SURVIVAL OF MICROENCAPSULATED PROBIOTIC BACTERIA IN DYNAMIC GASTROINTESTINAL CONDITIONS AND THE EFFECT OF FOOD MATRICES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028047
• Grant No.: 2022-67017-36583
• Cumulative Award Amt.: $596,050.00
• Proposal No.: 2021-09559
• Project Start Date: Jan 1, 2022
• Project End Date: Dec 31, 2025
• Grant Year: 2022
• Program Code: [A1364]- Novel Foods and Innovative Manufacturing Technologies

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
(N/A)

Non Technical Summary
Microencapsulation of probiotic bacteria to improve cell survival in the gastrointestinal (GI) tract has been studied extensively. In vitro studies using the static digestion models are commonly employed to investigate release kinetics of probiotic embedded microcapsules (PEMs), but the experimental conditions are oversimplified due to a lack of simulation of complex physiological processes present in the human body. Besides, PEM fortified foods are available to consumers but the influence of the food matrices on probiotics remains to be elucidated.The goal of this study is to understand the effect of dynamic GI conditions and food matrices on transit, release, mucoadhesion, and survival of microencapsulated probiotics. Six microcapsules with different wall materials will be prepared with extrusion, spray drying, and emulsification methods. The impact of dynamic GI conditions (pH, viscosity, and contraction force) on the release kinetics of PEMs will be studied. Dynamic stomach and intestine models simulating realistic physiological conditions will be used to investigate the GI transit of the microcapsules and the cell viability as affected by food matrices and wall materials. Ex vivo porcine mucosal model will be used to explore the mucoadhesive properties of the PEMs. In vivo swine model will be used to reveal the cell survival and colonization from different PEMs, and the effect on microbiota changes. The information obtained from this study will contribute to developing effective microencapsulation delivery systems for probiotic bacteria and other bioactive targeting improved health benefits.

Animal Health Component

100%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	5010	2010	100%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
5010 - Food;

Field Of Science
2010 - Physics;

Keywords

probiotics

cell viability

dynamic gastrointestinal model

food matrix

microencapsulation

Goals / Objectives
Microencapsulation of probiotics for improving human health has been extensively studied in the last decade. Most of the past studies focused on microencapsulation techniques and carrier materials. The cell release and survival studies and related mechanisms were mostly studied with the static digestion model. The information is limited about how the dynamic changes in pH and rheological conditions of GI media, as well as contraction forces, could modulate the transit and release properties of probiotic-embedded microcapsules (PEMs). The effect of the food matrices and wall materials on the transformation, transit, mucoadhesion, and release properties of PEMs in dynamic GI conditions remains mostly unknown.The overall goal of this project is to understand how dynamic GI conditions, food matrices, and wall materials affect transit, release, mucoadhesion, and cell survival of PEMs in the human GI tract using in vitro, ex vivo, and in vivo models.Specific objectives are 1) Investigate the impact of dynamic GI conditions (pH, viscosity, and contraction force) on release and viability of microencapsulated probiotics; 2)Study the protective effect of selected wall materials with different preparation methods on probiotics at realistic physiological conditions; 3)Determine the effect of food matrices and composition on the behavior of PEMs in the human GI tract; 4)Study the mucoadhesive property of PEMs as affected by wall materials and food matrices; 5) Assess the cell release and colonization in pigs and the effect on gut microbiota changes.The information obtained from this study will contribute to developing effective microencapsulation delivery systems for probiotic bacteria and other bioactive targeting improved health benefits.

