The Postbiotics, Totipro PE0401, and Probiotic Mixture, PF1001, Modulate the Gut Microbiota and Ameliorate Diarrhea in Weaning Piglets

Most piglets are weaned between three and four weeks of age, which is very soon after birth...

materials such as probiotics, prebiotics, organic acids, and plant extracts have been tested as effective alternatives to antibiotics [3].
Recently, it has been shown that weaning stress not only changed microbial composition and function, but also altered the microbial metabolic profiles in the intestine [5]. Gut microbiota is symbiotically residing in digestive tracts, and fecal microbiota transplantation are often used to regulate gut microbial colonization [6]. In human, supplements of probiotics have been recommended in pediatric gastrointestinal diseases, and considered for prevention of antibiotic associated, Clostridium difficile-induced, or nosocomial diarrhea [7]. Therefore, postbiotic or probiotic products might provide a new insight in alleviating weaning stress and facilitating disease prevention during the period of weaning in piglets [8,9]. In this study, effects of not only the mixture of ten probiotic strains,PF1001, but also the postbiotics, Totipro PE0401, on the growth and health in weaning piglets were evaluated in 4-week old weaning piglets.

Materials and Methods
All experiments were performed in accordance with approved guidelines. The animal protocol was approved by the Agricultural Technology Research Institute. (Project number AAI24-E-P10708).

Preparation of Supplementation
The dry product of Totipro (postbiotics product number PE0401) and probiotic mixture (probiotics product number PF1001) were prepared and provided by glac Biotech Co., Ltd.
(Tainan City, Taiwan). Totipro PE0401 was the fermentation of four alone (1x10 7 CFU/g). Food and water were offered ad libitum throughout the experimental period. The body weight, daily feed consumption, daily weight gain, diarrhea incidence analysis and feed conversion ratio were recorded every 2 week. After 6 weeks of feeding, the serum of weaning piglets was collected for immunity analysis (IgA and IgG). The stool was collected for fecal microbiota analysis and odor analysis.

Diarrhea Incidence Analysis
Diarrhea incidence was recorded daily in each stall throughout the entire experiment through direct observation conducted by 2 evaluators. The diarrhea was determined based on fecal consistency using a modification of the method described by a previous study [10]. The feces were ranked on the following scale: 0 = solid; 1 = semi-solid; 2 = semi-liquid; and 3 = liquid. The piglets were considered to have diarrhea when the fecal consistency was level 2 or 3, as described previously by Cheng et al. [11] and calculated as follows: Diarrhea incidence (%) = [the number of pigs with diarrhea in each stall / total numbers of pigs / days] x 100%. loaded onto a Miseq cartridge, then onto the instrument. Automated cluster generation and a 2 x 300 bp paired-end sequencing run was performed. The sequences generated went through a filtering process to obtain the qualified reads. Total reads were merged, removed low-quality sequence, removed chimera sequence and clustered the OTU at 97% similarity with the Greengenes database. All OTU sequences and diversity analysis were using CLC Microbial Genomics Module (Qiagen, Germany), basespace (illumine, USA) and

Bacterial DNA Extraction and 16S rRNA Sequencing
Graphpad prism 7 (Graphpad Software, USA). A p value less than 0.05 is considered statistically significant.

Statistical Analysis
Data were analyzed using one-way ANOVA through the GLM

Fecal Microbiological Analysis
Fecal samples of weaning piglets were plated in triplicate on NA (Nutrient agar), PCA (plate count agar) and MRS with 0.05% cysteine agar broth for detecting Escherichia coli, total gut bacteria and Lactic acid bacteria (Lactobacillus and Bifidobacterium) respectively. CFUs on each plate were evaluated to study microbiota in gut of weaning piglets.

Odor Analysis
The fecal of weaning piglets 250 g was collected in the sample bag for ferment 30 minutes. The odor analysis was detected by Gastec GV-100 machine, injected 100 ml above gas into the machine for 30 secs. Identify the odor concentration (NH 3 , N 2 S and R-SH) based on color change.

