Whey preparation for improving brain development

Abstract

The present invention relates to a bioactive sweet whey protein concentrate or composition for increasing e.g. cognitive functions particularly in young mammals such as pre-term or term infants, toddlers, children or young adults. The present invention further disclose a new concept for large-scale industrial production method of bioactive whey protein concentrates or compositions by use of mild heat treatment and pressure driven membrane separation.

Claims

1. A method for producing a mildly-heated treated bioactive sweet whey WPC80 preparation comprising a high level of bioactive compounds with reduced denaturation and aggregation, said method comprising the sequential steps of: a) providing a sweet whey; b) applying a first heat-treatment of the sweet whey between 60 C. and 68 C. for between 10 and 20 seconds, wherein the heat treatment is for reducing cheese starter culture bacterial growth and preventing further pH drop in the whey; c) cooling the sweet whey to a temperature of 5 C. to 18 C.; d) applying a separation and/or concentration step at 5 C. to 18 C., which separation is ultrafiltration and which concentration is reverse osmosis or nano filtration to obtain WPC35; e) applying a micro filtration on the retentate from step d) to prevent particles from entering into the permeate, wherein the particles are sediment, algae, protozoa or large bacteria; f) applying a separation on the permeate from step e) at 5 C. to 18 C. to obtain WPC80, and wherein, the separation is ultrafiltration; g) applying a second heat-treatment of between 60 C. and 65 C. for between 10 and 20 seconds on the retentate of step f); h) optionally, cooling the product obtained in g) to less than 15 C. for storage of the bioactive sweet whey WPC80 preparation; and i) optionally drying said product, thereby obtaining a dried bioactive sweet whey WPC80 preparation.

2. The method of claim 1, wherein the separation in step d) is ultra-filtration.

3. The method of claim 1, wherein the microfiltration is performed using a ceramic membrane with a pore size of 0.5 to 2 m.

4. The method of claim 1, wherein the concentration in step d) is reverse osmosis or nano filtration.

5. The method of claim 1, wherein the sweet whey is derived from a milk base that has not been exposed to standard industrial processing steps selected from the group consisting of thermal processing, ultra-high temperature processing (UHT), hydrolysis and irradiation.

6. The method of claim 1, wherein the heat treatment under step b) is applied at a temperature of 60 to 67 C.

7. The method of claim 1, wherein the heat treatment under step b) is applied for less than 19 seconds.

8. The method of claim 1, wherein the method comprises cooling the preparation obtained in g) to less than 15 C.

9. The method of claim 1, wherein the method comprises drying said preparation.

10. The method of claim 1, wherein the microfiltration is performed using a ceramic membrane with a pore size of 0.8 to 1.5 m.

11. The method of claim 1, wherein the microfiltration is performed using a ceramic membrane with a pore size of 1.0 to 1.5 m.

12. The method of claim 1, wherein the microfiltration is performed using a ceramic membrane with a pore size of 1.0 to 2 m.

13. The method of claim 1, wherein the heat treatment under step b) is applied at a temperature of 60 to 63 C.

14. The method of claim 1, wherein the heat treatment under step b) is applied for less than 15 seconds.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1

(2) Whey drainage and processing at the cheese plant for Bioactive WPC.

(3) FIG. 2

(4) Bioactive WPC production diagram.

(5) FIG. 3

(6) Standard WPC 80 production diagram.

(7) FIG. 4

(8) Open field assessment scores of locomotion (distance travelled within 3 min sessions) in BioWPC (N=8) and ConWPC (N=8) group. Data are meanSEM. P<0.05. Walking distance in open field test on day 4. Values are means+SEM. Values indicated by * differ between each other, P<0.05. Bio, formula containing bioactive WPC; Con, formula containing conventional WPC.

(9) FIG. 5

(10) Schematic diagram displaying the production flow of bioactive WPC and conventional WPC. BioWPC, bioactive WPC; Bio, BioWPC containing formula; Con, ConWPC containing formula; ConWPC, conventional WPC; WPC, whey protein concentrate.

(11) FIG. 6

(12) Protein composition in BioWPC and ConWPC with and without aggregate removal by centrifugation analysed by SDS-PAGE.

(13) FIG. 7

(14) Relative quantification of protein band volume for LF, BSA, -Lg and -La. Values (meansSEM, n=3 for triplicates of sample preparation) indicated by * differ between each other, P<0.05. BioWPC, bioactive WPC; ConWPC, conventional WPC; -Ig, -lactoglobulin; -La, -lactalbumin; BSA, bovine serum albumin; LF, lactoferrin.

