SCIENTIFIC FRONTIERS ABSTRACTS

1 A comparative study of packaging configurations on the stability of Bifidobacterium lactis Bl-04™

Frank Haack, Sarah J.Z. Hansen, and Connie Sindelar, DuPont Nutrition & Health, Madison, Wisconsin

USA

Dietary supplement manufacturers and distributors must make many decisions to plan for the

launch of a new product, including maintaining product integrity and marketing specifications.

Careful consideration should be given to the packaging configuration for probiotic capsules, because

it will have an impact on maintaining the viable cell count of the product. High Density Polyethylene

(HDPE) is the most popular bottle type used for probiotic products because of its low cost, but when

stored in high humidity storage conditions, water activity (Aw) can rise in the capsule and viability

declines. This study compares the stability of probiotic strain Bifidobacterium lactis Bl-04™ when

stored in either HDPE bottles or CSP™ desiccant-infused bottles over the shelf life of 2 years. The

desiccant in CSP™ bottles was able to mitigate any moisture ingress, lowering Aw. It improved

survivability of the probiotic culture compared to capsules in HDPE bottles when stored at conditions

above refrigerated temperatures. Another variable rarely considered is the number of capsules per

bottle relative to the bottle size. Often a monthly supply of capsules is placed in an oversized bottle

to allow for more label space for marketing. The greater the bottle surface area and headspace, the

greater the moisture ingress into the capsules. When there are fewer capsules in the bottle, there is

less product to absorb the moisture which results in faster increase in Aw. This study shows that

effective desiccation and decreased bottle surface area and headspace relative to the capsule count

reduces Aw and improves survivability.

2

Probiotic Standards from the United States Pharmacopeial (USP) Convention

Seong-Jae Yoo, Maria Monagas, and Gabriel Giancaspro

Science Division-Dietary Supplements and Herbal Medicines, United States Pharmacopeia (USP)

Convention, Rockville, MD, USA SJY@USP.org; Phone: 301-230-6366

USP standards for probiotics are publically established to assure quality and supply chain integrity of

probiotic products through appropriate identification, assay methods, acceptance criteria, and

controlling limits for contaminants. USP has published 5 monographs of 7 probiotic strains in the USP-

NF (USP40-NF35) with the collaboration of major manufacturers as listed below. Currently, the two

draft monographs (Bacillus coagulans and its capsules) undergo public comment. Also, a general

chapter, <64> PROBIOTIC TESTS, for probiotics has been proposed in Pharmacopeial Forum (PF) 43(2)

to provide general information and testing procedures for identification, enumeration, contamination

and other requirements for probiotics. USP is seeking feedback on those drafts of monographs and

general chapter from stakeholders from industry, regulatory agencies and academia.

Lactobacillus acidophilus LA-14

Lactobacillus acidophilus NCFM

Lactobacillus rhamnosus HN001

Lactobacillus paracasei Lpc-37

Bifidobacterium animalis ssp. lactis (Bi-07, Bl-04, & HN019)

DRAFT - Bacillus coagulans GBI-30, 6086 and Capsules

DRAFT - General Chapter of <64> PROBIOTIC TESTS

The present poster summarizes the benefits that USP’s public standards offer stakeholders from

industry, academia and regulatory agencies; compendial standards developed for FCC and USP-NF;

monograph requirements, and examples of tests and acceptance criteria in probiotic monographs.

The United States Pharmacopeial Convention (USP) is a scientific standard-setting organization that

brings together stakeholders and interested parties in support of its mission to protect public health

and help ensure the quality, safety, and benefit of all medicine, food, and dietary supplements through

establishing quality standards. USP develops standards using science-based, public process guided by

its Council of Experts - volunteer experts from government, academia and industry.

3

Efficacy of Hyperbiotics Pro-dental probiotic in treatment of chronic periodontitis

Aim:The aim of this randomized placebo-controlled clinical trial was to evaluate the effects of

Hyperbiotics pro-dental probiotic lozenges as an adjunct toscaling and root planing (SRP).

Material and Methods: Forty chronic periodontitis patients were recruited and monitored clinically

and microbiologically at baseline, 2 and 4 months after non-surgical periodontal therapy i.e. SRP. All

patients received one-stage full-mouth disinfection and randomlyassigned over a test (SRP + probiotic,

n = 20) or control (SRP + placebo, n = 20)group.

Results: At study end point i.e.4 months, all clinical and periodontopathic bacterial level (A.

