Casein Hydrolysate with Glycemic Control Properties: Evidence from

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Bioactive Constituents, Metabolites, and Functions

A casein hydrolysate with glycaemic control properties: evidence from cell, animal models and humans. Elaine Drummond, Sarah Flynn, Helena Whelan, Alice B Nongonierma, Therese Anne Holton, Aisling Robinson, Thelma Egan, Gerard Cagney, Denis C Shields, Eileen R. Gibney, Philip Newsholme, Celine Gaudel, Jean-Christophe Jacquier, Nessa Noronha, Richard J Fitzgerald, and Lorraine Brennan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05550 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Journal of Agricultural and Food Chemistry

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A casein hydrolysate with glycaemic control properties: evidence from cell, animal

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models and humans.

3

Elaine Drummond1*, Sarah Flynn1*, Helena Whelan1, Alice B Nongonierma2, Thérèse A

4

Holton3, Aisling Robinson4, Thelma Egan1, Gerard Cagney4, Denis C Shields3, Eileen R

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Gibney1, Philip Newsholme4, Celine Gaudel4, Jean Christophe Jacquier1, Nessa Noronha1,

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Richard J FitzGerald2, Lorraine Brennan1#

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Affiliations:

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1

School of Agriculture and Food Science, Institute of Food and Health and Food for Health

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Ireland (FHI), University College Dublin, Belfield, Dublin 4, Ireland

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2

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Health Ireland (FHI), University of Limerick, Limerick, Ireland.

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3

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Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin

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4, Ireland.

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4

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Conway Institute of Biomolecular and Biomedical Research, University College Dublin,

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Belfield, Dublin 4, Ireland.

Department of Biological Sciences, University of Limerick, Limerick, Ireland; Food for

School of Medicine, Food for Health Ireland (FHI) and UCD Conway Institute of

School of Biomolecular and Biomedical Sciences, Food for Health Ireland (FHI) and UCD

19 20

# Corresponding author:

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Professor Lorraine Brennan, Institute of Food and Health, University College Dublin,

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Belfield, Dublin 4, Ireland.

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Email: [email protected]

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Phone: 00353 1 7162811

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Fax: 00 252 1 716000

1 ACS Paragon Plus Environment


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*Both authors contributed equally to the manuscript

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Abstract

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Evidence exists to support the role the dairy derived proteins whey and casein in glycaemic

30

management. The objective of the present study was to use a cell screening method to

31

identify a suitable casein hydrolysate and to examine its ability to impact on glycaemia

32

related parameters in an animal model and in humans. Following screening for ability to

33

stimulate insulin secretion in pancreatic beta cells a casein hydrolysate was selected and

34

further studied in the ob/ob mouse model. An acute postprandial study was performed in 62

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overweight and obese adults. Acute and long term supplementation with the casein

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hydrolysate in in vivo studies in mice revealed a glucose lowering effect and a lipid reducing

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effect of the hydrolysate (43% reduction in overall liver fat). The postprandial human study

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revealed a significant increase in insulin secretion (p=0.04) concomitant with a reduction in

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glucose (p=0.03). The area under the curve for the change in glucose decreased from 181.84

40

± 14.6 to 153.87 ± 13.02 (p=0.009). Overall, the data supports further work on the

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hydrolysate to develop into a functional food product.

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Keywords: Casein, Hydrolysate, Postprandial Glycaemia, insulin, nutrition

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Introduction

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Poor metabolic health increases the risk of the development of Type II Diabetes Mellitus

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(T2DM) (1) and the associated frequent hyperglycaemic episodes greatly increase

52

cardiovascular disease risk, which occurs 2-4 times more often in the diabetic population (2).

