ARIFinal Report

A. Date : August 31, 2002

B. Reporting Period: July 1, 2000 – August 31, 2002

C. Project Number: 46980 (CDRF project 46380)

D. Project Title: Cell count – probiotic bacteria

E. Principal Investigator (s): Name (first and last) Mary Ellen SandersAddress 7119 S. Glencoe Ct., Centennial, CO 80122-2526Phone and fax 303-793-9974/303-771-6201/805-756-6998University affiliation Dairy Products Technology Center, Visiting Research Professor

Name (first and last) Rafael Jimenez-FloresAddress Cal Poly State University, San Luis Obispo, CA 93407Phone and fax 805/756-6103 / 805-756-6998University affiliation Dairy Products Technology Center

F. Prepared by: Name (first and last) Mary Ellen SandersAddress 7119 S. Glencoe Ct., Centennial, CO 80122-2526Phone and fax 303-793-9974/303-771-6201/805-756-6998Project affiliation: PI

Name (first and last) Rafael Jimenez-FloresAddress Cal Poly State University, San Luis Obispo, CA 93407Phone and fax 805/756-6103 / 805-756-6998Project affiliation: PI

G. Executive Summary:

The fundamental premise of this study is based on the observation that the colony counts after commercial production of an industrial strain-in-development, Lactobacillus crispatus HP101 (Northeast Nutraceuticals, South Boston, MA), were determined to be inadequate in industrial settings. This inadequacy was primarily exhibited as low colony counts after growth in broth and after freeze-drying. This study was undertaken to determine if procedures yielding higher colony counts could be developed.

The results from this study led to the following conclusions:1. This study confirmed the industry observation that Lactobacillus crispatus HP101

demonstrates poor colony forming yield when grown in laboratory broth media. Compared to another industrial probiotic strain, Lactobacillus acidophilus NCFM, 2-3 log cycle fewer CFU/ml were recovered of HP101.

2. Growth under conditions that yielded poor colony forming units yielded equivalent optical density.

3. Growth under conditions that yielded poor colony forming units resulted in cells with long, spindly cell morphology as observed under a light microscope. Control NCFM cells were short, compact rods. The HP101 morphology resulting from growth in MRS broth is associated with ‘unhealthy’ cells.

4. A staining procedure that distinguishes between live and dead cells demonstrated that HP101 grown under conditions that yielded poor colony forming units resulted in many dead cells.

5. Growth of HP101 in milk reversed all these negative growth parameters, resulting in colony yields, cell morphology, levels of live/dead cells equivalent to the NCFM control strain. Therefore, HP101 appeared well adapted to growth in milk.

6. Attempts to mimic milk-grown HP101 results included many media and growth condition manipulations, including growth in tween, organic honey, alcalase-digested milk, oxgall, lecithin, CaCl2, milk permeate, whey protein concentrate and casein. Only growth in milk permeate supplemented with ≥0.3% casein resulted in CFU yield and cell morphology that mimicked milk-grown cells. MRS supplemented with casein showed improved CFU yields, but not to milk-grown levels.

7. This focus of this study was on improved CFU/ml in static growth. There are no data provided for CFU/gm yield after freeze drying.

H. Major Accomplishments:

1. This study confirmed the industry observation that Lactobacillus crispatus HP101 demonstrates poor colony forming yield when grown in laboratory broth media. Compared to another industrial probiotic strain, Lactobacillus acidophilus NCFM, 2-3 log cycle fewer CFU/ml were recovered of HP101.

2. This study found that HP101 exhibited excellent growth in milk. However, as milk is not a practical growth medium for industrial application, we defined an industrially feasible growth medium which supported growth of HP101 equivalent to growth observed in milk. This medium was comprised of milk permeate supplemented with ≥0.3% casein. It was also found that MRS supplemented with casein showed improved CFU yields, but not to milk-grown levels.

I. Impact Statements:

The primary impact of this study is the improved understanding of the nature of the growth inadequacies of HP101.

An improved medium, suitable for commercial scale-up, is proposed.

J. Dissemination, publications and presentations of research: 1. The results of this study were presented as a poster at the 2002 American Dairy Science

Association meeting, Quebec City, Quebec, Canada.

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The following presentations on probiotics were made since 2001. These provided opportunity to disseminate results obtained from research at Cal Poly, especially the importance of maintaining high cell count of probiotics for physiological relevance and the importance of dairy ingredients in enhancing probiotic functionality.

