dri project report - ice cream mix - roberts dairy research... · national ice cream mix...
TRANSCRIPT
![Page 1: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/1.jpg)
NICMA Meeting
January 19, 2016Dr. Bob Roberts
Professor and Head of Food Science
1
![Page 2: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/2.jpg)
Thank You!National Ice Cream Mix Association Dairy Processing Internship Award
First Award will be made in May of 2016
If you are interested in interns for your operations please contact me
2
![Page 3: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/3.jpg)
Update on Dairy Research at Penn State
What’s going on in Happy Valley
3
![Page 4: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/4.jpg)
Current Areas of Research• Dairy Chemistry
– Casein Micelle Structure-Dr. Federico Harte• Food Safety/Quality
– Development of Food Safety Training Programs for Artisanal Cheese Manufacturers-Dr. Cathy Cutter
– Improving Safety and Quality for Small Dairy Processors-Dr. Kerry Kaylegian
– Does Listeria Grow During Thawing of Ice Cream?-Dr. Sara Milillo• Manufacturing
– Consumer Acceptability of Ice Creams Made Replacing Fat with Maltodextrin-Dr. Bob Roberts/Dr. John Coupland/Dr. John Hayes
– High Pressure Jet Processing of Milk and Milk Products-Dr. Federico Harte
• Human Health– Assessment of Probiotic Delivery Vehicles-Dr. Bob Roberts– Casein as a delivery vehicle for hydrophobic molecules-Dr. Fede Harte
4
![Page 5: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/5.jpg)
Fede Harte’s Research Areas
5
Biology of the Casein Micelle- structure and function
Effect of Environment- solvent effects
Analytical Tools
Effect of Processing- structure and function- novel interactions / applications
![Page 6: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/6.jpg)
High Pressure Jet
xchanger
nozzle
HP line
HP Pump (600 MPa)
Polycarbonateenclosure
![Page 7: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/7.jpg)
Pressure (MPa)0 100 200 300 400
G' (
Pa)
250
300
350
Yogurt from Homogenized milk
RAW
THERMAL
HPH
HPH + THERMAL
J. Dairy Sci. 2008, 91(10):3761-3767
20
![Page 8: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/8.jpg)
Delivery Systems
- Hypothesis: We can make the casein micelle a delivery system for “things” other than calcium phosphate
Casein micelle
![Page 9: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/9.jpg)
Delivery Systems
- Hypothesis: We can make the casein micelle a delivery system for “things” other than calcium phosphate
Casein micelle
Hydrophobic probe
![Page 10: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/10.jpg)
Delivery Systems
- Hypothesis: We can make the casein micelle a delivery system for “things” other than calcium phosphate
Casein micelle
Hydrophobic probe
Final System
![Page 11: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/11.jpg)
NIH-NICHD“Milk-based nano-delivery system for
hydrophobic drugs in infants and children”
Overall objective: Use modified casein micelles from bovine milk as carrier systems for delivering the low
MW hydrophobic drug RITONAVIR
![Page 12: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/12.jpg)
The case for Ritonavir
• Antiretroviral against HIV by Abbott (Norvir®)• Very low solubility in water (1 μg/mL at pH 6.8 for Form I)• High hydrophobicity (LogP~4.94)• Current formulation used in infants > 1 month contains 43%
ethanol• AWFUL flavor• Direct effect on stomach (mostly nausea, vomiting)• Exhibits polymorphism
![Page 13: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/13.jpg)
RITONAVIR
1 μm
>300 MPa30 μm
RITONAVIR
SKIM MILK
0 MPa
![Page 14: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/14.jpg)
Ritonavir release and digestion
0.0
10.0
20.0
30.0
40.0
50.0
60.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
110.0
0 1 2 3 4 5 6 7 8 9 10
Released R
IT (%)
Tota
l RIT
(rec
over
y %
)
Gastric digestion Duodenal digestion
5 ‘0 ’ 15 ‘ 30 ‘ 120 ‘ 15 ’ 60 ‘ 120 ‘30 ‘
Total ritonavir
From micelles after >300 MPa
Pharmaceutical Research 2015, 32(3):1055-1071
![Page 15: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/15.jpg)
Other Areas of Interest
• Influence of HPJ processing on foaming• Influence of HPJ on ice cream functionality• Understanding effects of various salts and
solvents on casein micelle structure
![Page 16: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/16.jpg)
A Study on the Effect of Delivery Vehicles on the
Efficacy of a Probiotic Strain Bifidobacterium
animalis subsp. lactis BB-12 in Humans
16
![Page 17: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/17.jpg)
Probiotics• Probiotics are “live microorganisms that,
when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO 2002, Hill et al. 2014)