Actinomycetemcomitans, F. Nucleatum, P. Gingivalis and P. Intermedia) were significantly reduced in

both groups, while there was highly significantly more pocket depth reduction (p < 0.05)

andattachment gain (p < 0.05) in moderate and deep pockets; more microbiological reduction was

observed in the SRP and hyperbiotic pro-dental probiotic group.

Conclusions:This study proved that daily oral administration of Hyperbiotics Pro dental probiotic

lozenges with SRP resulted in significant additional clinical and microbiological improvements

primarily for initially moderate to deep pockets when compared to SRP alone.

Key Words: Periodontitis, Oral Microbiome, Dental Biofilm

Corresponding Author:

Dr Rajiv Saini

Associate Professor,

Department of Periodontology & Oral Implantology

Pravara Institute of Medical Sciences, Loni

Maharashtra, India.

Email: sainiaccess@gmail.com

Cell: +91-9923206789

4

Novel probiota reducing inflammation in the entire body and persisting in the

intestines at least 30 days

Robert H. Schiestl

Department of Pathology, Environmental Health Sciences and Radiation Oncology UCLA, 10833 Le

Conte Ave, 71-295 CHS, Los Angeles, CA 90095, USA, 310-267-2087, rschiestl@mednet.ucla.edu

When our lab moved from Harvard to UCLA we found a huge difference in genetic instability and

longevity in our Atm deficient mice after 5 years. When we changed the intestinal microbiota

back to conventional microbiota we could reproduce the phenotype at Harvard. We tested Atm

deficient mice for genotoxicity, genetic instability, DNA damage, inflammation markers, cancer

latency and longevity and high throughput sequencing of the intestinal microbiota. Isogenic mice

with different microbiota showed a four fold difference in life expectancy, a 4.5 fold difference in

genetic instability and DNA damage. The onset of lymphomas was significantly 2.5 fold different.

We sequenced the microbiota and found a Lactobacillus johnsonii strain as dominant bacterial

strain in the health beneficial microbiota.

Just this bacterium by itself reduced genotoxicity, reduced inflammatory cytokines, induced

anti-inflammatory cytokines and significantly reduced levels of cytotoxic T, CD3 and natural killer cells

in the spleen, liver and blood. We also found similar differences in Trp53 deficient and even in

wildtype mice. The underlying mechanisms is due to inflammation promotion or suppression

mediated by the intestinal microbiota. We did a clinical trial with this Lactobacillus strain that makes

a great yogurt. 13 people took it for 7 days and 12 of them had an increase of Lactobacilli in their feces,

7 of them had the same increase after 30 days, which is unique. Inflammation is involved in most

deadly diseases such as heart disease, cancer, neurodegenerative disease, inflammatory bowel

disease, ulcerative colitis, Lupus, Celiac disease, arthritis, asthma, and Diabetes.

5 Evaluation of an Acoustic Focusing Flow Cytometer for Enumeration of Probiotic Organisms

Andrzej A. Benkowski1 and Jean L. Schoeni1, 2

1Covance Laboratories Inc.

2201 Wright Street

Madison, WI 53704

2Corresponding author; E-mail - jean.schoeni@covance.com; Phone - 608.417.9528

Introduction: Probiotics manufacturers and consumers are demanding to know more about the

identity and quantity of organisms in probiotic products. Enumeration provides the most basic

information required and methodologies are evolving to provide results with improved accuracy,

precision, and turnaround times. Flow cytometry has shown promise in rapidly providing

quantitative data.

Objective: This study evaluated the use an acoustic focusing flow cytometer, in accordance with the

standardized method ISO 19344/IDF 232 (2015), to estimate the number of probiotics in samples.

Acoustic focusing is a newer innovation in the field of flow cytometry.

Methods: Use of the acoustic focusing flow cytometer (Invitrogen™ Attune™ NxT, ThermoFisher

Scientific) and ISO enumeration method was verified according to USP 39<1225>/ICH Q2R1 for

accuracy, precision, linearity, specificity, limit of quantification, range, and robustness. Powder

samples containing freeze dried probiotics were evaluated. Cytometric values were compared

to direct microscopic counts and plate counts derived from the same sample preparations.

Results: Functionality of the cytometer was confirmed by recovering counts >90% of applied BD

Liquid Counting Beads. Performance of the method was verified by meeting or exceeding

selected criteria: Accuracy and robustness ≥70% recovery compared to conventional methods;

precision showing ≤15% RSD; and specificity and linearity data producing R2 ≥0.95.