(15) FIG. 8

(16) Western-blotting analysis of TGF-2 in the two WPCs (triplicates) with and without removal of aggregates by centrifugation. The relative abundance of TGF-2 is presented as meansSEMs. Values indicated by * differ between each other, P<0.05. BioWPC, bioactive WPC; ConWPC, conventional WPC.

(17) FIG. 9

(18) Effects of BioWPC and ConWPC on the cell cytotoxicity (A) and proliferation (B) of IPEC-J2 cells in vitro. Values are meansSEM, n=3 representing triplicates in three different cell passages. Values indicated by * differ between each other, P<0.05. Means without a common letter differ, P, 0.05. BioWPC, bioactive WPC; ConWPC, conventional WPC; CON, negative control in the proliferation assay.

(19) FIG. 10

(20) Intestinal digestive and absorptive functions and permeability. Plasma galactose levels at 20 min after administration of oral boluses of galactose solution on day 3 and at 40 min after administration of oral boluses of lactose solution.

(21) FIG. 11

(22) Intestinal permeability presented as measured by the urinary lactulose to mannitol ratio (L/M ratio) after oral administration of lactulose/mannitol solution.

(23) FIG. 12

(24) Brush border enzyme activity in the proximal region of small intestine. Values are meansSEM. Values indicated by * differ between each other, P<0.05. Bio, formula containing bioactive WPC; Con, formula containing conventional WPC

(25) FIG. 13

(26) Villous height and crypt depth in the proximal small intestinal region.

(27) FIG. 14

(28) General physical activity. Trend of proportion of active time during postnatal day 3 and 4 showing in every half day.

EXAMPLES

Example 1Manufacturing of Bioactive WPC

(29) Bioactive WPC is produced from whey drainage from cheese production. After whey drainage, cheese fines are removed and whey cream is also removed in a continues centrifugal separator.

(30) Heat treatment between 60-68 C. is performed in order to reduce cheese starter culture bacterial growth and preventing further pH drop in the whey. The whey is cooled to below 5 C. and stored in a silo tank before further processing preferably less than 2 days.

(31) The whey is concentrated into WPC 35 using a 5 or 10 Kd UF Spiral Wound membrane. The temperature is kept in the range of 5-18 C.

(32) The feed pressure is in the range of 0.5-3.5 bar and a pressure drop per element ranging from 0.5 bar to 1.3 bar.

(33) A second filtration reduces the bacterial count in the WPC 35. The bacterial reduction is performed using a Ceramic Membrane with a pore size ranging from 0.5 m to 2.0 m. The temperature is kept at 5-18 C. and the Trans Membrane Pressure (TMP) should be between 0.1 and 2.5 bar. The VCF is in the range of 2-100.

(34) Permeate from the ceramic filtration is further processed into WPC 80 using a 5 or 10 Kd UF Spiral Wound membrane. The temperature is kept in the range of 5-18 C.

(35) The feed pressure is in the range of 0.5-3.5 bar and a pressure drop per element ranging from 0.5 bar to 1.3 bar. An SW membrane that can be sanitized at temperatures above 70 C. is preferable in order to keep the high microbial status after MF filtration.

(36) The WPC 80 is heat treated at 63 C. for 15 sec. prior to spray drying.

Example 2Manufacturing of Bioactive WPC80

(37) Bioactive WPC 80 is produced using whey drainage from Tistrup Dairy, which produces Gouda cheese.

(38) After whey drainage, cheese fines are removed in a Sweco vibrating sieve and whey cream is removed in a continues centrifugal separator.

(39) The whey is heat treated at 63 C. for 15 sec. to reduce cheese starter culture bacterial growth and preventing further pH drop in the whey.

(40) The whey is cooled to below 5 C. and stored in a silo tank.

(41) 20,000 kg whey is concentrated into WPC 35 using a 5 Kd KOCH HKS 328 UF Spiral Wound membrane. The temperature is 10 C. The feed pressure is 3.0 bar and the pressure drop per membrane element is 1.0 bar.

(42) The second filtration reduces the bacterial count in the WPC 35 retentate. A ceramic Tami 1.4 m isoflux membrane is used. The filtration is performed at 15 C. and the TMP is 1.5 bar. The VCF is set to 50.