– Alleviation of diarrhea– Modulation of host immune system– Reduction of allergic responses– Decreased bowel transit time– Prevention of infections
(de Vrese et al. 2008; Sherman et al. 2009, Hill et al. 2014)
17
en.wikipedia.org/wiki/Élie_Metchnikoff
![Page 18: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/18.jpg)
Probiotics• Attributes of a successful probiotic:
– Survival in delivery vehicle – Tolerant to oxidative stress– Survival in human gastrointestinal tract – Health benefit associated with consumption– Non pathogenic
• Majority belong to the genera Lactobacillus or Bifidobacterium
• Global probiotic market is projected to reach $46.55 billion by 2020 (marketsandmarkets.com 2015)
18
![Page 19: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/19.jpg)
The genus Bifidobacterium• Gram positive
• Anaerobic
• Non-spore-forming
• Commonly added to fermented dairy products due to putative health benefits
• Currently contains 38 species and subspecies
– B. animalis subsp. lactis
– B. longum
– B. infantis
– B. breve
www.activia.com
With BifidusRegularis
With BB-12
www.delish.com/food/grocery-products
19
![Page 20: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/20.jpg)
20
B. animalis subsp. lactis BB-12
• Oxygen & bile tolerant (Garrigues et al. 2005, Jungersen et al. 2014)
• Generally Recognized As Safe (GRAS)• Safe in population ranging from infants to the elderly (Ouwehand et al.
2004)
• Modulate intestinal microbiota (Savard et al. 2011)
• Improve bowel function (Pitkala et al., 2007; Uchida et al., 2005; Nishida et al. 2004)
• Reduce gut transit time (Larsen et al. 2006)
• Improve immune function (Rizzardini et al. 2012)
www.chr-hansen.com
![Page 21: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/21.jpg)
foodbizdaily.com
www.foodmayhem.com
Probiotic Delivery Vehicles
phyto-care.com
www.aligngi.com/21
• BB-12 survived and performed better in low fat milk than in orange juice (Saarela et al. 2006)
• Milk might be the preferred delivery matrix for certain probiotic strains (Lee et al. 2015)
![Page 22: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/22.jpg)
22
Probiotic Functions of Interest
Gut transit time
Gut microbiota
Fecal SCFAs
![Page 23: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/23.jpg)
23
Gut Transit Time (GTT)
• Gut transit time is simply the amount of time (hours) it takes for a meal to travel from the mouth, through the digestive tract and for its waste by-products to be eliminated through a bowel movement (Zaslavsky et al. 1998)
– Gastric emptying time
– Small bowel transit time
– Colonic transit time
• Negatively correlated with fecal SCFAs concentration (Lewis and Heaton, 1999)
http://northshorecolonics.com/the-digestive-system/
![Page 24: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/24.jpg)
24
Gut Microbiota and Gut Microbiome
• “Gut microbiota” - the diverse and dynamic community of microorganisms in the gut (Grice and Segre 2012)
• “Fecal microbiota” - the diverse and dynamic community of microorganisms in the feces
• “Gut/fecal microbiome” - catalog of microbes and their genes present in the gut/feces (Human Microbiome Project Consortium 2012)
![Page 25: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/25.jpg)
25
Gut Microbiota and Health
• Alter host metabolism (Vijay-Kumar et al. 2010, Koren et al. 2012)
• Transmit colitis (Garrett et al. 2007)
• Modulate type I diabetes (Wen et al. 2008)
• Modulate host immune function (Clemente et al. 2012)
• Associated with susceptibility to influenza, colon cancer, retrovirus transmission, autoimmune demyelination, irritable bowel disease (IBD), and even behavior (Finegold et al. 2010, Gerritsen et al. 2011,
Marsland and Salami 2015)
![Page 26: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/26.jpg)
26
0 500 1000 1500 200019951997199920012003200520072009201120132015
Publications
Year
Recent Gut Microbiota Publications
![Page 27: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/27.jpg)
• The vehicle used to deliver probiotic bacterium B. animalis subsp. lactis BB-12 (BB-12) into the body influences the performance of the probiotic in vivo
27
Overall Hypothesis
![Page 28: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/28.jpg)
Specific Aims• Aim 1: Develop three different BB-12 delivery systems (BB-12 added
before yogurt fermentation, BB-12 added after yogurt fermentation and BB-12 containing capsules) and study the viability of BB-12 in the yogurt drinks over 4 weeks of storage at 4°C
• Aim 2: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ gut transit time (GTT)
• Aim 3: Study the effect of the different interventions on participants’ fecal short chain fatty acid (SCFA) production
• Aim 4: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ fecal microbiota
28