Conclusions: ISO 19344/IDF 232 (2015), conducted with an autofocusing flow cytometer, provides

accurate, reliable enumerations of probiotics in powders. This study supports the use of flow

cytometry as a strong, next step in the evolution of enumeration.

6 Title: Probiotics and Cognitive Function: Translation to Clinical Science

Author name: Matthew A. Roberts, Ph.D., MBA

Institution: Chief Scientific Officer - KGK Science Inc.

USA, Cell: (516) 864-7088, Email: mroberts@kgkscience.com

Proposed Session Abstract:

Objective: Probiotics play a major role in the bidirectional communication between the gut and

brain, termed the gut-brain axis. While preclinical evidence to elucidate these effects has been

robust, translation to humans is still emerging. Currently, numerous probiotic products exist on the

market, yet few are specifically targeted at cognitive health. Development of products that include

probiotics for cognition will require an understanding of the specific strains that act on cognition as

well as populations who respond to intake. Moreover, these ingredients will require rigorous

scientific evaluation for claim substantiation.

Methods: To date, clinical trial designs for probiotics and cognitive functioning have been plagued by

uncharacterized strains, a lack of viability assessments, and the unique context of cognitive testing.

KGK proposes the use of an Augmented randomized clinical trial© encompassing a two-stage

protocol design to better evaluate the effects of probiotics on cognition. This study design accounts

for the multifaceted nature of probiotics and personalizes trial conditions, providing valuable

information for a larger RCT aimed at claim substantiation.

Conclusion: This talk will focus on the current state of research into the effects of probiotics on

cognition and provide insight into optimal trial designs for their assessment in healthy populations.

Learning Objectives:

1. Clinical evidence of probiotics and the gut-brain axis

2. Issues in trial design for probiotics and cognition studies

3. Challenges of claim substantiation for probiotics

4. Insight into optimal trial designs to study the effect of probiotics on cognitive function

7

Comparison of lactobacilli species in two different populations of patients with atherogenic risk

José Antonio Vergara Cruz 1,2, Sergio Tecpanecatl Xihuitl1, Lilia Cedillo Ramírez1, 2

1.-Centro de Detección Biomolecular, Benemérita Universidad Autónoma de Puebla. 2.- Posgrado en Microbiología, Centro de investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Pue., México E-mail: josantonio17987@gmail.com Cell phone: 011 52 2225473942 Abstract:

It has been proved that lactobacilli have diverse beneficial effects on human beings. One of these is

the immune system modulation through regulation of pro-inflammatory and anti-inflammatory

cytokines, avoiding thus atheroma progression. Another described mechanism is bile salt hydrolase

increasing, which reduces serum cholesterol levels. On the other hand, atherosclerosis is featured by

cholesterol levels increase and their subsequent deposition into the vascular endothelium.

Furthermore, in people performing physical activities, dyslipidemia and even death by stroke have

been reported.

By having Apolipoprotein E3/E4 (Apo E3/E4) genotype an individual might genetically have a risk of

developing such condition.

The goal for this study is evaluating the reducing role of lactobacilli in patients featuring a genetical

risk to atherosclerosis and also comparing intense physical activity population with sedentary life

leading individuals.

First, we can identify the Apo E3/E4 genotype from a blood sample due to the PCR in real time, next,

an interrogation is performed in order to find out the person’s nutritional state, health and

atherogenic risk. Simultaneously, a lipids profile is performed.

Regarding the stool, thanks to the end-point PCR, 8 species of lactobacilli are searched (Lactobacillus

acidophilus, L. casei, L. fermentum, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus y L. salivarius).

These species have showed major hypocholesterolemic effects.

These assays are carried out every month for 3 months.

The results obtained until now show a heterogeneity in the presence of lactobacilli species among

populations.

8

Evaluation of the survival of probiotic bacteria in a coated enteric triple layer tablet under elderly

conditions

S.Courau1, L. Espinosa2 and K. Venema3

1 Merck Medication Familiale, France

² Merck KGAA, United Kingdom

3 Maastricht University – campus Venlo, The Netherlands

Modulation of the gut microbiota of senior population (microbiota and SCFA) may improve health

parameters. This study reports the findings of TNO in vitro gastro-intestinal model experiments for

the evaluation of the survival of Lactobacillus gasseri PA 16/8, Bifidobacterium bifidum MF 20/5 and

Bifidobacterium longum SP 07/3 in an enteric coated triple layer tablet under healthy elderly

conditions.

