Progress 06/01/23 to 05/31/24
Outputs
Target Audience:Project Title: In Vitro And In Vivo Evaluation Of Bacteria From Swine-Related Sources As Direct-Fed Probiotics For Enhanced Feed Utilization The main objective of this project is to isolate, identify, and characterize potential probiotic bacteria that can facilitate enhanced feed utilization in swineto allow for betterfeed efficiency and weight gain. This project targets a variety of audiences that includes food animal producers (swine and others),consumers, students, food industry workers, professionals, scientific academicians, and the general public. Scientific presentations and journal articles target scientific/academic professionals, food industry management, and the general public. Animal probiotics that facilitate enhanced feed utilization are aiming toimproveweight gains through more efficient utilization of feed by converting feed to moremass, or obtainingweight gains withless feed.In the past, providing low levels of antibiotics as growth promotants in animal feed to suppress intestinal infection and improve weight gain, howeverthis practice promotes the enrichment of intestinal bacteria harboringantimicrobial resistance and is no longer encouraged. The availability of alternate methods of enhancing weight gain (probiotic bacteria for enhanced feed utilization) should be well received by industry, consumers, and the scientific community. Changes/Problems:Isolation methodology. Isolation by direct plating of intestinal samples may not be the most efficient way of isolating preferred bacteria. Direct playing allows you to work with the last series of dilution plates (plates with >1,000 cfu are not likely to allow you to distinguish activities on differential agars and/or recover the bacteria). However, the method of GIT liquor/fermentation enrichment may allow usto specifically enrich certain metabolic types with the specific substrate of interest provided in the fermenter media. Uncertainty in bioassay results. Should the differential agar methods not work as intended, each assay has an alternative protocol that could be used. For example, the lipase assay is currently using Rhodamine B-Olive Oil agar to assess the presence of lipase activity. However, Spirit Blue agar with the addition of Tributyrin and Tween 80 could be alternatively used. Tributyrin in this case will be used as the lipid source and the Tween 80 is an emulsifier to homogenize the lipid with the agar. Additionally, each agar-based assay is being conducted with both TSA and MRS agar to test for not only LAB that might be present but all culturable bacteria. We are aware that there are multiple agar formulationsthat can be used in our bioassay agars and perhaps others are better. Probiotic inoculum preparation for animal trials. We intend to give the probiotic inoculum to pigs in their drinking water (we have defined the minimal cfu/ml that we would establish). One of the problems is that as the pigs grow, they eat and drink more feed and water; we may find it hard to provide the same level of probiotic per amount of feed or water consumed by the pigs as they get larger. However, if this is untenable, we are examining if we can obtain freeze-dried bacteria to the solid feed and/or have freeze-dried cultures micro-encapsulated that would better survive travel into the GI tract as alternative approaches if needed. Selection of prebiotic substances. Many published works with prebiotic additives have noted the difficulty of using some prebiotics that are easily degraded in the intestinal tract limiting their efficacy and the number of substances available to test. This proposed work will focus on prebiotic ingredients that have been determined to survive in the animal GI tract including non-digestible oligosaccharides, MOS, and other plant cell wall carbohydrates. Effect of low pH in the stomach as a barrier to bacteria that may reside in the small intestine. The stomach of swine is about the lowest pH that ingested bacteria will experience (pH 2.3-4.5) and then increases as you get to the small intestine (pH 3.5-6.7; duodenum, jejunum, ileum). The bacteria selected for animal studies will be 'acid-adapted' to provide some degree of adaptation to low pH before making them available to animals in drinking water. While each bacterial strain will be assessed for resistance to bile and low pH, each agar assay to assess for biochemical activity will not be acidified. However, there could be a pH-related effect on the biochemical activity of the bacteria. Therefore, the assays can be conducted on agar with an adjusted pH (pH 6.0) to evaluate if there is a difference in enzymatic activity compared to the unadjusted agar assays. The identification of spore-forming probiotics may eliminate this potential problem. What opportunities for training and professional development has the project provided?Aside from academically oriented food microbiology research, graduate and undergraduate students who work on this project are located in the Robert M. Kerr Food & Agricultural Products Center. Aside from their involvement in this research, they have an opportunity to assist food processing companies through multiple projects that matriculate through my lab, or the Center, to carry out our mission to help Oklahoma and national-based companies with issues/problemsthey have regarding further processed foods. The