(43) Permeate from the ceramic filtration is further processed into WPC 80 using a 10 Kd KOCH hpht 131 UF Spiral Wound membrane. Before filtration, plant and membranes are sanitized at 70 C. just before filtration. During the production, the temperature is 10 C. The feed pressure is 3.0 bar and the pressure drop per membrane element is 1.0 bar.

(44) The Bioactive WPC 80 retentate is heat treated at 63 C. for 15 sec. prior to spray drying.

Example 3Manufacturing of Standard WPC 80

(45) A standard WPC 80 is produced using whey drainage from yellow cheese production. After whey drainage, cheese fines are removed and whey cream is removed in a centrifugal separator.

(46) Pasteurization at 73 C. for 15 sec. is performed to inactivate cheese starter culture and prevent further pH drop in the whey.

(47) The whey is cooled and stored in a silo tank before further processing.

(48) WPC 80 is produced using a 5 kD Koch HKS 328 membrane. The temperature is 10 C., the feed pressure is 3.0 bar and there is a pressure drop per membrane element at 1.0 bar.

(49) The WPC is heat treated at 71 C. for 15 sec. prior to spray drying.

Example 4Composition of Bioactive WPC 80 and WPC 80

(50) The present inventors compare mildly-heat-treated WPC (Bioactive WPC, Arla Foods Ingredients, AFI) with a conventionally-heat-treated WPC (Lacprodan DI-8090, AFI)

(51) TABLE-US-00003 Powder Bioactive WPC 80 WPC 80 (DI-8090) Total Solid [%] 95.36 93.96 Protein [%] 79.35 75.6 Lactose [%] N.A. 4.42 Galactose [%] 0.28 0.76 Fat [%] 5.52 6.89 Ash [%] 2.79 2.67 Calcium [%] 0.417 0.372 Phosphate [%] 0.302 0.3075 Magnesium [%] 0.0726 0.0563 Sodium [%] 0.156 0.205 Potasium [%] 0.611 0.5425 Chloride [%] 0.04 0.08 pH 6.52 6.44 Alpha -La [%] 10.86 8.90 Beta-Lg [%] 38.91 31.71 GMP [%] 14.16 13.14 IgG [%] 5.30 3.80 Lactoferrin [%] 0.270 0.085 a-la/protein [%] 13.7 11.8 B-lg/protein [%] 49.0 41.9 cGMP/protein [%] 17.9 17.4 IgG/protein [%] 6.7 5.0 Lactoferrin/protein [%] 0.3 0.1 Other protein + denatured [%] 12.4 23.8 PROT/TS (WPC) [%] 83.2 80.5 Beta:CGMP [%] 2.75 2.41

Example 5Settings in Preterm Neonates Piglet Model

(52) Preterm term infants suffer from impaired growth and brain development postnatally. Early enteral feeding with high quality formula plays an important role to stimulate growth and organ maturation, which may be important for long-term neurodevelopment.

(53) Here we investigate the effects of gentle treatment of WPC on physical activity and motoric control in preterm pigs.

(54) Materials and Methods

(55) Pigs, Nutrition Protocol, and General Experimental Design

(56) Nighty-two preterm pigs were delivered from four sows by caesarean section at 105-106 d gestation (Large WhiteDanish LandraceDuroc, Askelygaard Farm, Roskilde, Denmark; term=1162 days). Oro-gastric feeding tube (6 F, Portex, Smiths Medical, St Paul, MN, USA) was placed into the stomach for enteral nutrition (EN), and vascular catheter (4 F, Portex) was inserted in the umbilical artery for parental nutrition (PN).

(57) Pigs were reared in temperature-regulated individual incubators with oxygen supply and given 16 ml/kg maternal serum during the first 24 h after birth to achieve passive immunological protection. All pigs received parenteral nutrition (PN) for the first 2 days as previously described (ref.). Pigs also received their respective milk diets as minimal enteral nutrition (MEN; 24-40 ml/kg/d) for the first two days and as full enteral nutrition (120 ml/kg/d) from day 3 to euthanasia.

(58) The studies were approved by the National Committee on Animal Experimentation in Denmark.

(59) Pigs Selected for BIO WPC Protocol

(60) Thirty one caesarean-delivered preterm pigs were assigned into two groups fed formula with gentle treatment of WPC (BioWPC, n=15) or conventional WPC treatment (ConWPC, n=16).