![Page 29: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/29.jpg)
Randomized, 4-period crossover intervention
Overview of the Study
Screened subject group A, B, C, D
Immune markers
Fecal microbiota
Gut transit time
Capsule with BB-12
BB-12 added after fermentation
BB-12 added before fermentation
SmartPill capsule
16S rDNA sequencing
Blood & enzyme test
WithoutBB-12
CAPPOST PREYS
29
Fecal SCFAs
GC
Lipids profile
Blood test
Log10 10 ± 0.5 CFU per 240 g serving or per capsule
![Page 30: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/30.jpg)
Study Design
Subjects with irregular bowel
function
18-40 yearsBMI: 22-40
kg/m3
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
All subjectsFree-living
(4 weeks)
= Randomized to treatment sequence
= Data collection: fecal sample, blood draw, blood pressure, diaries & questionnaires
= 2-week compliance break; change to next treatment
Screening Period 1 Period 2 Period 3 Period 4
= Data collection: fecal sample only 30
![Page 31: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/31.jpg)
Specific Aims• Aim 1: Develop three different BB-12 delivery systems
(BB-12 added before yogurt fermentation, BB-12 added after yogurt fermentation and BB-12 containing capsules) and study the viability of BB-12 in the yogurt drinks over 4 weeks of storage at 4°C
• Aim 2: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ gut transit time (GTT)
• Aim 3: Study the effect of the different interventions on participants’ fecal short chain fatty acid (SCFA) production
• Aim 4: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ fecal microbiota
31
![Page 32: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/32.jpg)
Milk selection
Dry matter standardization
Pasteurization
Homogenization
Cool down to 110oF
Heat treatment
32
Flow Diagram of Yogurt Smoothies
Blend
Inoculation (YF-L702)
Pectin, CSS, sugar, water
Fermentation
Stir and cool Yogurt to 70oF
Homogenization
Package
Slurry
Cool to 105oF
Heat to 185oF
Strawberry flavor
BB-12 for POST
BB-12 for PRE
Draw mix for PRE
![Page 33: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/33.jpg)
Fermentation
Inoculate BB-12 (0.011% inoculation rate) into the PRE treatment
33
![Page 34: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/34.jpg)
Slurry Production
34
![Page 35: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/35.jpg)
Mixing and Homogenization
35
![Page 36: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/36.jpg)
Sequence of Treatments
YS
POST
PRE
A
B
C
36
![Page 37: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/37.jpg)
Packaging
37
![Page 38: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/38.jpg)
Storage
38
![Page 39: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/39.jpg)
Analyses of the Yogurt Smoothie Interventions
• Microbial analyses– Coliform– Survival of yogurt starter cultures– Survival of BB-12 over the 4-week shelf life
• Compositional analyses– Fat– Total solids
39
![Page 40: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/40.jpg)
• Following manufacture of the yogurt smoothies and weekly throughout shelf life
• Track BB-12 concentration (Target: Log10 10 ± 0.5 CFU/240 g serving)
– Selective media• MRS-NNLP (MRS agar supplemented
with nalidixic acid, neomycin sulfate, lithium chloride, paromomycin sulfate, and cysteine hydrochloride
– Confirm BAL by PCR
40
Enumeration of BB-12
![Page 41: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/41.jpg)
41
Specific Aim 1 - Results
![Page 42: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/42.jpg)
42
BB-12 Increased Modestly During Yogurt Fermentation
Log10 CFU/gBB-12 in PRE before yogurt fermentation 8.01 ± 0.05b
BB-12 in PRE after yogurt fermentation 8.10 ± 0.04a
BB-12 in POST after yogurt fermentation 8.10 ± 0.03a
Data shown were mean ± SD from at least 4 replicates. Values in a column without a common letter are significantly different, P < 0.05
![Page 43: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/43.jpg)
43
Component YS PRE POSTTotal solids (%) 20.92 ± 0.36b 21.58 ± 0.53a 20.96 ± 0.32b
Fat (%) 0.68 ± 0.07b 0.68 ± 0.07b 0.71 ± 0.07a
Data shown were mean ± SD from 30 batches. Values in a row without a common letter are significantly different, P < 0.05
Composition of the Yogurt Smoothies
![Page 44: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/44.jpg)
44
TreatmentBB-12 concentration (Log10 CFU/serving)
Week 0 Week 1 Week 2 Week 3 Week 4
PRE 10.55 ±0.12Aa
10.43 ±0.13Ba
10.42 ±0.13Ba
10.34 ±0.13Ca
10.24 ±0.13Da
POST 10.50 ±0.14Ab
10.16 ±0.15Bb
10.03 ±0.20Cb
9.77 ±0.20Db
9.54 ±0.25Eb
Data shown were mean ± SD from 27 batches. Values in a column without a common lowercase letter are significantly different. Values in a row without a common UPPERCASE letter are significantly different, P < 0.05.
