly used species, are incorporated into food, and then 5042 | RSC Adv.,2018,8,5042–5057 This journal is © The Royal Society of Chemistry 2018 View Article Online Paper RSC Advances survive in the host digestive tract where they modulate the gut Probiotics and prebiotics treatment microbiota and bring host benecial effects. Rats in the CON group were administered with physiological Prebiotics, as a kind of indigestible component in food, can saline, while probiotics (L-NCFM and B-LBi07) was administered enter the lower gastrointestinal tract and selectively stimulate 11 À1 at a nal concentration of 1.0 Â 10 cfu kg (1 : 1), LBP, PCPs the growth of health-promoting bacteria.15,16 Dietary prebiotics and Lentinan (1 : 1 : 1) were used as prebiotics at a concentra- treatment, as one of the representative strategies, selectively À1 tion of 24 g kg . Each treatment was administered for 28 days. change the substrates that can be utilized by gut microbiota, Probiotics and prebiotics were provided by Innitus (China) 17 and have a great impact on host metabolism and physiology. Company Ltd. Lycium barbarum polysaccharides (LBP), Poria cocos poly- saccharides (PCPs) and Lentinan, the three main types of polysaccharides came from Chinese herbal medicine, have Sample collection and extraction many biological activities, including immunomodulation, anti- On post-dose day 14 and 28, rats were randomly individually 18 tumour, antioxidant, anti-diabetes, anti-virus, and anti-fatigue placed in a metabolic cage aer 12 h of symbiotic treatment. 19 functions. Then urine samples and feces samples were collected in The combination of probiotics and prebiotics is termed Eppendorf tubes on ice from 20:00 pm to 8:00 am. The samples ‘ ’ synbiotics . Synbiotic refers to a nutritional supplementation from each group were stored at À80 C for NMR analyses. which combines probiotics and prebiotics in a form of syner- Urine samples were thawed at room temperature. A volume 20 ff gism. However, few studies have assessed the e ects of syn- of 600 mL urine samples and 60 mL phosphate buffer solution biotics regarding the changes of metabolism in weaned ¼ (1.5 M, K2HPO4/NaH2PO4 4 : 1, pH 7.4, 0.1% NaN3, 0.05% animals. In nutritional research, metabonomic is the dynamic TSP, 99.9% D2O) were mixed and then centrifuged at 16 000g at complement of the endogenous metabolites within the inte- 4 C for 10 min to remove precipitates. A volume of 600 urine grated biological systems and is a powerful top-down systems supernatants were transferred to 5 mm NMR tube. Feces Creative Commons Attribution-NonCommercial 3.0 Unported Licence. biological tool in nutritional research that can provide the end samples were thawed at room temperature and extracted point of consequences of nutritional intervention, which can according to an optimized sample preparation procedure help us understand how did metabolic balances disturbed by described before.21 interventions.9 In addition, metabonomic is useful in exploring the complex relationship between nutritional intervention and metabolism, and guring out the effect of dietary components NMR measurement and preprocessing on health maintenance and disease development. 1H-NMR spectra were obtained using a Bruker AVIII 600 MHz In the present study, we investigated the metabolic proles spectrometer (Bruker Biospin, Germany) at 600.13 MHz and of biouids and feces in weaned rats fed with probiotics (L- 298k. The acquisition parameters were essentially mentioned in This article is licensed under a NCFM and B-LBi07) and prebiotics (LBP, PCPs and Lentinan) by previous study.22 A one-dimensional pulse sequence was used 1 using H-NMR integrated with multivariate data analysis. The [recycle delay (RD)–G1–90 –t1–90 –tm–G2–90 –acquisition]. The correlation of metabolic alteration in multiple biological water signal was suppressed by irradiation during RD of 2 s, and matrices including urine and feces was systematically analyzed. mixing time (t ) of 80 ms. For each sample, the 90 pulse length Open Access Article. Published on 30 January 2018. Downloaded 9/27/2021 5:54:34 AM. m The purpose of this study was to discover the systematic was adjusted to about 10 ms and a total of 64 transients were metabolites changes and supply a molecular mechanism of PP accumulated into 32k data points over spectral width of 20 ppm. regulating the metabolism in weaned rats. Two-dimensional (2D) NMR experiments (1H–1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), Materials and methods total correlation spectroscopy (TOCSY), 1H–13C heteronuclear single quantum correlation spectroscopy (HSQC) and 1H–13C Animals heteronuclear multiple bond correlation spectroscopy (HMBC)) 21 days old male specic-pathogen-free Sprague-Dawley rats were performed to assign NMR signal as previously reported.23,24 (body weight ¼ 35–45 g, n ¼ 24)