(61) Feeding

(62) The two milk formulas were made by mixing the following ingredients and contained similar macronutrient compositions and energy level (Table 1): BioWPC, and ConWPC, Arla Foods Ingredients, AFI, Viby J. Denmark); lactose (Variolac 960, AFI); maltodextrin (Ross Polycose; Abbott Nutrition, Columbus, USA); lipids (Liquigen and Calogen; Nutricia, Allerde, Denmark) and vitamins and minerals (SHS Seravit; Nutricia).

(63) After five days of feeding, organ weights, gut functions and incidence of necrotizing enterocolitis (NEC) were evaluated. Time for acquisition of basic motor skills (i.e. first opening of eyes, first stand, first walk) was noted and physical activity was recorded by cameras and analyzed (PIGLWin application). Motoric skills were further investigated by measuring the distance travelled in an open-field arena (EthoVision XT10 analyses).

(64) Clinical Evaluation and Sample Collection

(65) Pigs were continually monitored and killed if clinical symptoms of NEC such as abdominal distension, lethargy, cyanosis or bloody diarrhea were observed. All remaining pigs were euthanized for tissue collection on day 5.

(66) Feeding intolerance was defined as withholding or reducing the amount of planned enteral feeding due to vomiting or abdominal distension.

(67) After anaesthesia (Zoletil 50, zolazepam/tiletamin; Boehringer Ingelheim, Copenhagen, Denmark), cardiac blood was collected into heparin- or EDTA-containing tubes, and subsequently pigs were euthanized with an intracardiac injection of pentobarbitone sodium (60 mg/kg).

(68) Individual weights of the heart, lungs, liver, kidneys, spleen, stomach, small intestine and colon were recorded.

(69) The length of the small intestine was recorded in a relaxed stage. The whole small intestine was divided into three sections of equal length, proximal (Prox), middle (Mid) and distal (Dist). Whole-wall tissue samples were taken at the middle of each region, and immediately snap-frozen in liquid nitrogen, and stored at 80 C. for further analyses. Whole-wall tissue samples were also taken from each region at the same position, and were immediately submerged into paraformaldehyde solution. A one-centimeter segment of Dist tissue from 10 cm prior to ileocecal junction and of colon tissue from the apex region was fixated in Clarke's solution for later goblet cell quantification.

(70) The mucosal lesions in the stomach, Prox, Mid, Dist, and colon were evaluated macroscopically and a lesion score was assigned to each region.

(71) The score was graded according to following criteria: 1 no or minimal focal hyperaemic gastroenterocolitis; 2 mild focal gastroenterocolitis; 3 moderate locally extensive gastroenterocolitis; 4 severe focal gastroenterocolitis; 5 severe locally extensive hemorrhagic and necrotic gastroenterocolitis; or 6 severe extensive hemorrhagic and necrotic gastroenterocolitis.

(72) Pigs with a score of three or above in any of the Prox, Mid, Dist and Colon regions, was diagnosed as NEC. The severity of inflammation in stomach and intestine was reflected by the mean of the lesion scores in stomach, colon, and the mean of the average lesion scores in Prox, Mid, Dist.

(73) Statistical Analysis

(74) Levels of proteins in WPCs were analyzed by student t-test (GraphPad Prism 5.0, La Jolla, CA, USA).

(75) Data of cell cytotoxicity and cell proliferation were analyzed by a linear mixed model with treatment and cell passage as fixed factors followed by post hoc Tukey test (JMP, SAS Institute, Cary, USA). Binary data such as NEC incidence were evaluated using logistic regression in R (version 3.1.1). NEC lesion scores were analyzed using non-parametric analysis with the nparcomp package.

(76) Continuous outcomes were compared among groups using general linear model with adjustments, e.g. sow, sex, birth weight. Repeated measurement of daily physical activity was analyzed using the Imer function for mixed modeling as repeated measures and comparisons were made with the Ismeans package.

(77) To confirm validity of modeled data, residuals and fitted values were assessed for normality and variance homogeneity, which for some outcomes required log-transformation of data before modeling. Resulting P-values were evaluated at a 5% significance level. The multcomp single-step method was used to adjust P values for multiple comparisons within each outcome measure and point of measurements.

(78) Data are presented as raw arithmetic mean and SEM, unless otherwise stated.