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In 2010, the International Diabetes Federation (IDF) estimated that diabetes care accounted

54

for €260 billion or 11.6 % of the total world expenditure on healthcare (3). It is understood

55

that altering environmental factors such as diet can modify disease risk and improve long-

56

term health outcomes (4) and accumulating evidence links dairy consumption with a reduced

57

likelihood of developing metabolic syndrome (5-13). Consumption of Fermented dairy

58

products, including low-fat cheese, buttermilk, and yoghurt has been associated with a

59

reduced likelihood of T2DM onset (14, 15). Multiple components of milk have shown a

60

propensity to improve postprandial glycaemic function (16-18), yet the key constituents

61

appear to be casein and whey protein fractions. In particular, most likely due to an elevated

62

rate of digestion and absorption (19, 20), hydrolysed casein and whey peptides have shown

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particular efficacy in optimising glycaemic management (21, 22). As a result of the

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demonstrated functional ability these are often referred to as bioactive peptides.

65

A large body of evidence supports the insulinotropic function of certain amino acids

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and peptides (23). Although the major protein in milk is casein (comprising approximately

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80%), to date there has been a greater focus on whey proteins in the literature. When ingested

68

intact, whey proteins have a rapid impact on insulin secretion, faster than micellar casein,

69

which coagulates in the acidic gastric environment and transits more slowly (24). Following

70

consumption whey exits the stomach more quickly and induces rapid increases in plasma

71

amino acid levels (25). Recently, focus on the behaviour of casein hydrolysates has shown

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that these adopt a quicker transit time, thereby achieving more rapid uptake of amino acids.

73

In 2013, Boutrou et al. (26) conducted a study on the gastrointestinal fate of casein,



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examining the release of various peptides in the jejunum of healthy human volunteers.

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Following ingestion of casein medium-sized peptides were released 6 hours later, while

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larger peptides were released from whey proteins at earlier time points.

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Much of the work examining bioactive peptides in the context of glycaemic

78

management has targeted T2DM populations and many studies have used the bioactive

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peptides in combination with amino acids with insulin secretion promoting effects such as

80

leucine. In a group of type II diabetic subjects consumption of casein hydrolysate/ leucine

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after a main meal demonstrated their ability to enhance insulin secretion compared to a

82

placebo beverage (21). Manders et al (27) reported that administration of a carbohydrate

83

bolus along with a casein hydrolysate plus added leucine in 10 T2DM males improved the

84

insulin response in comparison to a carbohydrate-only control. Furthermore in a separate

85

study it was shown that such a response could be maintained even without the addition of

86

amino acids (28). Elsewhere, in a randomised controlled trial (RCT) of 13 T2DM patients (5

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females), a 50g oral glucose tolerance test was carried out using three test treatments: 0, 6 or

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12g casein hydrolysate (18). In this case the 12g casein hydrolysate had a significant, positive

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effect on the postprandial insulin and glucose AUC (area under the curve) values. The

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evidence for casein hydrolysates achieving a reduction in postprandial glucose in T2DM

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patients is growing; however, further studies on the long term effects are needed.

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The effects of dairy protein ingestion by non-diabetic, at risk overweight and obese

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human subjects have not been extensively studied. However, there is some evidence to

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support a functional role of hydrolysates in this population. In an RCT of 70 healthy,

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overweight or obese men and women, whey protein was compared to casein or glucose

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supplementation over a 12 week period (29). Significant reductions in fasting insulin and

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homeostasis model assessment of insulin resistance (HOMA-IR) levels were observed in the

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whey treated group compared with the control. No significant effects were demonstrated for


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the casein group, however it is noteworthy that the study utilised an intact casein. However,

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more favourable results were obtained when a hydrolysed casein was employed: Bendsten et

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al. (2014) (25) recruited 24 healthy, overweight men and women to compare the acute effects

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of hydrolysed casein (DH:37%), intact casein and intact whey on biochemical and perceptual

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markers of glycaemic and appetite regulation. Glucose concentrations were higher after

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ingestion of intact casein than after hydrolysed casein. In this case the intact whey was

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similar to the hydrolysed casein. No differences were seen in 24 hour or postprandial energy

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expenditure or appetite regulation. Animal studies have also highlighted the potential positive

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effects of hydrolysed casein: following consumption of the hydrolysed casein for 8 weeks the

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animals displayed an array of preferable metabolic characteristics such as lower respiratory

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exchange ratio and higher spontaneous locomotor activity (30). Furthermore, the mice fed

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with the hydrolysate had lower plasma glucose concentrations and concomitantly strongly

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reduced insulin levels.