Pending: Sanders, M. E. 2003. Health effects of probiotics and their field of application. Canadian Dietetics Association 2003 Annual Meeting.

Pending: Sanders, M. E. 2002. Safety of oral probiotics in human health: what is known? Montreal International Symposium Probiotics and Health: Biofunctional Perspectives. October 24-25, 2002, Montreal, Quebec

Pending: Sanders, M. E. 2002. Probiotics: what are they and how are they used? American College of Nutrition 43rd Annual Symposium on Advances in Clinical Nutrition, October 3-6, 2002, San Antonio, Texas.

Pending: Sanders, M. E. and Guarner, F. 2002. Convene round table on: Yogurts and Fermented Milks. Benefits of Live Cultures. September 25, 2002. World Dairy Congress, Paris.

Sanders, M. E. 2002. L. acidophilus NCFM®: functional properties of a unique probiotic strain. Vitafoods International 2000 Exhibition and Conference, Geneva, Switzerland.

Sanders, M. E. 2002. Probiotics for humans: status and future of the science and marketplace. FDA National Center for Toxicological Research, Jefferson, AK.

Sanders, M. E. 2002. The scientific basis and clinical effects of probiotics. Michigan State University Malcolm Trout Lecture Series, E. Lansing, MI.

Sanders, M. E. 2002. Marketing opportunities for nutraceuticals and probiotics. SmartMarketing 2002 (sponsored by IDFA), San Diego, CA.

Sanders, M. E. 2001. Probiotic delivered through foods. Probiotics, Prebiotics and New Foods Conference. Rome, Italy.

Sanders, M. E. 2001. Regulatory aspects of health claims associated with probiotics. International Dairy Federation Nutrition Conference. Auckland, New Zealand.

Sanders, M.E. 2001. Probiotics: New Strains and Strain Specific Research. Nutricon 2001, San Diego, CA.

Sanders, M. E. 2001. Probiotics in the human diet: what is their role? American Dietetic Association Annual Meeting, St. Louis, MO.

Sanders, M. E. 2001. Probiotics as foods. Probiotics, Prebiotics and New Foods Conference. Rome, Italy.

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Sanders, M.E. 2001. Probiotics: New Strains and Strain Specific Research. Nutricon 2001, San Diego, CA.

Sanders, M. E. 2001. Probiotics: efficacy and applications to dairy products. 2001 Annual Meeteing of Institute of Food Technologist, New Orleans.

Sanders, M. E. 2001. Regulatory considerations for labeling foods and supplements with health statements in the U.S. First International Bio-Minerals Symposium: Trace Elements in Nutrition, Health and Disease. Salt Lake City, UT.

Sanders, M. E. 2001. Dairy foods and those friendly bacteria important to your health. California Dairy Industry Conference, Pomona, CA.

Sanders, M. E. 2001. The emerging role of probiotics in health. Food 3000, Rome, Italy.

2. Include all names of those presenting and/or coauthoring material.Kevin Bourzac, Ann Bernard, Rafeal Jimenez-Flores, Mary Ellen Sanders

3. State name of publications and identify as refereed journal/paper or trade publication.Manuscript in preparation

4. State names and places where presentation(s) were or will be made. The results of this study were presented as a poster at the 2002 American Dairy Science Association meeting, Quebec City, Quebec, Canada.5. Attach copies of all disseminated materials including senior projects and graduate theses.

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ARI Final Report: Cell count – Probiotic Bacteria

A. Introduction

The fundamental premise of this study is that the colony counts after commercial production of an industrial strain-in-development, Lactobacillus crispatus HP101 (Northeast Nutraceuticals, South Boston, MA), were determined to be inadequate in industrial settings. This inadequacy was primarily exhibited as low colony counts after growth in broth and after freeze-drying. This study was undertaken to determine if procedures yielding higher colony counts could be developed.

B. Materials and Methods

Growth studies All bacterial strains were kept as purified stock cultures at -80C. Before the start of each experiment,

scrapings from a frozen seed were added to MRS+ 0.05% cysteine broth and incubated at 37C overnight. Cultures were then inoculated into the medium required for the specific study. Plating was conducted using a pour plate technique. MRS agar was melted and kept at 46C. 0.1 ml of appropriate dilution of cells was added to each plate in duplicate. 12-15 ml of molten and tempered MRS agar was added to the plates and the plate was gently swirled to evenly disperse the cells through the molten agar. Plates were incubated in anaerobe jars for 48 hrs.