Probiotic bacteria administered orally are exposed to 2 stresses: gastric acid, and bile and digestive

enzymes contained in pancreatic juice. This is why it is important to test resistance to these stressors

in a dynamic manner, such as in TIM-1, in order to predict the delivery of viable cells to the gut.

Probiotic survival was high after the gastric compartment (97–257%; 3.52–5.38 * 107 CFU for L.

gasseri; 2.38–6.29 * 107 CFU for the bifidobacteria). Combined, survival was 98% (4.5 * 107 CFU).

After complete TIM-1 passage, survival for L. gasseri and the bifidobacteria was 7–8% (2.56–

2.88*106 CFU) and 11.9–15.1% (2.91–3.7*106 CFU), respectively; combined: 10.5% (3.01*106 CFU).

The high percentage of survival of the bacteria:

- confirmed the efficiency of the enteric coated triple layer tablet technology in delivering the

bacteria alive under elderly conditions.

- suggests maximum impact of L. gasseri PA 16/8, B. bifidum MF 20/5 and B. longum SP 07/3 in

the intestine, with metabolic activities such as their ability to produce metabolites as SCFA

and vitamins, which has been demonstrated in vitro, and which are of nutritional interest for

the elderly population.

9

Title: Lactobacillus fermentum ME-3 Produces Glutathione/Biomarker of

Aging

ABSTRACT: Lactobacillus fermentum ME-3 is a strain of probiotic bacteria that has been found to

synthesize glutathione. Glutathione is known as the “Master Antioxidant” and it is also a key

regulator of detoxification and immune health. All common disease states are associated with low

glutathione levels. Studies also report that elevated glutathione levels are associated with better

health and longer survival in all species tested, including humans. These studies confirm the

Glutathione Deficiency Hypothesis, which suggests that glutathione is a reliable biomarker of aging.

Oral glutathione supplements are not absorbed well. However, in human clinical trials,

individuals taking ME-3 gained a remarkable 49% increase in the ratio of reduced to oxidized

glutathione. Having an effective, reliable method of increasing glutathione levels daily is a significant

breakthrough in health and medicine.

ME-3 also synthesizes Manganese Superoxide Dismutase (MnSOD), which is an important

intracellular antioxidant. Glutathione produced by ME-3 is also capable of recycling and regenerating

other oxidized antioxidants such as vitamin C, vitamin E, lipoic acid and coenzyme Q10. Humans

taking ME-3 exhibited a 26% increase in total antioxidant activity.

ME-3 is also active against harmful intestinal bacteria, reduces inflammation, and supports

cardiovascular health and liver detoxification.

This presentation (or poster) will review the human clinical trials conducted with Lactobacillus

fermentum ME-3 and discuss the health benefits associated with increased glutathione levels.

Author: Ross Pelton, Essential Formulas

(541) 601-1492

ross@naturalpharmacist.net

10

Probiotics In Gingivitis Management: A Randomized Clinical Trial

Maria Tintoré1, Jordi Espadaler1 and Jordi Cuñé1*

1AB-Biotics, S.A. *Corresponding author: cune@ab-biotics.com Tel. +34 93 580 01 97

Objective: To evaluate the efficacy of a probiotic combination in the treatment of gingivitis and its

impact on the subgingival microbiota.

Methods: A double-blind, placebo-controlled, clinical trial, with a 6-week follow-up was conducted in

59 gingivitis subjects. After a professional mechanical plaque removal (PMPR), subjects were randomly

assigned to test (n=30) or control (n=29) groups. Test treatment consisted on tooth brushing b.i.d plus

oral tablets containing strains Lactobacillus plantarum CECT 7481, Lactobacillus brevis CECT 7480 and

Pediococcus acidilactici CECT 8633 b.i.d; control group received the same treatment, with placebo

tablets. The main endpoint was the change in gingival index (GI). Subgingival samples were analyzed

by qPCR for five putative periodontal pathogens.

Results: Both treatment groups experienced a significant clinical improvement in mean GI (p<0.001),

without intergroup differences, all patients being in remission at the end of the study (mean GI < 1.3).

The test group achieved a significantly higher reduction (p<0.001) in the number of sites with

spontaneous bleeding (GI=3). In subgingival samples, after correcting for multiple comparisons, a

significant reduction of Aggregatibacter actinomycetemcomitans occurred in both groups, while the

reduction in Tannerella forsythia was significant only in the test group (p<0.008). Only levels of T.

forsythia correlated to the number of sites with GI=3 throughout the study.