PI's Food Microbiology lab is structured so that graduate students not only work on their specific research projects but also provide company assistance by contributing efforts to industry projects to help solve food microbiology-related problems that local companies may be experiencing. This provides a great opportunity for undergraduate and graduate students to enhance theirproblem-solving abilities with actual problems incurred by the food industry. Because of this, >90% of my MS/PhD-degreegraduates are well sought after and find placement in the food industry; several of my Ph.D. students have found academic faculty positions as well. I would say that our work provides good training and excellent opportunities for those students who spend the time to learn additional skills while earning their degree. In several recent years, I had 3 undergraduates in my lab who worked on research, and that helped them land positions in veterinary medicine. Our center also puts on many food safety workshops each year (HACCP, Food Defense, Preventive Controls for Human Foods, SQF, FSMA Compliance, etc) for industry, and both graduate and undergraduate students are encouraged to take these workshops to enhance their knowledge and capabilities. Students taking these courses/workshops get accredited by the appropriate organizations and they are well sought after by industry who would otherwise have to pay to send employees for these trainings. I have led a team to initiate the "FAPC Certificate of Training as a Food Safety Professional" for industry workers and/or students who accumulate sufficient credits of workshop training (12 credits) requiring a minimum of 2 workshops in each of three workshop categories: Basic, Advanced, and Regulatory. The certificate helps identify those employees who have achieved sufficient training that they should be considered strategic assets within their organization. Likewise, students who attain the certificate during their undergraduate/graduate degrees would be considered valuable job candidates by food companies that don't have to 'retrain' them once they are hired. The industry has responded well to our certificate program and we have now 'graduated' approximately 26 people (total) who have achieved these certificates of training (http://fapc.biz/workshops/foodsafetyprofessional How have the results been disseminated to communities of interest?The results of our research work are disseminated via peer-reviewed research publications (publications in preparation), departmental/university magazines, and the R.M. Kerr Food & Ag Product Center (FAPC) website. Also, information is disseminated through seminars or presentations: we had a poster presentation at the Arkansas Association of Food Protection (September 2023) and the FAPC Food Science Research Symposium (April 2024) and additional updated poster presentations will be made at both the Project Directors' Mtg (Alberta, Canada)and the 2024 Annual Meeting of the International Association of Food Protection in Long Beach, CA (July 2024). We have an in-house communication specialist who helps us put out short bulletins/articles (FAPZ.biz website, Food Safety blog articles, and FAPC podcasts) and other extension-related publications. This data will be of interest to swine producers and will be disseminated by our newsletters to stakeholders of the Department of Animal and Food Sciences. What do you plan to do during the next reporting period to accomplish the goals?Prior swine samples and probiotics used in initial swine trials were obtained by direct plating. The prior swine trials were performed with potential probiotic bacteria displaying selected metabolic enzymatic activitiesisolated from swine intestinal and fecal samples by direct plating of samples. The current approach is to use GIF liquor/anaerobic fermentation supplemented with swine feed for enrichment. We have now initiated the use of anaerobic fermentation with nitrogen gas of residual bacterial populations obtained from centrifugal clarification of swine gastrointestinal (small intestine) and stomach contents, supplemented with piglet feed (Phase 1 vs.Phase 4 feeds) that can enrich to higher levels for detection and recovery of those bacteria that can grow in these conditions which simulate the swine intestinal tract. Spore-forming isolates (Bacillus) as probiotics. Further, we are also screening for spore-forming (Bacillus?) strains by heating the dilutions made for plating unheated samples and plating themto identify spore-forming probiotics which would provide a more resistant viable probiotic strain for animal dietary supplementation. Prebiotics for enhancement of probiotic cultures in vivo. With respect toBacillus, we have already identified a potential quorum-sensing effect wherebyBacillus sp.only produces copious antimicrobial inhibitors in the presence of another foodborne pathogen. If we can define this factor that causes this (it also induces copious amounts of excretedBacillus inhibitory activity in vitro), this may be a 'prebiotic' that can be given directly to animals being fed these probiotic cultures (to inhibit animal pathogens in the gut). Microbiome analysis. Finally, weare taking samples from our fermenter studies to perform microbiome analysis of the clarified GIF vs post-fermented GIF to identify the transition of microorganisms during our fermenter approach.