Immunity Analysis of Weaning Piglets (IgA and IgG)
IgA and IgG concentrations in serum samples were evaluated using Pig IgA and IgG ELISA kit (abcam, ab190536 and ab190813).
Each sample was analyzed in triplicate.     CFU/mL, respectively), of which group C and E displayed the best inhibitions ( Figure 5B). On the other hand, the level of total bacteria in each group showed no significant difference between week 0 and 6 ( Figure 5C). These results indicated the supplement of 0.1% Totipro together with PF1001 (1×10 7 CFU/g) significantly increased the number of lactic acid bacteria and reduced the number of E. coli with comparable efficiency to Tylosin 0.05% treatment in weaning piglets.      Note: Data are expressed as mean of fecal microbiota change rate for n = 24 weaning piglets per group. Values with different superscript letters are significantly different at *p < 0.05; **p < 0.01 by t-test.  Weaning is a sudden, complex and highly stressful event in pig's life. It is a transition from liquid to solid food, and needs to be taken smoothly step by step to make sure the health of the piglet is in a good condition. The starter feed is very crucial to a successful transition and the taste is important to piglets.  From our odor analysis of the feces, components of ammonia, hydrogen sulphide, and thiol derivatives are reduced upon the addition of Totipro or/and PF1001 in the feeding. The nitrogenand sulphur-containing gas contributes the most to the stinky smell of feces. Therefore, supplements of Totipro PE0401 or/and PF1001 decrease the production of strong odors and improve the environment of the piglet housing.

Discussion
If the composition of gut microbiota is roughly divided into two ends of beneficial or pathogenic populations, the balance can be dynamically affected by the external condition of the host. Gut bacterial dysbiosis has emerged as a leading cause of post-weaning diarrhea [16], and treatment with capsulized fecal microbiota transplantation is proved to ameliorate the symptom in piglets [6]. From the analysis of fecal microbiota, additions of Totipro PE0401 or/and PF1001 do not affect the size but the proportion of microbiota. Take two populations as the example, the change of proportion showed an increase in the population of Lactic acid bacteria, which represents the beneficial bacteria, and a decrease in the population of Escherichia, which represents the major pathogenic bacteria which causes diarrhea.
The NGS analysis shows the change of microbiota community in a more comprehensive aspect [17]. The bacteria culture on petri dish grows all lactic acid bacteria, and shows an increase of the community upon the treatment. The richness of Lactobacillus, but not Bifidobacterium, in NGS analysis increases significantly, and the increase is contributed especially by the specie of L.
johnsonii. The population of pathogenic bacteria, Escherichia, decreases significantly on petri dish, although the richness of the bacteria shows only a moderate, but not significant, reduction in NGS analysis. On the other hand, richness of another pathogenic bacteria, Clostridium, decreases significantly upon the supplement of Totipro PE0401 together with PF1001 in NGS analysis. Taken all together, our treatments modulate the microbiota toward a more beneficial composition in weaning piglets.
In piglets, the amount of immunoglobulin A (IgA) is associated with the intestinal mucosal immunity against pathogens, and is especially important during weaning when a new form of feed is introduced [18]. The level of immunoglobulin G (IgG) represents the anti-infection immunity and is strongly associated with the survival of piglets [19]. Recent evidences have demonstrated that some probiotics facilitate increasing the host sIgA levels and some probiotics contribute to increase the host serum IgG levels [20].
Supplements of Totorpo or/and PF1001 elevate the level of IgA, and especially the supplement of Totipro together with PF1001 increases the level of IgG. This result indicates a fundamental enhancement of intestinal immunity is achieved by either Totipro or PF1001 supplements. Preferably, a more comprehensive protection feasible when Totipro PE0401 and PF1001 are given together.
Weaning diarrhea is one of the most frequent causes of heavy economic losses in pig herds. Although the antibiotic treatment was Volume 28-Issue 1 sufficient to solve this single problem, it raised more environmental issues and was limited for this use. Our previous study has demonstrated that the fermentation products of lactic acid bacteria have significant antibacterial activity against fish pathogens. Diet supplementation with lactic acid bacteria fermentation products in aquaculture significantly alters the gut microbiome of fish. In this study, supplements of Totipro or/and PF1001 reduce the occurrence of diarrhea, and stabilize the gut health sooner than the antibiotic treatment. Preferably, the best alleviation of the symptom is performed when Totipro and PF1001 are given together. One of the important characteristics of probiotics is the antibacterial activity, which is possibly achieved through the production of bacteriocins [21]. Moreover, researches about postbiotic applications on diarrhea in children have obtained positive effects [9], so as our results in piglets. Taken all together, a combination of the postbiotics, Totipro PE0401, together with probiotic mixture, PF1001, is able to modulate gut microbiota and alleviate diarrhea sufficiently in weaning piglets.

Conclusion
The supplement of the postbiotics, Totipro PE0401, together with probiotic mixture, PF1001, is an alternative solution to weaning diarrhea in piglets. Supplements of Totipro PE0401 or PF1001, or especially together, give better feed conversion rates and less odor of feces. The composition of gut microbiota was modulated, and immune response was enhanced toward a better defense against pathogens. The improvement on diarrhea were comparable with the antibiotic treatment.