To conduct growth curves, 100ul of MRS-grown strains were transferred to fresh 10ml MRS + cys broth and grown to stationary phase (~11 hrs). 100ul of MRS broth (Blank 1) was transferred into a tube labeled “Blank 2”. A small Tupperware container was filled with ~1cm water and a plastic petri dish lid was placed inside. A sterile ELISA plate was placed on top of the lid (above the water) and two Alka-Seltzer® tablets were added to create a local CO2-rich environment. Once the tablets had dissolved completely, ELISA wells were filled with 300ul of MRS+cys broth. Appropriate wells were inoculated with 1% (3ul) culture, followed by a 70ul sterile mineral oil cap according to Figure 1. The plate was then covered with sterile adhesive ELISA plate sealer.

Figure 1: General layout of ELISA plate for growth curve studies. Orange wells contain MRS+0.05% cys and are inoculated as indicated.

The plate was then loaded into a Molecular Devices SpecraMax Plus ELISA plate reader. Optical density readings at 650nm were taken every 30 minutes for 24 hours at 37C. The ELISA plate was removed at 4 hours and the plastic covering row 12 was cut away. A pipette tip was inserted below the oil level and the sample was

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carefully mixed via up and down pipetting before 100ul was removed for dilution and plating. An additional 50ul was removed and spread on a microscope slide for observation. This was done for each sample at indicated sample times. Dilutions were made in MRS+cys and plated on MRS+cys agar. Plates were incubated at 37C for 48hrs in anaerobe jars before counting. For microscopic visualization, slides were heat fixed and Gram-stained for observation. Observations were recorded with the digital CCD camera of a BioRad GelDoc 1000™ gel documentation system. Images of the plates were made using a BioRad Scanning Densitometer. Growth curve data from the SpecraMax were exported to Microsoft EXCEL for analysis. Optical density data from wells 1-4 were averaged to create graphs. Colony count data was averaged for plates containing between 25 and 300 colonies.

Live/Dead bacteria detectionIn an attempt to further demonstrate the health of HP101 cells in MRS versus milk media, the cells were stained with a fluorescent kit. HP101 was grown up twice from frozen and inoculated at 1% into 40ml of MRS and 11% NFDM. After 24hrs growth, 9ml of each sample was transferred to a fresh 15ml tube. The NFDM sample was titrated slowly with 10N NaOH to make proteins soluble. Cells were pelleted by centrifugation and super was discarded. Cells were resuspended in 9ml 0.22um filter-sterilized DI water. A 1ml portion of each was transferred to a small eppendorf and cells were pelleted again. A total of 3 washes was performed with filter sterilized DI water (to completely remove all media which might interfere with staining), the final resuspension was let to sit for 30 minutes to equilibrate cells. After this, the cells were pelleted and resuspended again. LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular Probes, Eugene, OR) reagents A & B mixed in equal volumes then 3ul of mix was added to each sample. Cells incubated with dye protected from light for 15 minutes at room temperature. Cells were visualized with a fluorescence microscope (brand?) using some sort of filter? Images captured with their software.

Capillary Gel ElectrophoresisHP101 and NCFM cells were grown up twice from frozen in MRS then inoculated at 1% into 10ml steam-sterilized non-fat milk (store bought, same as above). Cells along with a control were incubated for 24hrs at 37C. After growth, a 500ul portion of each sample was pipetted into a centrifugal filter (Micron YM-10, Millipore Corporation, Bedford, MA) and all proteins over 10kD were removed by a 30 minute spin at 14,000g. Approximately 100-200ul of collected protein-free supernatant was transferred to a fresh 0.6ml vial and mixed 1:1 with a 1:10 dilution of 0.1M phosphate buffer, pH 2.5 (BioRad, Hercules, CA). After a 3-minute de-gassing spin at maximum RPM, samples were loaded into a BioFocus 3000 Capillary Electrophoresis System (BioRad, Hecules CA). Running conditions:Inlet/outlet buffer 0.1M phosphate pH 2.5 (biorad), pre-inject buffer 120s. Low pressure sample injection 20psi*sec. Polarity: + - Run voltage: 20.00kV, 250.00uA current limit. Cartridge @ 20C.