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Overall, the current literature supports the concept of the use of casein hydolysate for

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blood glucose regulation. However, the type and extent of peptides and amino acids released

114

during enzymatic hydrolysis (and consequently the bioactivity) is dependent on the

115

parameters employed (i.e., temperature, time, pH, enzyme concentration, total solids, etc.)

116

during the hydrolytic reaction (31). The objective of the present was to use a cell screening

117

method to identify a suitable casein hydrolysate and to examine its glycaemic reduction

118

ability in animal models and in humans.

119 120

Materials and Method

121

Chemicals

122

All chemicals were purchased from Sigma unless otherwise stated. Ultrasensitive Rat Insulin

123

ELISA, Mouse insulin ELISA, High sensitivity insulin ELISA, C-peptide ELISA kits were



from

Mercodia.

RPMI

1640

culture,

Foetal

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purchased

Calf

Serum

(FCS),

125

Penicillin/streptomycin (100 units/ ml, 0.1 mg/ml) were purchased from Invitrogen

126

Haematoxylin, Eosin were purchased from Clin-Tech. GIP ELISA, Glucagon-like peptide-1

127

(GLP-1) ELISA kits were purchased from Merck Millipore. Rodent Islet isolation

128

formulation was purchased from Roche. Bicinchoninic (BCA) assay kit was purchased from

129

Pierce, Thermo Scientific.

130 131

Bioactive hydrolysate

132

A sodium caseinate hydrolysate, referred to as the “casein hydrolysate” was prepared as

133

previously described by Nongonierma and FitzGerald using food-grade gastro-intestinal

134

enzymes (32). Briefly, the starting milk protein substrate (sodium caseinate, Kerry Group Plc,

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Listowel, Ireland) was suspended at 10 % (w/w) on a protein basis in water and dispersed

136

under agitation at 50°C for 1 h using an overhead stirrer (Heidolph RZR 1, Germany) and

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hydrolysed for 1 h at 50°C. A control sample without enzyme was removed from the protein

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dispersion and maintained at 50°C for the duration of the hydrolysis reaction. Hydrolysis was

139

carried out at a constant pH of 8.0 or 7.0 using a pH Stat (Titrando 843, Tiamo 1.4 Metrohm,

140

Dublin, Ireland). The gastro-intestinal enzyme preparation was inactivated by heating the

141

hydrolysate samples at 90°C for 20 min.

142

The casein hydrolysate was fractionated using an ultrafiltration (UF) unit (Sartoflow Alpha

143

filtration system, Sartorius, Germany). Fractionation was carried out using membranes

144

having 5 and 1 kDa molecular weight cut-off (MWCO) values for the casein hydrolysate. The

145

hydrolysates and the four UF fractions (permeates and retentates for 5 and 1 kDa cutoffs)

146

collected were freeze-dried (FreeZone 18L, Labconco, Kansas City, U.S.A.) and stored at -

147

20°C until further analysis.

148


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Reversed-phase ultra-performance liquid chromatography and molecular mass distribution

150

of peptides and proteins in the casein hydrolysates

151

The sodium caseinate and corresponding hydrolysate (UL 291) and UF fractions were

152

analysed by liquid chromatography using an ultra-performance liquid chromatograph (UPLC

153

Acquity - Waters, Dublin, Ireland) as described by Nongonierma and FitzGerald (32). The

154

molecular mass profile of the proteins and peptides was determined by gel permeation

155

chromatography using high performance liquid chromatography (GPC-HPLC) essentially as

156

described by Spellman et al. (33). Each sample was analysed in duplicate.