Project Methods
Species of Lactobacillus or Bifidobacterium will be used as the probiotic bacteria for microencapsulation. Several wall materials with high potential in food applications will be used for the study. Different technologies including emulsification, extrusion, and spray drying will be used to create PEMs.Six PEMs with different wall materials will be prepared with > 8 Log CFU bacteria/g microcapsule. The particle size will be 1-2 mm, 10-50 μm, and 100-200 μm for the PEMs made with extrusion, spray drying, and emulsification methods, respectively.Study 1. Effect of pH, viscosity, and contraction forces on PEM release and cell viability.We will use dynamic digestion models DGSM and HDMto study the release kinetics of PEMs in the stomach and intestine.The effect of pH, viscosity, and contraction on the release of PEMs will be studied.This study will provide kinetic information on the release and survival of probiotics as affected by wall materials and various GI conditions. Mathematical models will be derived to relate cell release from PEM with the pH, viscosity, and contraction forces.Study 2. Effect of food matrix and composition on the properties of digesta, and transit and release of PEMs.Individual food components (protein, lipid, and carbohydrate), and matrices (liquid, semisolid, and solid) will be mixed with PEMs. The transit and release of PEM in the human stomach as affected by the food matrix will be tested in the dynamic stomach and intestinal models (GSM and HDM) to evaluate the GI transit of PEM, cell release and survival as affected by realistic physiological conditions.This study will provide information about the relationships among food composition/structure and properties of digesta (pH, viscosity, and water diffusivity), and the effect of food matrices on transit and release of different PEMs in the human GI tract and cell viability, including the gastric retention time and intestinal transit rate.Study 3. Ex vivo evaluation of mucoadhesive properties of PEMs as affected by food matrix.Freshly excised porcine GI tissues (stomach, duodenum, jejunum, ileum, and colon) will be used. Tensile test and flow-through test will be performed to assess the mucoadhesion properties ofdifferent PEM in the stomach and intestine as affected by food matrices.Study 4. In vivo study using a swine model.Sixty domesticated barrowswill be used for5 treatments (two harvest groups/treatment)which will consist of 3 PEMs selected based on the results of in vitro studies, free cells in water, and untreated control (gavage with water). During the harvesting procedures, samples will be collected from each pig GI tract including tissue and digesta for analysis of PEM and cell contents. Fecal samples will be collected to analyze microbiota changes.The results from the study will demonstrate the distribution of PEMs and the cell release/survival along the different sections of the GI tract, and show the colonization status of probiotics and the microbiota changes. All together, the results will allow for quantification of probiotic transit and transit rate, release, survival, adhesion, and colonization in an in vivo model that is similar to the human digestive system.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:The primary target audience for this project includes probiotic manufacturers, food companies, governments, regulatory agencies, researchers and scientists seeking to understand the survival of probiotics in the gastrointestinal tract and the efficacy of microencapsulation systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students have contributed to the project by conducting experiments and preparing reports and manuscripts for publication. They also attended professional conferences and presented their findings. How have the results been disseminated to communities of interest?Two papers were submitted topeer-reviewed journal and under review. A poster was presented at 2024 Conference of Food Engineering (COFE). Seattle, WA. August 25--28, 2024. What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we will: 1) investigate the mucoadhesive properties of the microcapsules; 2) complete the pig feeding trials and analyze the data; and 3) disseminate our findings through manuscripts and conference presentations.