BB-12 Population in Yogurt Smoothies Throughout Shelf Life
![Page 45: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/45.jpg)
BB-12 Population in Capsule Throughout Shelf Life
45
Values in a row without a common UPPERCASE letter are significantly different, P < 0.05
BB-12 concentration (log10 CFU/cap)Month 0 Month 4 Month 12 Month 14 Month 19
10.4 ± 0.0AB 10.4 ± 0.0A 10.3 ± 0.1AB 10.1 ± 0.2BC 10.0 ± 0.0C
![Page 46: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/46.jpg)
46
Conclusions
• Yogurt smoothies were of comparable composition• BB-12 population declined in yogurt smoothies
throughout shelf life• BB-12 population decreased slower in the PRE than
in the POST intervention• BB-12 remained at the specified dose level in all
interventions
![Page 47: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/47.jpg)
Specific Aims• Aim 1: Develop three different BB-12 delivery systems (BB-12 added
before yogurt fermentation, BB-12 added after yogurt fermentation and BB-12 containing capsules) and study the viability of BB-12 in the yogurt drinks over 4 weeks of storage at 4°C
• Aim 2: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ gut transit time (GTT)
• Aim 3: Study the effect of the different interventions on participants’ fecal short chain fatty acid (SCFA) production
• Aim 4: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ fecal microbiota
47
![Page 48: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/48.jpg)
Recruiting Participants
48
![Page 49: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/49.jpg)
49
Assessed for eligibility (n=136)
Excluded (n=100)• Did not meet inclusion criteria (n= 41)• Declined to participate (n=0)• Other reasons (n=59)
Randomly allocated to receive interventions, including YS, PRE, POST, or CAP (n=36)• Received allocated intervention (n=29)• Did not receive allocated intervention (n=7)
o Schedule conflict (n=2)o Lost to follow-up (n=1)o Other person reasons (n=4)
Allocation
Randomized (n=36)
Enrollment
Expressed interest (n=203)
![Page 50: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/50.jpg)
50
Analysed (n=28)Excluded from analysis (n=1)
Lost to follow-up (n=0)Discontinued intervention after allocation (n=6)• Pregnancy (n=2)• Diagnosed of Irritable Bowel Syndrome (n=1)• Schedule conflict (n=1)• Other personal reasons (n=2)
Analysis
Follow-Up
Crossover
Crossed over to receive other interventions, including YS, PRE, POST, or CAP (n=29)• Received allocated intervention (n= 29)• Did not receive allocated intervention (n=0)
![Page 51: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/51.jpg)
51
Participant CharacteristicsCharacteristics Values (n = 28)Age (yr) 28.2 ± 0.6Male, n (%) 11 (39.3%)Body mass index (kg/m2) 24.1 ± 0.2
≤24.9 16 (57.1%)25.0 -29.9 11 (39.3%)≥30 1 (3.6%)
Waist Circumference (cm) 85.2 ± 0.6
Blood pressure (mm Hg)Systolic 107.7 ± 0.8Diastolic 72.8 ± 0.6
Glucose (mg/dL) 86.6 ± 0.8Insulin (mg/dL) 5.4 ± 0.4hs-CRP (mg/L) 2.0 ± 0.5Physical activity (METs)2 3.0 (2.3 – 4.3)
![Page 52: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/52.jpg)
52
Methodology – Gut Transit Time• Baseline transit time measured using blue dye and SmartPill
• SmartPill measurement were taken immediately after each intervention period– Gastric Emptying Time (GET)
– Small Bowel Transit Time (SBTT)
– Colonic Transit time (CTT)
– Whole Gut Transit Time (WGTT)
www.givenimaging.com
![Page 53: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/53.jpg)
53
SmartPill Administration
• Fast for 8 hours
• Eat a SmartBar prior to SmartPill ingestion (drink ½ cup blue dye solution at baseline)
• Ingest the capsule
• Nothing to eat for the next 6 hours
• Passed capsule within a few days
• Collect data recorder and download data
• Analyze data using MotiliGI software