157 158

BRIN-BD11 cell culture and Insulin secretion assay

159

The BRIN-BD11 cell line is a functional rat clonal insulin-secreting pancreatic β-cell line.

160

BRIN-BD11 cells were maintained in GI-1640 containing 11.1 mM glucose, supplemented

161

with 10 % (v/v) foetal calf serum, 2 mM glutamine, 50 IU/ml penicillin, 0.05 mg/ml

162

streptomycin and incubated at 37 ˚C in a humidified atmosphere containing 5 % CO2 and 95

163

% air. BRIN-BD11 cells were seeded at a density of 2 x 105 cells/well in 1 ml of media in 24-

164

well plates for 24 hour in GI-1640 media containing 11.1 mM D-glucose. The cells were

165

incubated with 1 ml Kreb’s Ringer Bicarbonate buffer (KRB) (pH 7.4) (115 mM NaCl, 4.7

166

mM KCl, 1.28 mM CaCl2, 1.2 mM MgSO4.7H2O, 1.2 mM KH2PO4, 10 mM NaHCO3, 0.1 %

167

BSA), supplemented with 1.1 mM glucose for 40 min. The cells were stimulated with 1ml

168

KRB supplemented with 16.7 mM glucose plus 1 mg/ml of solution of interest (casein

169

hydrolysate) for 20 minutes at 37 ˚C. The positive control 16.7 mM glucose plus 10 mM

170

alanine was used. Insulin secretion was determined using the Mercodia Ultrasensitive Rat

171

Insulin ELISA kit (Mercodia AB, Uppsala, Sweden). For the insulin secretion assays with

172

synthetic peptides, the peptides were supplied by Peptide 2.0, USA at > 98% purity.



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3T3-L1 adipocyte cell culture and conditioned media collection and preparation

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The adipocyte cell line was maintained in DMEM containing 25 mM glucose, supplemented

175

with 10 % (v/v) foetal calf serum, 50 IU/ml penicillin, 0.05 mg/ml streptomycin and

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incubated at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air. 3T3-L1

177

differentiation was induced in fully confluent 3T3-L1 pre-adipocytes by adding 3-isobutyl-1-

178

methylxanthine (500 mM), dexamethasone (1 µM) and insulin (0.01 mg/ml) for 48 hours.

179

Media was retrieved from the adipocytes both pre and post differentiation. The adipocyte

180

conditioned media was removed from the cells 72 hours following the addition of the

181

differentiation media. DMEM media was used as the control. All media were diluted 1 in 2

182

with sterile water prior to cell treatment.

183

BRIN-BD11 cell treatment with of differentiated conditioned media

184

BRIN-BD11 cells were seeded in T175 flasks at a density of 3 x 106 cells and allowed to

185

grow for 48 hours. Cells were treated with 15 ml of differentiated conditioned media,

186

differentiated conditioned media with casein hydrolysate or DMEM as a control for 2 hours.

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Following treatment, cells underwent a methanol:chloroform metabolite extraction and,

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subsequently, a bicinchoninic (BCA) assay to determine protein content (Pierce, Thermo

189

Scientific, Rockford, 1L61105, USA). Acute insulin secretion was measured as described

190

above, following 2 hour incubation with the adipocyte conditioned media and conditioned

191

media supplemented with either the casein hydrolysate or the backbone synthetic peptide.

192

Islet isolation, culture and acute insulin secretion assay

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The pancreas was digested with 3 ml of isolation buffer consisting KRB buffer (pH 7.4)

194

supplemented with 1.38 mg/ml liberase Rodent Islet isolation formulation (Roche, Welwyn

195

Garden City, U.K.) and 1.83 mg/ml each of egg white and soybean trypsin inhibitors. The

196

pancreas was digested by incubating in a water bath at 37 ˚C while shaking. Islets were

197

washed twice in KRB buffer, resuspended in KRB buffer and place on ice until the islets


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were collected. Individual islets were isolated and cultured in batches of 20 in untreated

199

culture plates overnight in RPMI 1640 culture medium, 10 % FCS (Invitrogen, Biosciences,

200

Dublin, Ireland) and penicillin/streptomycin (100 units/ ml, 0.1 mg/ml; Invitrogen,

201

Biosciences, Dublin, Ireland) and maintained at 37 ˚C in a 5 % CO2 humidified atmosphere.