Impacts
What was accomplished under these goals?
Bacterial metabolic activity agars. Intestinal and fecal samples were collected from freshly harvested pigs in the abattoir at the Food and Ag Products Center. Samples were transferred to the microbiology lab in sterile bottles, diluted 1:10 with BPW, serially diluted and plated on bioassay agars that were incubated anaerobically at 37 °C for 48-72 hours and examined for enzymatic activity. Amylase assay: TSA/MRS plates containing 0.25% starch to assess amylase activity confirmed by clear halo zones around the bacterial colonies following iodine treatment. Protease assay: TSA/MRS plates containing 1% casein sodium salt and 10% skim milk was used to demonstrate proteolytic activity by the presence of clear zones around colonies. Lipase assay: TSA/MRS media supplemented with 3g of yeast extract, 5g of peptone, 2.5 mM of Ca++, and 5 mM of Mg++ was autoclaved; after autoclaving, a mixture of 5 mL/L of Tributyrin and 5 mL/L of Tween 80 was added. Lipolytic activity was determined by clear zones of hydrolysis around colonies. Cellulase activity: TSA/MRS plates containing 0.5% cellulose powder. Following incubation, plates were flooded with 0.1% Congo Red and poured off after 15 minutes. Plates were flooded with 1M NaCl and poured off after 15 min. Clear zones were indicative of cellulase activity. Isolation of probiotic cultures. Isolates demonstrating positive assay results on the specific differential agars were carefully streaked onto TSA/MRS media plates. Streaked plates were incubated anaerobically for 24-48 hours at 37°C and individual colonies were selected and patched to bioassay agar plates to confirm their activity and then evaluated for other bioassay activities. Identification of swine-related probiotics (16S rRNA PCR/sequencing). Strains showing positive metabolic activities were confirmed for identity by 16S rRNA RT-PCR and DNA sequencing. The purified isolates were grown from frozen stock in TSB broth media and incubated anaerobically at 37 °C for 18 hours. Following DNA extraction and purification, DNA amplicons from each sample was sent to the OSU DNA/Protein Core Facility for sequencing. The resulting forward and reverse sequences for each isolate was trimmed and aligned using MEGA-X software, and further compared to the known bacterial sequences utilizing the Basic Local Alignment Search Tool (BLAST; NCBI; Bethesda, MD). The selected isolates were identified as probiotic strains; Bifidobacterium porcinum, Bifidobacterium thermophilum, Streptococcus lutetiensis and Limosilactobacillus reuteri. Preparation of culture cocktail for animal trials. Individual strains were grown anaerobically overnight in 9 mL MRS broth at 37 °C. Cultures were individually reinoculated into 250 mL centrifuge bottles containing 150 mL MRS or TS broth followed by overnight anaerobic incubation at 37 °C . Cultures were harvested through centrifugation (6000 rpm, 15 min) and resuspended in 10 mL of freezing media containing 10% non-fat dry milk, 2% trehalose, 0.2% yeast extract and 5% sucrose per 100 mL. Subsequently, the four probiotic strains were combined and stored in an ultra-low freezer (−80 °C) until used. Animal trials with probiotics directly isolated from swine. Swine trials were conducted at the OSU Swine Research Center. Pigs utilized in the trial were sourced from a commercial sow farm in Oklahoma. The trial involved 280 crossbred pigs distributed among four treatments (28 pens; 10 pigs/pen; 7 pens/treatment). A total of 280 pigs were distributed into 28 pens based on their weight, with each pen housing 10 pigs. Beginning their nursery phase at 20 days, starting with an average weight of 5.77 kg, the pigs were fed corn-soybean meal-based diets throughout the 43-day nursery period. The diet was a common 4-phase nursery diet that meets or exceeds the NRC (2012) recommendations, with feed provided ad libitum. The diet did not contain antibiotics or pharmacological levels of zinc or copper. The experimental trial included 4 treatments: Control treatment with feed alone (Treatment 1), experimental treatments included bacteria added to feed (Treatment 2), bacteria added to water (Treatment 3). and bacteria added to feed and water (Treatment 4). For the water-based probiotic treatments, a stock solution was prepared