Results

Growth studies. In this study, growth was assessed and compared using two different methods: (1) following changes in optical density using a microtiter assay in an ELISA plate reader and (2) colony counts on MRS agar. The control strain used throughout these evaluations was the commercial strain NCFM®, marketed worldwide by Rhodia Inc. (Madison, WI). This strain is the most widely distributed probiotic strain in fermented dairy foods and dried dietary supplements in the U.S. This strain is acknowledged to demonstrate acceptable growth characteristics in an industrial setting.

Our initial objective was to document the growth characteristics of HP101 and determine if low colony counts occurred in our laboratory setting. Table 1 shows the raw data from several experiments on colony counts. As can be seen from this table, on average HP101 demonstrated lower colony counts than NCFM by 2, 1 and 3 log cycles at samplings of 4, 8 and 24 hours, respectively. Optical density data are summarized in Figure 2. Growth curve data in Figure 1 indicate that HP101 takes longer to reach stationery phase than NCFM (exhibiting a slower growth rate), but achieves a comparable maximum OD. These results confirm findings from industry.

Table 1. Average colony counts (CFU/mL) of HP101 and NCFM after growth in MRS broth for 4, 8 and 24 hrs.4 hrs 8 hrs 24 hrs

HP101 4.65E+061 9.35E+072 3.40E+063

NCFM 3.25E+084 9.57E+084 5.27E+095

1Average of 5 experiments; 2Average of 4 experiments; 3Average of 3 experiments; 4Average of 6 experiments; 5Average of 3 experiments.

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Figure 2. Growth of NCFM and HP101 in MRS media. Data represent averages of 4 independent experiments for HP101 and 7 experiments for NCFM.

To further characterize the growth of HP101, the final pH for growth of this organism in different media was determined. Table 2 shows that HP101 is capable of dropping the pH of milk-based media very low after 24 hr growth. The ΔpH for HP101 in 11% NFDM exceeded that for NCFM (data not shown).

Table 2. Final pH of HP101 after growth in MRS, 11% reconstituted non fat dry milk (NFDM) and non-fat (NF) milk purchased from the grocery store.

Media Media pH pH after 24 hr growth of HP101 ΔpHMRS 5.90 4.41 1.49NF Milk 6.50 3.59 2.9111% NFDM 6.57 3.52 3.05

Microscopic observations. Observations of HP101 and NCFM using a light microscope demonstrated distinct differences in morphology between HP101and NCFM when grown in MRS broth. HP101 was found to form long (1x15 m), Gram-positive rods that were often present in clumps. In contrast, NCFM formed short (~1x5 m), Gram-positive rods. Examples of cell morphology are shown in Figure 3. Furthermore, clumping of HP101 was often observed with material that stained pink from the Gram-stain procedure. The nature of this material is not known, although some type of long chain polysaccharide may be produced by this strain. The presence of this material in clumps of cells suggests that it may serve as an aggregation agent for HP101 cells. Aggregation of large groups of cells could significantly lower cell count.

Figure 3. Images showing NCFM and HP101 grown 24 hours in MRS broth. 10x magnification images demonstrate cell density or dispersion. Images at 100x magnification provide more detail into cell shape and structure. NCFM cells are smaller than HP101 (~1x5um compared to 1x15, respectively).

Magnification HP101 NCFM

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10x

100x

Additional microscopic observations were conducted using a method designed to distinguish between live and dead cells. Results (Figure 4) demonstrate the same cell morphology observed under the light microscope, but also indicate a large percentage of dead or membrane-damaged cells among the MRS-grown cells. Milk-grown cells are smaller with fewer dead cells. Although these results are qualitative, they suggest that milk-grown cells are healthier. The high percentage of dead cells in the MRS-grown preparation may partially explain why MRS-grown HP101 cells achieve high cell density as measured by optical density but low colony count results.

Figure 4. HP101 cells grown in milk or MRS broth for 24 hours stained with BacLight™ Bacterial Viability Kit to determine the presence of dead or membrane permeable (red) and live (green) cells. These images demonstrate that milk-grown cells are much smaller and more numerous than MRS-grown cells (both images are same magnification).

Media 10xMRS

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Milk

Attempts to improve CFU yield by HP101. HP101 appears to be adapted quite well to growth in milk. Colony forming units met or exceeded CFU achieved by NCFM. Cell morphology showed compact, small rods, similar to NCFM. However, since milk is not an acceptable commercial fermentation medium because cells cannot be harvested from it easily, a medium that mimicked milk-grown cells was needed. Therefore, a variety of manipulations of media content and conditions were conducted to assess their effect on HP101 growth. A summary of these manipulations is shown in Table 3.