202

Islets in groups of three were incubated in 3 mmol glucose/l KRB for 30 minutes at 37˚C.

203

Following this, islets were incubated in KRB buffer containing 20 mmol glucose/l plus casein

204

hydrolysate or 3 mmol glucose/l KRB plus casein hydrolysate for 2 hours at 37˚C. Islets

205

were treated with casein hydrolysate at concentrations of 0.1 mg/ml, 0.5 mg/ml and 1.0

206

mg/ml. Islets were centrifuged, the supernatant was collected and the amount of secreted

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insulin was determined by Mouse insulin ELISA (Mercodia, Uppsala, Sweden). The data is

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presented as fold difference in insulin secretion between isolated islets which were treated

209

with low (3.3 mM) and islets which were treated high glucose (20 mM).

210

Animal studies

211

All procedures were performed with the approval from the UCD research Ethical board

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(AREC-P-09-26). All mice were maintained in a controlled environment with 12h/12h

213

dark/light cycles at 22˚± 2 and have specific pathogen free (SPF) status. They were given free

214

access to standard pelleted laboratory rodent chow and filtered water. For all animal

215

experimental studies, ob/ob and C57BL/6 male mice, at the age of 12 weeks were used.

216

Ob/ob mice were sourced from Charles River, UK and C57BL6 mice were sourced in house

217

from the UCD Biomedical facility, Dublin.

218

Acute Treatment with casein hydrolysate

219

Animals were fasted overnight for 16 hours prior to the acute experiment. The following day,

220

commencing at 10am, fasting blood glucose levels were measured using a Medisense Optium

221

Xceed (Abbott, Ireland) glucometer before the treated animals received 100 µl of casein

222

hydrolysate at 100 mg/kg body weight via oral gavage (Instech PA, USA) 1 hour prior to the



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glucose tolerance test, control mice received 100 µl distilled H2O via oral gavage (Instech

224

PA, USA) 1 hour prior to the glucose tolerance test. Animals were sacrificed by CO2

225

asphyxiation and cervical dislocation.

226

Long term treatment with casein hydrolysate

227

Animals were administered 100 µl of 100mg/kg body weight casein hydrolysate diluted in

228

sterile distilled H2O using a 1 ml syringe and a 18ga (0.7x1.2mm) gavage feeding needle.

229

Animals received the treatment every second day via oral gavage for a period of 12 weeks.

230

Control mice received 100 µl sterile distilled H2O. Animals were fasted overnight for 16

231

hours prior to the experiment. Fasting plasma glucose levels were measured using a

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Medisense Optium Xceed (Abbott, Ireland) glucometer at 0 minutes prior to administering

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each mouse with 2 g/kg body weight of glucose and plasma glucose levels were measured at

234

15, 30, 60, 90, 120 minutes. Animals were sacrificed by CO2 asphyxiation and cervical

235

dislocation.

236

Liver histology

237

Liver sections were stored in formalin 10% formaldehyde at room temperature before they

238

were embedded in paraffin wax overnight. Liver sections were dehydrated in a series of

239

increasing ethanol concentrations: once in 50 % (v/v) for 15 min, twice in 70 % ethanol for

240

15 min, twice in 90 % ethanol for 10 min and three times in 100 % ethanol for 15 min.