Impacts
What was accomplished under these goals? The emulsification-based microencapsulation techniques using soy protein isolate (SPI) were evaluated for their efficacy in enhancing the delivery and storage of Lactobacillus rhamnosus GG (LRGG). Five different types of microencapsulation were employed using 3% (w/w) SPI, including a water-in-oil-in-water (W/O/W) emulsion (MD), external gelation with sodium alginate (ME), a water-in-oil (W/O) emulsion with pectin (MP), a W/O/W emulsion with pectin (MDP), and a W/O/W emulsion with external gelation (MDE). The analyses performed included particle size distributions, encapsulation efficiency, microscopic observations, creaming index, and LRGG survival during cold storage and simulated gastrointestinal (GI) digestion. The results showed that particle size distributions were highly influenced by the microencapsulation technique used. Enhanced storability of LRGG during cold storage was observed in the MD, ME, MDP, and MDE samples compared to free LRGG. Furthermore, MD, ME, and MDE significantly improved the survival rate of LRGG during 2 hours of simulated gastric digestion (pH 2.5) and 4 hours of intestinal digestion (pH 8.1) compared to free LRGG. In contrast, the W/O single emulsion and the addition of pectin did not contribute to the enhancement of probiotic survival during cold storage or GI conditions. Pig feeding studies (Rounds 1 and 2) were conducted to evaluate the survival and colonization of LRGG incorporated into their diet. Four different microcapsule formulations were tested. Samples were collected for future analysis. A third trial (Round 3) is scheduled for Spring 2025.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Udo T. Kong F, Singh R. Impact of food matrices and dynamic gastrointestinal conditions for survival and release of encapsulated Lactobacillus rhamnosus GG. 2024 Conference of Food Engineering (COFE). Seattle, WA. August 25--28, 2024
• Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Vanare, S. A. N. K. E. T. (2024). DEVELOPMENT OF A DOUBLE EMULSION MICROENCAPSULATION SYSTEM FOR LACTOBACILLUS RHAMNOSUS GG USING PEA PROTEIN AND CELLULOSE. (Master's Thesis)

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The primary target audience for this project includes probiotic manufacturers, food companies, governments, regulatory agencies, researchers and scientists seeking to understand the survival of probiotics in the gastrointestinal tract and the efficacy of microencapsulation systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students have worked on the project. They havecarried out experiments and prepared reports and manuscripts for publication. They also attended professional references and presented their results. How have the results been disseminated to communities of interest?One paper has been published in peer-reviewed journal. A poster was presented atIFT Annual Conference. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we continue to study as follows; 1) developing an in vitro dynamic intestinal model for the assessment of microencapsulated probiotics; 2) exploring novel wall materials and other encapsulation technologies to enhance the performance of microcapsules; 3) preparing an assessment of microencapsulated probiotics using ex vivo swine models.