![Page 54: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/54.jpg)
54
GET SBTT CTT
Pres
sure
pH
Tem
pera
ture
WGTT
![Page 55: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/55.jpg)
55
Specific Aims 2 & 3 - Results
![Page 56: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/56.jpg)
56
Baseline WGTT as Measured by Blue Dye and SmartPill (ρ = 0.67, P < 0.0001)
0
20
40
60
80
100
120
140
PRO
036
PRO
030
PRO
016
PRO
013
PRO
006
PRO
012
PRO
035
PRO
003
PRO
002
PRO
025
PRO
010
PRO
014
PRO
021
PRO
028
PRO
009
PRO
019
PRO
032
PRO
033
PRO
008
PRO
027
PRO
029
PRO
015
PRO
034
PRO
020
PRO
022
PRO
026
PRO
004
PRO
023
Who
le g
ut tr
ansi
t tim
e (h
)
Participant's ID
SmartPill Blue Dye
![Page 57: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/57.jpg)
57-120
-100
-80
-60
-40
-20
0
20
40
60
80
2 3 4 6 7 8 9 12 13 14 15 16 17 19 20 21 22 24 25 26 28 29 30 32 33 34 35
Tim
e (h
)
Participant ID
YS PRE POST CAP
Changes in Colonic Transit Time (CTT) from Baseline after each Treatment
![Page 58: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/58.jpg)
58
Regional Transit Times (h) of the Participants
WGTT CTT SBTT GET
YS (A) 36.08 ±24.95
27.76 ±24.90
5.40 ±3.40
2.91 ±0.94
POST (B) 35.02 ±17.12
27.51 ±16.26
4.42 ±1.81
3.07 ±1.32
BL 40.78 ±24.50
33.21 ±24.25
4.69 ±1.38
2.86 ±0.89
PRE (C) 38.51 ±26.63
30.80 ±26.21
4.54 ±1.65
3.04 ±1.13
CAP (D) 35.54 ±14.74
27.71 ±14.07
4.40 ±1.46
3.41 ±1.27
Values shown are mean ± SD
![Page 59: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/59.jpg)
59
Conclusions• Blue dye method was proven a generally reliable cost-efficient
method for baseline screening• No significant treatment effect was observed on either gut
transit time or fecal short chain fatty acids• Predominant fecal SCFAs negatively correlated with WGTT,
CTT, and GET, but did not correlate with SBTT
![Page 60: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/60.jpg)
Specific Aims• Aim 1: Develop three different BB-12 delivery systems (BB-12 added
before yogurt fermentation, BB-12 added after yogurt fermentation and BB-12 containing capsules) and study the viability of BB-12 in the yogurt drinks over 4 weeks of storage at 4°C
• Aim 2: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ gut transit time (GTT)
• Aim 3: Study the effect of the different interventions on participants’ fecal short chain fatty acid (SCFA) production
• Aim 4: Assess the influence of consumption of yogurt smoothie (with and without BB-12) and BB-12 containing capsules on participants’ fecal microbiota 60
![Page 61: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/61.jpg)
61
Glossary
• 16S rDNA: The genes coding for 16S ribosomal RNA, a component of the 30S small subunit of prokaryotic ribosomes
• Operational Taxonomic Unit (OTU): A cluster of sequence reads with 97% similarity, motivated by the expectation that these correspond approximately to species
• Alpha-diversity: diversity within a sample
• Beta-diversity: dissimilarity between microbial communities based on their composition (Whittaker 1972, Caporaso et al. 2010)
http://drive5.com/usearch/manual/otu_definition.html
![Page 62: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/62.jpg)
62
Methodology for Microbiota Assessment
• Stool samples were collected at baseline, after each treatment, and after final washout
• Store in a home freezer prior to long-term storage at -80°C at the CRC
• DNA was extracted using MOBIO PowerSoil®
Kit– BAL specific PCR compliance check
– 16S rDNA amplified for V4 region
• Sequenced by Illumina® GAIIx
• Data processed using QIIME
– Bif-TRFLP
![Page 63: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/63.jpg)
63
DNA extraction
Stool DNA
PCR Compliance
checkBif-TRFLP
Amplify V4 region of 16S
rDNA
Barcode
Verify amplicons
Purify and combineSequence
Data Demultiplex and QA data Pick OTUs
OTU table