241

Samples were left overnight in 100 % ethanol at 4 °C before cutting at 4 µm. The

242

haematoxylin and eosin (H&E) staining process consisted of 10 min at 62 ˚C, 3 x 5 min in

243

xylene, 2 x 5 min in 100 % ethanol, 3 min in 95 % ethanol, 3 min in 70% ethanol and 5 min

244

in PBS Leica. Sections were placed in haematoxylin (Clin-Tech, Guilford, UK) for 10 min,

245

washed in ddH2O and 1 % acid ethanol, and incubated with eosin (Clin-Tech, Guilford, UK)

246

for 90 sec. Sections were washed and dehydrated in PBS for 5 min, 70 % ethanol for 3 min,

247

95 % ethanol for 3 min, 100 % ethanol for 10 min and in xylene for 3 x 5 min. Slides were


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scanned at 40x magnification using a ScanScope XT Digital slide scanner (Aperio

249

Technologies CA, USA), digital images were analysed by k means clustering using image J

250

to separate the fat globules from the remainder of the tissue. 6 images of each liver section

251

was analysed, the area (µm) of fat in each 40x image of liver was measured. The average of

252

the 6 images for each liver section from each animal was calculated and was presented as

253

n=1.

254

Human study participants and design

255

The present study was a randomised, controlled, crossover design conducted according to the

256

guidelines laid down in the Declaration of Helsinki and all procedures involving human

257

subjects or patients were approved by the University College Dublin Human Ethics Research

258

Committee (LS-12-92_BRENNAN). Written, informed consent was obtained from all

259

participants. Eligible participants were healthy Caucasian men and women aged between 40-

260

65 years with a BMI >25kg/m2 were recruited in the Dublin region by poster and radio

261

advertising. Participants were excluded if they were taking hormone replacement therapy

262

had a chronic or infectious disease, were pregnant or lactating or had an allergy or intolerance

263

to dairy or wheat products.

264

Anthropometric measurements were performed including body weight and height to

265

calculate BMI (kg/m2), waist circumference, blood pressure and heart rate using an Omron

266

M6 Comfort digital automatic blood pressure monitor (Omron Healthcare Europe). A single

267

study coordinator was responsible for obtaining the random allocation sequence, enrolment

268

and assignment of subjects. Subjects were assigned to treatment or control on their first visit

269

using a randomization table. This was method was used to assign subjects to crossover from

270

treatment to control, or vice versa, in a non-biased order. Controls and treatments were coded

271

as A and B, respectively. The study coordinator was not blinded to controls and treatments,

272

which were both plain white powders, however, study subjects were. Individuals were given



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de-identified codes using a random number generating tool (www.randomizer.org). The

274

sample size was calculated using data from our unpublished previous studies (AUC for

275

glucose curves) with similar primary outcomes and intervention products. Based on these

276

data we expected a change in AUC glucose of approximately 80 units. A sample size of 50

277

was calculated as sufficient to detect the difference with a level of significance of 0.05 for a

278

difference of 80 units. Accounting for a 30% potential dropout rate we aimed to recruit 72

279

individuals. Recruited subjects were invited to attend the study centre on two separate

280

occasions, at least 7 days apart but no more than 21 days. Subjects fasted for 12 hours prior to

281

each visit and were fitted with a peripheral 20-22 gauge cannula (BD Nexiva, Becton

282

Dickson, Oxford, UK) before giving fasting blood samples. They then consumed a prescribed

283

breakfast containing 74 g carbohydrate (Nutrient breakdown provided in Table S1 in

284

supplementary material based upon on pack information) along with a 10 % (w/v) solution

285

(100 ml) of casein hydrolysate or a sodium caseinate (intact control protein) in a randomised,

286

crossover fashion. Subjects were instructed to consume the meal and beverage within 10

287

minutes. The meal comprised 2 slices of white bread toasted with 40 g strawberry jam and

288

100 ml of orange juice from concentrate (Table S1, supplementary material, provides the

289

nutrient composition of the test meal).