Table 3. Manipulations tested in the laboratory to increase cell yield of HP101. Note that all MRS media also contained 0.05% cysteine.

Manipulation Result(assessed by CFU, OD and/or morphology)

Net Effect

MRS control 10e6 – 10e7 cfu/ml11% NFDM 10e8 – 10e9 cfu/ml; cell count and

morphology equivalent to NCFMImproved

Tween (0.01-1%) added to MRS No effect seen on growth curve, CFU or cell morphology

none

0.2% CaCl2 added to MRS0.2% bovine oxgall added to MRS0.05% lecithin added to MRS

10e6 CFU HP101, with MRS morphologyNCFM was unaffected compared to MRS growth

inhibitory

1.5% organic honey1 added to MRS No effect on cell count (10e6-10e7 CFU/ml); no morphology data

none

MRS + 0.2ug/mL B12 and 500ug/mL NaFormate

No effect on cell count (10e6-10e7 CFU/ml); no morphology data

none

10 ppm hydrogen peroxide in diluent No impact on cell count none10% NFDM buffered with phosphate No growth InhibitoryBead beating to break clumps Damaged cells negativeMilk permeate2 No change over MRS noneMRS dissolved in permeate No impact on cell count noneMRS + 3% whey protein concentrate Not usable media – couldn’t be autoclaved Not testedMRS + 3% casein Increased cfu by ~1 log, but not to milk

levels Improved

10% NFDM in MRS No difference over milk NoneHydrolyzing milk - alcalase Cell counts very low (<10e5) InhibitoryCaseinate medium3 No growth InhibitoryMilk permeate + 3% casein Increased cfu to milk levels Improved

1 Shamala, et al. (2000)

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2Milk permeate (Cheryan, 1998) is the resulting serum from ultra filtration of skim milk through a membrane with molecular weight cut off of 10,000 Da. Any molecular species of a molecular weight higher than 10,000 is excluded from the permeate. Milk permeate is an isotonic solution with all the buffering and ionic strength of milk, with added lactose, peptides (smaller than 10k) and other nitrogen substances available in milk (such as urea). 31.0 g ammonium citrate, 2.5 g sodium acetate, 0.5 g magnesium sulfate, 0.05 g manganous sulfate, 1.0 g dipotassium phosphate, 23 g lactose, 16.2 g sodium caseinate.

To determine if milk proteins were responsible for the growth enhancement seen when HP101 was grown in milk, casein and whey protein concentrate were tested as medium additives. Unfortunately, MRS media prepared with whey protein concentrate added did not maintain its integrity after autoclaving (coagulation and clumping resulted). MRS media prepared with 3% casein was prepared from 2X MRS and 2X casein in water, then autoclaved, then cooled and mixed 1:1 for use. This medium did show stimulation of CFU, but not to milk levels. The results with casein did suggest that the milk protein component was at least partially responsible for growth enhancement. Milk could be used as a commercial medium if it was clarified to allow centrifugation to recover cells. Therefore, milk was treated with alcalase, a high-pH proteolytic enzyme. This enzyme effectively clarified the milk, but CFU counts were very low (<10e5 est). This suggests that some property of whole protein or peptides is required for growth enhancement of HP101, with digested milk proteins being inadequate as a growth stimulator. To determine if there was a concentration effect for milk components on growth, 5, 10 and 15% NFDM were tested as growth media. Final CFU counts were 4.7e8, 1.1e9 and 1.6e9, respectively. Although the CFU differences were not large, results did suggest a dependency of CFU on milk concentration (Figure 5).

Figure 5. Relationship between NFDM concentration (5, 10 and 15%) and CFU/ml achieved after 24 hr growth.

Among the media tested, the one showing the most promise was milk permeate supplemented with 0.3% caseinate (Figure 6). Milk permeate alone did not show growth improvement. CFU/mL greater than 10e9 were obtained in this medium. This medium was prepared from 3% NaCaseinate dissolved in permeate. This stock solution was diluted with permeate to proper concentration (0.3%) after steam-sterilization and cooling. Low concentrations of permeate were found to precipitate when autoclaved. Although not reported here, it would be of interest to test the effect of 0.3% casein supplemented into different basal media on CFU/mL yield of HP101.