α-diversity β-diversity
Phylogenetic tree
LEfSe
QIIME
Experimental Procedure and Data Analysis
![Page 64: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/64.jpg)
64
Specific Aim 4 - Results
![Page 65: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/65.jpg)
65
Compliance Result• 73/78 fecal samples were BAL positive after receiving
BB-12 interventions
• All samples were BAL negative after receiving control yogurt smoothie when the corresponding baselines were negative
• Samples from 5 individuals were BAL positive regardless of treatment
![Page 66: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/66.jpg)
66
Alpha Rarefaction Curves of the Sequences
![Page 67: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/67.jpg)
67
Compositional Characteristics of the Fecal Microbiota of Participants
![Page 68: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/68.jpg)
Compositional Characteristics of the Fecal Microbiota of the Participants
68
![Page 69: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/69.jpg)
69
Simpson α-Diversity Index of the Fecal Microbiota of the Participants Before and After each Treatment
![Page 70: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/70.jpg)
70
Weighted UniFrac Distance PCoA of the Fecal Microbiota Group by Treatment
![Page 71: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/71.jpg)
71
Weighted UniFrac Distance PCoA of the Fecal Microbiota Group by Gender
![Page 72: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/72.jpg)
72
Unweighted Pair Group Method with Arithmetic mean (UPGMA) Tree Based on Weighted UniFracDistanceDemonstrating the Hierarchical Relationships Between Fecal Samples
![Page 73: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/73.jpg)
73
Relative Proportion of Bifidobacterium Species in the Stool Samples as Determined by Bif-TRFLP
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Baseline YS (A) POST (B) PRE (C) CAP (D) FinalWashout
Rel
ativ
e pr
opor
tion
Treatment
B. adolescentisB. animalisB. breveB. bifidum/pseudocatB. longumB. pseudocatenulatumUnknown
![Page 74: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/74.jpg)
74
Conclusions
• No significant treatment effect on gut microbiota was detected although yogurt interventions tended to exhibit a higher abundance of Streptococcus
• A significant gender effect was observed when comparing the gut microbiota of present study cohort
• BB-12 containing yogurt smoothies resulted in higher relative abundance of B. animalis
![Page 75: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/75.jpg)
75
Overall Conclusions
• Consumption of BB-12 delivered in yogurt or capsule did not change the gut transit time, fecal short chain fatty acid concentration, or the composition of the gut microbiota of the study cohort.
• Daily consumption of BB-12 in yogurt smoothie for 4 weeks may have an anti-inflammatory effect (data not shown) and result in higher relative abundance of B. animalis than capsule, control yogurt smoothie, baseline, and final washout.
![Page 76: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/76.jpg)
76
Acknowledgements
• DMI/DRI for Research Support
• Dr. Byron Ba• Dr. Rodolphe Barrangou
• Dr. Ryan Elias
• Dr. Penny Kris-Etherton
• Dr. James Rosenberger
• Berkey Creamery Staff
• Dr. Emily Furumoto
• Dr. Connie Rogers
• Jennifer Fleming
• Dr. Huicui Meng
• Yujin Lee
• Dr. David Mills
• Dr. Zac Lewis
• Morgan Bennett
• PSU Clinical Research Center
• Volunteers
![Page 77: DRI Project Report - Ice Cream Mix - Roberts Dairy Research... · National Ice Cream Mix Association ... • Global probiotic market is projected to reach $46.55 ... (Human Microbiome](https://reader036.vdocuments.us/reader036/viewer/2022062413/5ac131b97f8b9ae45b8d1318/html5/thumbnails/77.jpg)
Thank you for your attention!Questions?
77