290

The casein hydrolysate was produced specifically for this study was scaled up in the GMP

291

manufacture facility of Moorepark Limited. As a control, sodium caseinate was provided by

292

Kerry Group Plc. Both powders were dissolved to form a 10 %w/v solution using 12 g of

293

protein dissolved in 120 ml of mineral water (Ballygowan, Ireland). Dissolution was aided

294

with gentle agitation using a handheld blender. Beverages were refrigerated for 6-7 hours

295

prior to consumption in order to improve palatability. The point of consumption of the

296

protein (immediately following the breakfast meal) was recorded as time point zero (t=0) and

297

blood was drawn at time points t = 15, 30, 60, 90 and 120 minutes. Blood was drawn into


Page 13 of 43


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serum separator, EDTA, Lithium Heparin and DPPIV inhibitor tubes (BD Vacutainer).

299

Serum samples were allowed to clot at room temperature (RT) for 30 minutes. Serum

300

separator and DPPIV inhibitor tubes were centrifuged at 1300 g for 10 minutes at RT. EDTA

301

and Lithium Heparin tubes were centrifuged at 1500 g for 10 minutes at 4 ˚C. All aliquots

302

were frozen immediately and stored at -80 ˚C.

303

Biochemical Measurements

304

The primary outcome measures were blood glucose and insulin concentrations at six time

305

points across the oral glucose tolerance test period of two hours. Secondary outcome

306

measures included c-peptide, Gastric inhibitory polypeptide (GIP), triacylglycerols and non-

307

esterified fatty acids (NEFA) concentrations. All measurements were made in accordance

308

with the manufacturers’ instructions. Plasma samples were analysed for insulin by high

309

sensitivity ELISA (Mercodia, Sweden) and by regular ELISA for c-peptide (Mercodia,

310

Uppsala, Sweden). Plasma samples protected by DPPIV inhibitor were analysed by ELISA

311

for GIP and Glucagon-like peptide-1 (GLP-1) (Merck Millipore, Cork, Ireland). Lithium-

312

heparin serum samples were analysed for glucose, NEFAs and trigylcerides using an Rx

313

Daytona autoanalyser (Randox Laboratories, Co Antrim, UK).

314

Statistical Analyses

315

The results are expressed as mean ± sem. The general linear model procedure under IBM

316

SPSS Statistics 20 was employed. Differences between treatment groups during the two hour

317

post prandial period were tested by repeated-measures analysis of variance using the mixed

318

models application. In each group, differences from baseline were measured using a t-test

319

with a Bonferroni post hoc test. In any cases where the data was not normally distributed,

320

non-parametric analysis methods were applied. The data is represented in the figures as the

321

delta change from baseline and, unless otherwise indicated, the statistics were run on these

322

values, rather than raw numbers. P values < 0.05 were considered as significantly different.



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The response of plasma glucose, insulin, c-peptide were calculated as area under the curve

324

(AUC) for the change in values from 0 h using GraphPad Prism 5.0 (GraphPad Software, Inc.

325

La Jolla, USA). Paired samples t-tests were used to assess the difference between treatment

326

groups here.

327 328

Characterisation of the Casein Hydrolysate

329

The molecular mass distribution profile of the intact and hydrolysed caseinate and its

330

associated UF fractions are illustrated in Supplementary Fig. S1A. The unhydrolysed sodium

331

caseinate, as expected, consists of relatively large molecular mass proteins, with more than 98

332

% of the sample being greater than 10 kDa (Supplementary Fig. S1A). In contrast, the

333

hydrolysate UL 291contains 2.2 % of proteinaceous material > 10 kDa (Supplementary Fig.

334

S1A). Upon hydrolysis, the proteins were broken down into peptides, resulting in a reduction

335

in the molecular mass of the components within the casein hydrolysate. Analysis of the

336

casein hydrolysate UF samples showed that the higher molecular weight components were

337

retained in the UF membranes resulting in enrichment of lower molecular weight peptides (

Casein Hydrolysate with Glycemic Control Properties: Evidence from

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