A Human Clinical Trial Comparing the Digestibility of Sheep and Cow Milk

Findings from the ‘ShinDig’ study:

A human clinical trial comparing the digestibility of sheep and cow milk

Dr Amber Milan PhD 2020 Sheep Milk NZ Conference March 31 2020 Outline

• Background • Our relationship with milk • Digesting milk – allergy, intolerance, nutrients

• Research • Different milk, different digestion • Nutrients (DiNGo Trial – Goat) • Intolerance (aMiGo Trial – a2 Milk™)

• Sheep Milk Digestion – ShinDig Trial

• The future: Adaptation and nutrient delivery Our relationship with milk: Good days and bad days

ChartsBin statistics collector team 2011, Current Worldwide Total Milk Consumption per capita, ChartsBin.com, viewed 18th October, 2017, . Thorning, Tanja Kongerslev et al. “Milk and Dairy Products: Good or Bad for Human Health? An Assessment of the Totality of Scientific Evidence.” Food & Nutrition Research 60 (2016): 10.3402/fnr.v60.32527. PMC. Web. 18 Oct. 2017. Dairy avoidance: Do alternatives make the cut?

“Many individuals with real or perceived lactose intolerance avoid dairy and ingest inadequate amounts of calcium and vitamin D, which may predispose them to decreased bone accrual, osteoporosis, and other adverse health outcomes. In most cases, individuals do not need to eliminate dairy consumption completely.”

Suchy, Frederick J., et al. "NIH consensus development conference statement: lactose intolerance and health." NIH Consensus and State-of-the-science Statements 27.2 (2010): 1-27. Allergy OR Intolerance?

2% 6-8% 45% ADULTS CHILDREN

Photo credit: yorktest.com. Are you Allergic or Intolerant? ; twelvewellness.com. Connecting Food Allergies & Addiction Lactose Intolerance: A key culprit

“Lactose intolerance is a real and important clinical syndrome, but its true prevalence is not known.”

Food Intolerance Network. Multiple sources. Worldwide prevalence of lactose intolerance in recent populations. November 2013. Suchy, Frederick J., et al. "NIH consensus development conference statement: lactose intolerance and health." NIH Consensus and State-of-the-science Statements 27.2 (2010): 1-27. When lactose isn’t the problem

● With LI ○ Without Δ Perceived LI

“Many individuals who think they are lactose intolerant are not lactose malabsorbers.”

Matthews, Stephanie B., et al. "Systemic lactose intolerance: a new perspective on an old problem." Postgraduate Medical Journal 81.953 (2005): 167-173. Suarez, Fabrizis L., et al. "Tolerance to the daily ingestion of two cups of milk by individuals claiming lactose intolerance13." Am J Clin Nutr 997.65: 1502-6. Suchy, Frederick J., et al. "NIH consensus development conference statement: lactose intolerance and health." NIH Consensus and State-of-the-science Statements 27.2 (2010): 1-27. Dairy proteins – complex and varied

Whey (20%) β-lactoglobulin 10% 90g κ-casein α-lactalbumin 10% water 5% albumin 5g 1% β-casein immunoglobulins carbohydrate 28% 3% 3.5g protein αs1-casein 33% 1.5g fat αs2-casein 10% Vitamins & • Allergy - IgE mediated Minerals Casein (80%) • Immune - Non-IgE mediated? • Digestive effects? Casein differences – species and individuals αs1-casein 7% κ-casein 9% αs1-casein 7% κ-casein κ-casein 12% αs2-casein αs1-casein 9% αs2-casein β-casein 14% 42% 23% 35% αs2-casein β-casein β-casein 11% 70% 61% Digestion: protein impacts rate PL: placebo CS: casein WH: whey

Fast protein → greater amino acid in blood → greater muscle response

Tipton, Kevin D., et al. "Ingestion of casein and whey proteins result in muscle anabolism after resistance exercise." Medicine & Science in Sports & Exercise 36.12 (2004): 2073-2081. Digestion: structure impacts release