Impacts
What was accomplished under these goals? Lactobacillus rhamnosus G.G. ATCC 53103 was encapsulated using extrusion-based microencapsulation with alginate and chitosan wall materials. The survivability of the probiotic microcapsules was investigated using an in vitro dynamic gastric digestion model (DGDM), which enabled to simulation of continuous gastric juice secretion and emptying in addition to an in vitro static digestion model (SGDM). Compared to SGDM, it was revealed that the number of viable cells and the weight of the microcapsules significantly declined by DGDM at pH 2.5. One of the possible reasons is that the acidity of the simulated gastric juice was maintained quite close to the adjusted pH, whereas SGDM shows a higher pH at the end of digestion tests. It is also implied that the presence of gastric pepsin and mucin significantly affected the survivability of probiotics, compared to gastric fluids without enzymes. The effect of model food matrices, including casein, corn starch, and soybean oil, was also tested with probiotic microcapsules in SGDM. The results indicated that casein significantly mitigated the acidity of gastric juice, leading to a significant increase of viable cells during gastric digestion. These findings highlighted that; 1) the assessment of microencapsulated probiotics based on in vitro digestion models would overestimate the number of viable cells more than those in human gastrointestinal tracts; 2) when microencapsulated probiotics come to be applied in food products, the nutritional components would have a significant impact on determining the survivability of probiotics in the gastrointestinal tracts. Microencapsulation of Lactobacillus rhamnosus GG was also achieved using the emulsification method. We encapsulated Lactobacillus rhamnosus GG cells using pea protein and cellulose nanocrystals (CNC) as wall materials inside a W/O/W double emulsion. The microencapsulated probiotics (MEPs) were tested for their particle characteristics, storage stability and viability in a simulated gastrointestinal system. The results indicated that the incorporation of CNC in the wall matrix along with pea protein resulted in a more stable emulsion system. The digestion studies revealed multiple aspects. The pea protein-CNC encapsulated probiotics had a better viability in both gastric and intestinal stages of digestion compared to control and pea protein encapsulated system. Moreover, pea protein-CNC probiotic capsules showed delayed release of probiotics in the intestinal digestion stage. These results demonstrate the potential of pea protein and CNC as encapsulation materials for probiotics, as well as the potential of double emulsion systems for targeted release of probiotics and similar functional entities. We also determinedthe baseline levels of various culturable microbial species in the swine guts.Domesticated barrows (castrated male swine; sus scrofa domesticus) were used in three independent trials of the study. In each trial, the swine (n = 3) with an average individual weight of 118 kg were housed in a secured, climate-controlled environment that had individual pens (~3.9×1.5 m), each with solid (1.5 m) and slated floors (2.4 m), a feed bin, and automatic waterer. The swine were randomly allocated to individual pens and fed a commercial grower meal containing 14% crude protein, 5% crude fat, and 4% crude fiber. Fecal samples (approx. 50 g) from each swine were collected once a month and transported to our laboratory under freezing conditions. The populations of beneficial and pathogenic microorganisms in 1 g of each well-mixed fecal sample were analyzed using growth media selective for each group of microorganisms.Results showed that the populations of two beneficial bacteria, Lactobacillus and Bifidobacterium were 7.4±0.5 and 3.2±0.9 log CFU/g, respectively. Pathogenic bacterial populations, including those of Bacteroides, Clostridium, Enterobacteriaceae, Staphylococci, and Streptococci were 3.5±0.7, 7.9±0.1, 6.3±1.3, 5.9±0.4, or 8.8±0.5 log CFU/g, respectively. The total anaerobic population was 9.2±0.5 log CFU/g, whereas the populations of non-sporing anaerobes and gram-negative anaerobes were 8.7±0.5 and 8.0±0.5 log CFU/g, respectively. The total facultative anaerobes were 6.5±1.0 log CFU/g.The study provides important baseline information for research that uses swine as an animal model to study the dietary influence on beneficial and pathogenic bacterial populations in human guts.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Udo T, Mummaleti G, Mohan A, Singh RK, Kong F. Current and emerging applications of carrageenan in the food industry. Food research international (Ottawa, Ont.). 2023 Nov;173(Pt 2):113369.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Udo T. Kong F, Singh R. Survival of microencapsulated probiotic bacteria in dynamic gastric condition: An in vitro study. 2023 IFT meeting. July 16  19, 2023.

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The primary target audience for this project includes probiotic manufacturers, food companies, governments, regulatory agencies, researchers and scientists seeking to understand the survival of probiotics in the gastrointestinal tract and the efficacy of microencapsulation systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students had worked on the project. One abstract was submitted to 2023 IFT annual conference. How have the results been disseminated to communities of interest?One abstract based on the results of the study was submitted to the 2023 IFT annual conference. What do you plan to do during the next reporting period to accomplish the goals?We will continue to study the performance of spray drying and other microencasulation technologies on probiotic survival during dynamic in vitro digestion, including extrusion and emulsification methods.

Impacts
What was accomplished under these goals? We microencapsulated L. Plantarum 4008 with maltodextrin, whey protein isolate, and gum Arabic as wall materials using a mini spray-dryer (Model B-290) with 130 °C inlet and 76 °C outlet air temperatures. The probiotic microspheres were tested in the dynamic gastric digestion models DGSM. The results indicated thatthe viable cells maintained a relatively constant value when the pH decreased from 6 to 3, then rapidly dropped to almost zero when pH further decreased to 2, indicating that vast cell death occurred at low pH during gastric digestion. It also shows a different effect of proteins on the viable cell counts, and a higher cell viability was shown when soy protein was present at the end of digestion.When the viscosity was increased (by adding guar gum), a more rapid decline in the viable count is shown indicating the cell death is accelerated by increasing viscosity. This phenomenon is somehow in disagreement with literature as it was reported that high viscosity can help sustain cell survival. These results emphasize the importance of dynamic changes in the pH and viscosity, as well as the food components on cell survival. It also indicates that the dynamic GI model can give more comprehensive information about cell viability as compared to the static model.

Publications