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 [email protected]; 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: [email protected]
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, [email protected]
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 - [email protected]; 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: [email protected]
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: [email protected] 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
10
Probiotics In Gingivitis Management: A Randomized Clinical Trial
Maria Tintoré1, Jordi Espadaler1 and Jordi Cuñé1*
1AB-Biotics, S.A. *Corresponding author: [email protected] 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 [email protected] 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 [email protected]
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 [email protected]
Top Related