Gels (e.g. curd/cheese) Curd/gel structure Milk structure

(casein) (whey) (whey) CS

Fat Protein WH

• Complex milk structures • Interactions with fats

Lorieau, Lucie, et al. "Impact of the dairy product structure and protein nature on the proteolysis and amino acid bioaccessiblity during in vitro digestion." Food hydrocolloids 82 (2018): 399-411. Photo credit: Ahmed, Ali Hassan. “Milk Hygiene.” (2013) Different milk, different digestion

DiNGo Trial: cow and goat milk – nutrient availability aMiGo Trial: a2 Milk™ for Gut Comfort – dairy intolerance DiNGo Trial Digestive and Nutrient Bioavailability Benefits of Goat Milk Formula Is protein digestion similar between goat and cow milk?

Dose: 11ml/kg body weight Lactose malabsorption

Goat milk Cow milk Hydrolysed Cow milk Digestive comfort (symptoms)

Nutrient response (proteins, fats, Start 5 h micronutrients)

Milan, Amber, et al. "Digestive Responses to Fortified Cow or Goat Dairy Drinks: A Randomised Controlled Trial." Nutrients 10.10 (2018): 1492. DiNGo Trial Goat and cow milk digestion similar

15 80 Goat and Cow:   • Similar digestive comfort    • Similar digestion speed  60   10 g/ml)  (ppm) 2 40

5 whole cow Breath H Breath

Paracetamol ( Paracetamol whole goat 20  Hydrolysed Cow: hydrolysed cow • Delayed emptying • Delayed malabsorption

0 0 0153045607590 120 180 240 300 0 306090120 180 240 300 Time (min) Time (min) φ denotes statistical significance p<0.05, hydrolysed cow vs. goat, and χ hydrolysed cow vs. cow, respectively. α increase with cow, β increase with goat.

Milan, Amber, et al. "Digestive Responses to Fortified Cow or Goat Dairy Drinks: A Randomised Controlled Trial." Nutrients 10.10 (2018): 1492. DiNGo Trial Hydrolysed proteins alter insulin and protein metabolism

1 5 0 6 0 0  Goat and Cow:     • Similar metabolism    w h o le c o w m ol/l)  5 0 0 1 0 0 w h o le g o a t   hydrolysed cow U /m l)

  

  Hydrolysed Cow:   4 0 0 • Exaggerated insulin

In su lin ( 5 0   • Rapid amino acid clearance BCAA Concentration (

3 0 0 3 0 0 0 0 0 30 60 90120 180 240 300 0 60 120 180 240 300

T im e (m in ) T im e (m in ) Importance of protein structure φ denotes statistical significance p<0.05, hydrolysed cow vs. goat, and χ hydrolysed cow vs. cow, respectively.

Milan, Amber, et al. "Digestive Responses to Fortified Cow or Goat Dairy Drinks: A Randomised Controlled Trial." Nutrients 10.10 (2018): 1492. The aMiGo Trial: a2 Milk™ for Gut Comfort • Non-Lactose Dairy Intolerance • Known that a proportion of self-reported lactose intolerant are not lactose malabsorbers

• Protein-mediated effects of milk intolerance may be independent of lactose malabsorption

• Symptoms and identification of protein-mediated milk intolerance are undescribed

• Digestive tolerance of a2 Milk • Lactose-mediated versus protein-mediated

(750 ml)

Milan, Amber, et al. “Comparison of the impact of bovine milk beta-casein variants on digestive comfort in females self-reporting dairy intolerance: a randomised controlled trial" American Journal of Clinical Nutrition 111.1 (2020): 149-160. Lactose Cramps Flatulence  25 25  * * Intolerance Non-Lactose Dairy Intolerant Lactose Intolerant 20 20 Tolerant * * * 15 15 Lower GI **** Delayed (90 min+) 10 10 symptom score (mm) score symptom symptom score (mm) score symptom 5 5