by mixing 200 mL of culture cocktail in 4L of water, continuously stirred using a magnetic stirrer. The stock solution was mixed with tap water using a Dosatron dispenser and the diluted stock solution (1:128) was delivered to a water station in each pen. The feed-based probiotic treatment included a total of 200 mL culture cocktail mixed into 10 lbs. feed which was then added to 500 lbs. of feed. The growth performance data was collected weekly by weighing the pigs and feed to calculate the average daily gain, average daily feed intake, and gain to feed ratio. Data was reported for d 0-21, d 21-43, and d 0-43 period. On day 43, the average weights for different treatments were 51.76 lbs. (Treatment 1), 51 lbs. (Treatment 2), 52.61 lbs. (Treatment 3) and 50.1 lbs. (Treatment 4). The treatment group receiving probiotics only in water demonstrated a higher average weight gain (P>0.10) and exhibited a 1.6% increase in body weight over control pigs, suggesting potential to improve swine weight gain and enhance feed efficiency. Overall, the pigs demonstrated robust performance and sustained a healthy status, potentially constraining any observable response to bacterial supplementation. Subsequent investigations will focus on optimizing bacterial strains and concentrations administered via water or feed. Neither water nor feed intake exhibited adverse effects attributable to bacterial sources. Using 'GIF-liquor' as selective medium may enhance our chances of enrichment and identification of improved probiotic strains. Isolation of probiotic cultures (fermentation of GIF fluid + swine feed). Clarified gastrointestinal fluid (GIF) 'liquors' sourced from swine intestinal and stomach fluids were clarified by centrifugation (without autoclaving) to retain bioactive components and supplemented with piglet swine feed and used in pH-controlled (pH 6.0, 5.0M NaOH) anaerobic fermentation (N2 gas). Clarified gastrointestinal (small intestine) and stomach fluids recovered from swine were mixed 2:1 (400mL intestinal fluid + 200mL gastric fluid = 600 ml GIF) and split equally for addition of 15% Phase 1 or 15% Phase 4 feeds; the GIF was stirred continuously while ground feeds were separately added to their fermenter jars; another 100 ml of sterile water was also added. Samples were taken on day 0 from GIF without feed, on day 0 after GIF+Phase 1 feed and GIF+Phase 4 feed were added, and again day 1 from Phase 1 and phase 4 feeds after 24 hours of fermentation. Samples were serially diluted and plated on TSA for plate counts and on bioassay agars for enzymatic activity. All the plates were incubated anaerobically at 37 °C for 48-72 hours. On day 1, sample dilutions were further heated (15 min, 80°C) and plated on TSA and bioassay agars to isolate spore-forming bacteria which may serve as probiotics due to their resilience and stability. Plate counts for samples plated on day 0 resulted in 2.79 log CFU/mL (GIF/without feed), 3.3 log CFU/mL (GIF/Phase 1 feed), and 3.28 log CFU/mL (GIF + Phase 4 feed). After 24 hours, plate counts resulting in 8.84 log CFU/mL (GIF/Phase 1 feed), and 8.82 log CFU/mL (GIF/Phase 4 feed). This increase is attributed to favorable anaerobic conditions and feed substrate supplementation providing nutrients to stimulate growth. Samples were screened for various enzymatic activities and positive isolates were recovered for activity confirmation, identification, and evaluation for additional bioassay activities. Furthermore, 16S rRNA PCR, DNA sequencing and NCBI Blast search tool will be used to identify promising probiotic strains while confirming their non-pathogenicity.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Gavai, K., Carter, S., and P. Muriana. 2023. In vitro evaluation of bacteria isolated from swine-related sources as direct-fed probiotics for enhanced feed utilization and improved growth performance in swine. Arkansas Association of Food Protection, Fayetteville, AR, September 26, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Gavai, K., Carter, S., and P. Muriana. 2024. Utilizing clarified gastro-intestinal fluid (GIF) for anaerobic fermentation, enrichment, and isolation of swine-specific probiotics. FAPC Food Science Research Symposium, Stillwater, OK, April 11, 2024.
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