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Figure 6. Colony forming units of HP101 grown in milk permeate (perm), alcalase-hydrolyzed milk (Hydro), MRS, milk permeate+ 3% casein (Perm CAS 3%), milk permeate+ 0.3% casein (Perm CAS 0.3%) and milk permeate+ 0.03% casein (Perm CAS 0.03%).

1.0E+06

1.0E+07

1.0E+08

1.0E+09

1.0E+10

Perm Hydro NF

MRS

Perm C

AS 3%

Perm C

AS 0.3%

Perm C

AS 0.03

%

CFU/mL

Microscopic observations further indicated the value of caseinate as an additive to growth media for HP101 (Figure 7). Cells isolated from milk permeate supplemented with 0, 3, 0.3 and 0.03% caseinate are shown. The presence of caseinate at the 3% level clearly induces a shift to short, healthy rod morphology. Cells grown at the 0.3% caseinate level are improved over 0%. However, the 0.03% level seems to show a similar morphology to unsupplemented permeate. This is consistent with the 0.03% level being unable to fully support the growth

Figure 7. Microscopic observations of HP101 grown in milk permeate (perm), milk permeate+ 3% casein (Perm CAS 3%), milk permeate+ 0.3% casein (Perm CAS 0.3%) and milk permeate+ 0.03% casein (Perm CAS 0.03%).

Media 10X 100XPerm

Perm CAS 3%

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Perm CAS 0.3%

Perm CAS 0.03%

The fate of protein in milk supporting the growth of HP101 or NCFM was measured using capillary gel electrophoresis. The two strains were grown 24 hrs at 37C in steam-sterilized non-fat milk (purchased at grocery store). After removal of proteins over 10,000 MW through a centrifugal filter, the supernatant fluid was evaluated. Figure 8 shows the different profiles resulting from control milk that did not support the growth of any bacteria and milk in which HP101 or NCFM were grown. Although these results are preliminary and need to be repeated, the diminishment of peaks from the control milk are evident, indicating digestion of peptides of these sizes. Differences in the NCFM and HP101 profiles could point to the importance of specific peptide fractions of milk by HP101, perhaps explaining its dependence on milk as a growth substrate. However, these experiments were not conclusive. A similar experiment conducted with caseinate would perhaps better provide indications of the important components for growth enhancement.

Figure 8. Absorbance units of protein from capillary gel electrophoresis of milk after growth of HP101 or NCFM.

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Conclusions:1. This study confirmed the industry observation that Lactobacillus crispatus HP101 demonstrates poor

colony forming yield when grown in laboratory broth media. Compared to another industrial probiotic strain, Lactobacillus acidophilus NCFM, 2-3 log cycle fewer CFU/ml were recovered of HP101.

2. Growth under conditions that yielded poor colony forming units yielded equivalent optical density.3. Growth under conditions that yielded poor colony forming units resulted in cells with long, spindly cell

morphology as observed under a light microscope. Control NCFM cells were short, compact rods. The HP101 morphology resulting from growth in MRS broth is associated with ‘unhealthy’ cells.

4. A staining procedure that distinguishes between live and dead cells demonstrated that HP101 grown under conditions that yielded poor colony forming units resulted in many dead cells.

5. Growth of HP101 in milk reversed all these negative growth parameters, resulting in colony yields, cell morphology, levels of live/dead cells equivalent to the NCFM control strain. Therefore, HP101 appeared well adapted to growth in milk.

6. Attempts to mimic milk-grown HP101 results included many media and growth condition manipulations, including growth in tween, organic honey, alcalase-digested milk, oxgall, lecithin, CaCl2, milk permeate, whey protein concentrate and casein. Only growth in milk permeate supplemented with ≥0.3% casein resulted in CFU yield and cell morphology that mimicked milk-grown cells. MRS supplemented with casein showed improved CFU yields, but not to milk-grown levels.

7. This focus of this study was on improved CFU/ml in static growth. There are no data provided for CFU/gm yield after freeze drying.

References

Cheryan, M. 1998. In, Ultra filtration and Micro filtration Handbook, Technomic Publishing, Bassel Switzerland.

Shamala, T. R., Shri Jyothi, Y., Saibara, P. (2000) Stimulating effect of honey on multiplication of lactic acid bacteria under in vitro and in vivo conditions. Letters in Applied Microbiology 30:453-455.

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