0 0 0 1 30 60 90 120 150 180 0 1 30 60 90 120 150 180 Belching Distension Non-Lactose 25 25 * Non-Lactose Dairy Intolerant ** * Lactose Intolerant * Dairy Intolerance 20 20 ** * * Tolerant * 15 * 15 Upper GI; Distension * * 10 10 symptom score (mm) score symptom Rapid (30-90 min) (mm) score symptom 5 5

0 0 Different mechanism? 0 1 30 60 90 120 150 180 0 1 30 60 90 120 150 180 Time (min) Time (min) Milan, Amber, et al. “Comparison of the impact of bovine milk beta-casein variants on digestive comfort in females self-reporting dairy intolerance: a randomised controlled trial" American Journal of Clinical Nutrition 111.1 (2020): 149-160. The aMiGo Trial: Lactose and dairy intolerant subjects respond differently to β-casein variants

Flatulence Faecal Urgency BreathB re a thH H 2 LactoseLactose Intolerant Intolerant Subjects fla tu le n c e faecal urgency 2 1 5 0  1 5 1 5 * * Lactose-free * Lactose-Free Conventional Conventional a 2 m ilk a 2 m ilk * * 1 0 0 * 1 0 1 0 * (p p m ) 2

***

b r e a th H 5 0 * 5 5 symptom score (mm) symptom score (mm)

0 0 0 Non-Lactose Lactose Intolerant Tolerant Non-Lactose Lactose Intolerant Tolerant 0 15 30 45 60 75 90 105 120 150 180 Dairy Intolerant Dairy Intolerant T im e (m in ) Lactose Intolerant: reduced malabsorption, faecal urgency and nausea (not shown) with a2 Milk™

Non-Lactose Dairy Intolerant: more flatulence with a2 Milk™ → Different mechanism?

• Protein may play a mechanical or immune role • Multiple aspects of milk impact nutrients and tolerance • Proteins, structure, interaction

• Dairy intolerance not limited to lactose • Mechanisms still unknown • Malabsorption/intolerance not static Sheep Milk Digestion Nutrients Tolerance ShinDig Trial: Sheep milk nutrient bioavailability and digestive comfort

Is protein digestion similar between sheep and cow milk?

Lactose malabsorption

Sheep milk Cow milk Digestive comfort (symptoms)

Nutrient response (proteins, fats, micronutrients)

Milan, Amber, et al. Unpublished. ShinDig Trial: Sheep milk nutrient bioavailability and digestive comfort

Participants – “dairy avoiders”

Underlying lactose/food intolerance

Milan, Amber, et al. Circulating branched chain amino acids are heightened in dairy avoiding females following an equal volume of sheep milk relative to cow milk. In preparation. ShinDig Trial:

Protein more readily digested BCAA @ 60 min

* 250 *** 200

* mol/L)  Nutrient response * 150 (proteins, fats, micronutrients) * 100

* 50 concentration ( concentration 0 Sheep Milk Cow Milk Time (min) Sheep milk elevated: • BCAAs • Lysine & methionine

* p<0.05 difference between sheep and cow

Milan, Amber, et al. Circulating branched chain amino acids are heightened in dairy avoiding females following an equal volume of sheep milk relative to cow milk. In preparation. ShinDig Trial: Same fat response, more medium chain fatty acids, 1.4 Sheep Milk Cow Milk  1.2

# * 1.0

Trigycerides(mmol/L) 0.8 

 0.0 0 30 60 90 120 180 240 Time (min) Sheep milk resulted in more: • Medium-chain fatty acids • Ruminant trans fatty acids, including CLA

* p<0.05 difference between sheep and cow

Samuelsson, Linda, Milan, Amber, et al. In preparation. ShinDig Trial: Less malabsorption – and less lactose 80 30 Digestive comfort Abdominal cramps Sheep Milk * 10000 Cow Milk 60 8000 20