Conclusions: Probiotic tablets containing L. plantarum, L. brevis and P. acidilactici reduced significantly

the number of sites with severe inflammation in gingivitis patients after a PMPR. Probiotic use also

demonstrated a significant microbiological impact by reducing the counts of T. forsythia.

KEYWORDS: gingivitis, probiotics, gingival index, Lactobacillus spp., subgingival microbiota.

11

Effect of a blend of XOS and green coffee extract on the gut microbiota composition and activity as studied in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) Pieter Van den Abbeele1, T. Alan Jiang2, Iris Pinheiro1 and Massimo Marzorati3

1 = ProDigest BVBA, Belgium; 2 = Plexus Worldwide, USA; 3 = University of Ghent, Belgium

Objectives and design The aim of the current work was to evaluate the potential prebiotic activity of a blend containing xylooligosaccharides (XOS) and green coffee extract (SLIM™, Plexus Worldwide, USA). The in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) was used to assess the impact of repeated daily doses of this test product (1593 mg/day) on gut microbiota activity (i.e. markers for saccharolysis and proteolysis) and composition (qPCR on different microbial groups). The use of mucosal microcosms (i.e. M-SHIME) and of co-cultures of enterocytes and macrophages allowed also to evaluate the effect of the test product on biofilm formation and on gut wall modulation (i.e. gut permeability and inflammatory parameters). Results Microbial fermentation of SLIM occurred along the entire GIT and was correlated to an increased production of acetate, propionate and butyrate in all colon regions. In the ascending colon (AC), the increase was mainly due to increased butyrate production (+7 mmol/L, or 58%), while in the transverse colon (TC), the increase resulted from an increased production of all three main SCFA (approx. +4 mmol/L each). Finally, in the descending colon (DC), propionate production mainly increased (+4.1 mmol/L, or 29%). The treatment with SLIM led to significantly increased lactate levels (55.5%) in the AC and to a mild decrease in ammonium production (-7.5%). In terms of effects on the microbiota, the health-related, propionate-producing Akkermansia muciniphila increased in both luminal and mucosal environment of the proximal colon (+2 Log/mL or 257-fold in the lumen, and +0.2 log/mL or 3.9-fold in the mucosa, respectively). Health-related lactic acid bacteria such as Lactobacilli and Bifidobacteria were also stimulated (approx. +2.5 Log/mL or 365-fold, and +2.1 Log/ml or 290-fold, respectively) in the luminal gut microbiome upon SLIM treatment. Finally, SLIM was also shown to have a protective effect on Caco-2 barrier function in the distal colon (TC and DC) - for which a significant increase (+18.5%) in transepithelial electrical resistance was observed. The resulting enhanced gut barrier may reduce gut permeability. Finally, upon fermentation, SLIM was found to have pronounced immune activating properties in vitro, resulting in the increase of several immune mediators. Conclusions SLIM was well fermented along the entire length of the colon and showed potential prebiotic characteristics, being able to modulate activity and composition of the gut microbiota. This fermentation profile was correlated with a protective effect on Caco-2 barrier function and pronounced immune activating properties in the distal colon.

12

Comparative Functional Analysis of Xylooligosaccharide (XOS) with

other Prebiotic Oligosaccharides

XF Yao1, LS Zhang1, J Gu2, and YX Yang1

1 China Center for Disease Control and Prevention, Nutrition and Food Security, Beijing, China

2 AIDP, City of Industry, CA 91748 Jennifer@aidp.com 626-964-6910

Objective:

This study was to compare the effects of the prebiotic XOS (Xylooligosaccharides) and other commercially

available oligosaccharides on their ability to regulate the intestinal function, the gut microbiota, as well as their

SCFA (Short Chain Fatty Acid) production profiles.

Methods:

Male Kunming weighing 18 ~ 21g were separated into 12 groups and fed via intragastric administrations, either placebo or prebiotic oligosaccharides XOS, FOS, GOS, IMO, and L-A for 14 days. They were then fasted for 16 hours with free access to water. 0.50g/L diphenoxylate formula was then intragastric administrated to mouse to create the animal model for constipation. After 30 minutes, different oligosaccharides suspended in acacia gum and black ink was intragastric administrated to the mice. Fecal output characteristics including time for initial fecal output, quantities of fecal amount after 5 hours, as well as the total fecal weight after 5 hours were recorded. Effects on Microbiome were compared for the changes of Bifidobacterium and Lactobacillus, as well as pathogenic Enterococcus and Enterobacteriaceae after 14 days. Production of SCFA by different prebiotic oligosaccharides treated groups were also quantified and compared.