6000 40

4000 10 symptom score (mm) score symptom (mm) score symptom

Breath H2 (AUC) 2000 20 Sheep Milk Cow Milk 0 Sheep Milk Cow Milk 0 0 -5 0 5 30 60 90120180240 -5 0 5 30 60 90120180240 More malabsorption from cow milk Time (min) Time (min) 1/3 higher lactose in cow milk No difference in pain → Are differences only due to content? → Same for lactose tolerant? Shrestha, Aahana, Milan, Amber, et al. In preparation. ShinDig Trial: Summary

• Sheep milk proteins more readily digested • BCAAs (leucine, isoleucine, valine), methionine, and lysine

• Sheep milk fats have a different response • Same overall rise in blood despite higher content • More medium-chain fatty acids

• Sheep milk may reduce lactose malabsorption • Lower malabsorption, but also lower lactose content The future: Adaptation and nutrient delivery Los aMiGoS: long-term a2 Milk™ for Gut Comfort New Zealand Milks Mean More – NZ3M Can tolerance be improved? Adapted?

“The majority of people with lactose malabsorption do not have clinical lactose intolerance.”

Savaiano, Dennis A., et al. "Improving lactose digestion and symptoms of lactose intolerance with a novel galacto-oligosaccharide (RP-G28): a randomized, double-blind clinical trial." Nutrition journal 12.1 (2013): 160. Suchy, Frederick J., et al. "NIH consensus development conference statement: lactose intolerance and health." NIH Consensus and State-of-the-science Statements 27.2 (2010): 1-27. Los aMiGoS: Is Lactose tolerance impacted by repeated exposure to a2 Milk™, with effects on Gut Comfort Symptoms

Does long term exposure to β-casein variants impact lactose malabsorption? Can lactose absorption be improved with elimination of intestinal inflammation?

Bad Good

2 weeks Milan, A. M., et al. Unpublished. Los aMiGoS Trial: the role of inflammation in lactose malabsorption. Photo credit: newyou.com. Wendy Plovmand. Pros of Probiotics.; Rcmathiraj. NZ3M: New Zealand Milks Mean More Validation of digestive and nutritional outcomes that support improved health and wellbeing

Programme Leader: Prof Warren McNabb NZ3M: Building evidence of dairy product impacts

Food structure Digestion Appearance of nutrients Functional outcomes (intervention) (mechanism) (outcome) (impact)

Physical state, complexity of structures/matrices

e.g. blunted vs enhanced, e.g. muscle growth e.g. native vs heated e.g. fast vs slow Rapid vs delayed Programme Leader: Prof Warren McNabb NZ3M: New Zealand Milks Mean More TUMMI Trial: Temperature treatment of Milk impacts on MRI digestion rates and nutrient delivery

Gastrointestinal function (by MRI) Digestive feelings

Nutrient/hormone analysis Stomach full of milk (MRI)

• Impact of processing/ differences in properties Start 5 h • Validate against in vitro and pre-clinical models • Inform product development

Programme Leader: Prof Warren McNabb Insights and questions • Milk digestion • Processing affects digestion and metabolic responses • Species-specific differences understanding required How can we use this to improve health?

• Dairy intolerances • Lactose malabsorption affects majority – others? • Mechanisms other than lactose intolerance? • Protein intolerances? • Adaptation? Can we understand how and provide solutions? Funding

Acknowledgments Thank you Participants!

Institutional Affiliations

Research Team & Support Prof David Cameron-Smith Prof Warren McNabb Aahana Shrestha, Pankaja Sharma, Utpal Prodhan, Sarah Prof Nicole Roy Mitchell, Clara Han, Jimmy Nilsson, Josefin Karlström, Jakob Prof Richard Mithen Martinsson, Jenny Mistretta, Matthew Bourke, Franny Dr Matthew Barnett Markstedt, Julia Ängemalm, Sofia Egelrud, Ville Nyberg, Erik Dr Linda Samuelsson Linden, Vivian Kim, Isabella Kreber, Emil Talani, Petter Dr Li Day Johansson, Nora Farnisa, Chris Keven, Janene Biggs, Hieke [email protected] Dr Ali Hodgkinson Schwendel, Michael Agnew, Hedley Stirrat, Arvind Subbaraj [email protected]