Results:

0.6g / kgBW, 1.0g / kgBW and 2.0g / kgBW of XOS (PreticX) can improve the function of intestinal flora, mainly

in the promotion of Bifidobacterium and Lactobacillus proliferation, while suppressing the pathogenic

Enterococcus and Enterobacteriaceae growth. 1.0g / kgBW and 2.0g / kgBW of XOS (PreticX) also showed

laxative effect. At 2.0 g / kg BW, XOS (PreticX) promoted the secretion of butyric acid in the intestine. Butyric

acid as the most important SCFA has been shown to promote cell differentiation and maturation, regulate

gene expression, maintain the stability of the intestinal environment and prevent the occurrence of colon

cancer.

Conclusion:

Prebiotic XOS promotes stronger Bifidobacterium growth and higher butyric Acid production as compared to

other prebiotic oligosaccharides in this animal model of constipation.

13 Flow Cytometry: An Improved Method for Health Evaluation and Enumeration of Probiotics

Objective

The CFU assay is a standard methodology for the enumeration of probiotics. However, this assay is

known to be variable. Flow cytometry is a highly sensitive, laser-based technology employed in

immunophenotyping and biomarker detection. The goal of this study was to determine the accuracy

of flow cytometry methodologies in the evaluation of bacterial health and enumeration for a variety

of probiotic formulations. Additionally, using a strain specific antibody, we evaluated the ability to

analyze individual bacterial strains within a mixed population.

Methods

Commercially available probiotics were hydrated and bacteria were stained with DNA-binding dyes

to evaluate membrane integrity. Samples were analyzed in triplicate by flow cytometry for live,

injured, and dead bacterial populations. A polyclonal antibody was raised against L.Rhamnosus.GG

in rabbits and tested on various strains.

Results & Conclusions

Clear populations of live, injured, and dead cells were identified and enumerated in all formulations

with less than 10% CV. Significant population variance and enumeration is observed in the

formulations evaluated. Flow Cytometry methods provided comparable or higher bacterial

enumeration than the CFU-assay. Additionally, antibodies specific for L.Rhamnosus.GG are able to

discriminate the strain in a mixed population. Given these results and the ability to perform flow

cytometry assays in minutes versus days, we conclude it is well suited to support probiotic

manufacturing, including product formulation and stability testing.

Corresponding Author

Dana Buckman

BioForm Solutions, Inc.

6760 Top Gun St.

San Diego, CA 92121

(760) 215-9269 dana@bioformsolutions.com

14 Flow Cytometry as a Superior Method to Enumeration of Specific Microbial Product Formulations

Objective

Use of all natural, direct-fed microbial feed additives for farm animals are a cost-effective way of

improving return on investment. Probiotic for such market applications can require particular

formulations, such as water-insoluble cereal foods. The CFU-plating assay is commonly used for

enumeration of microbial content of such manufactured products. However, this assay can provide

variable results, depending on bacterial formulation and the growth condition requirements of

certain strains. Here we study the accuracy of two additional enumeration methodologies for

certain microbial formulations: flow cytometry (laser-based technology for the detection of the

health of individual bacterial) and real-time PCR (genomic based detection).

Methods

Ninety-day stability studies were performed on direct-fed microbials from BioWish Technologies

formulated in water-insoluble cereal (MB3P) and water-soluble powder (MB3PS). For flow

cytometry analysis, bacteria were stained with DNA-binding dyes to evaluate membrane integrity.

Samples were analyzed in triplicate for live, injured, and dead bacterial populations. For PCR

analysis, nucleic acids were extracted and the quantitative expression of a bacterial marker was

determined.

Results & Conclusions

The flow cytometry method identified clear populations of live, injured, and dead cells with less than

10% CV. These data where highly concordant with the PCR data. Neither assay correlated well with

the CFU-assay results. We conclude that the CFU-assay may not be appropriate for enumeration of

certain microbial products.

Corresponding Author

Dana Buckman

BioForm Solutions, Inc.

6760 Top Gun St.

San Diego, CA 92121

(760) 215-9269 dana@bioformsolutions.com