Example 6Whey Protein Concentrate Products and Analysis of Bioactive Markers

(79) ConWPC and BioWPC used in experiments were manufactured at AFI from sweet whey by similar processing procedures with differences only in the number of pasteurization steps and temperature of spray-drying (FIG. 7).

(80) Protein aggregation, protein compositions before and after removal of aggregates, and levels of LF, IgG, IGF-I, and TGF-2 were measured as the indicators of bioactivity.

(81) The level of total protein was measured by BCA protein assay (Thermo Scientific) before and after removal of the aggregated protein (centrifuged at 15000g, 4 C. for 30 min). WPCs before and after centrifugation (15 g protein in each sample) were loaded in 15% SDS-PAGE gel in non-reducing conditions for major protein analysis. LF and IgG were also analyzed by Eurofines.

(82) TGF-2 was analyzed by Western blot using TGF-2 antibody (Sc-90, Santa Cruz Biotechnology, CA, USA).

(83) Aggregated protein in BioWPC was negligible, whereas approximate 20% protein was aggregated in ConWPC.

(84) The levels of native LF and IgG measured by Eurofines were 3 and 1.3 times more in BioWPC than those in ConWPC (Table 1).

(85) By non-reducing SDS-PAGE, LF and BSA levels were higher in BioWPC vs. ConWPC for both samples before and after aggregate removal (P<0.05, FIG. 7).

(86) After aggregate removal, BioWPC also had higher levels of -Lg and -La than ConWPC (P<0.05, FIG. 7).

(87) For growth factors, two WPCs had similar levels of IGF-1 (170-180 ng/g), but BioWPC contains 10 times more of TGF-beta than ConWPC (FIG. 8).

(88) At low concentrations of 0.01-0.1 g/L, both BioWPC and ConWPC were negligibly cytotoxic whereas at 1 g/L, cytotoxicity, indicated by DNA release, was induced at higher levels by ConWPC than BioWPC (P<0.05).

(89) BioWPC-induced cell proliferation was greater than ConWPC-induced cell proliferation at 0.01 g/L, whereas at 1 g/L, ConWPC decreased cell proliferation (P<0.05).

Example 7Clinical Outcomes

(90) There was no difference between the Bio and Lac groups in terms of birth weight (101446 g), NEC incidence (14/31), and lesion scores in the stomach (1.40.2), small intestinal (1.70.1), and colon (2.10.2).

(91) The relative weights of organs and the relative length of the small intestine did not differ among groups (data now shown).

(92) Interestingly, feeding intolerance was observed in seven pigs in the Con group and none in the Bio group.

(93) Feeding intolerance was defined as feed-reduction or complete with-holding of feed at any time point for any reason. The decision to reduce or with-hold feeding (at each feeding time point), was based on a clinical assessment including the following criteria: signs of pain or discomfort, vomit, abdominal distension, bloody stools.

(94) The incidence of feeding intolerance was calculated as the number of pigs in each group experiencing feed-reduction, divided by group size.

(95) On this background there was an incidence of 44% in Lac and 0% in Bio.

Example 8In Vivo Gut Functions and Ex Vivo Brush Border Enzyme Activities

(96) To determine the intestinal digestive and absorptive capacity, the increment of plasma galactose in response to oral boluses of galactose and lactose was measured.

(97) On day 3 before full EN, pigs were given a bolus (15 ml/kg) of 5% galactose via the oro-gastric feeding tubes. Heparinized blood samples were taken through the umbilical artery catheter at before and 20 min after administration of the bolus.

(98) On day 4, pigs were given a bolus (15 ml/kg-1 body weight) of 10% lactose, and blood samples were sampled into heparin-containing tubes prior to, and at 20 min and/or 40 min after administration of the bolus. Concentrations of galactose in plasma were measured by spectrophotometry as described previously (13).

(99) To test the intestinal permeability, pigs received an oral bolus (15 ml/kg-1 body weight) containing 5% lactulose and 2% mannitol 3 h prior to euthanasia. Post-mortem urine samples were taken to measure the concentrations of lactulose and mannitol. The ratio between lactulose and mannitol concentrations was calculated to determine the intestinal permeability.

(100) Snap-frozen tissue samples from Prox, Mid, and Dist were homogenized in 1.0% Triton X-100 (10 ml/g tissue) using gentle MACS Dissociator (Miltenyi Biotec, Auburn, CA, USA).

(101) After centrifugation (2000g, 10 min, 4 C.), the supernatant was isolated and used for determining the brush border enzyme activities. The activities of lactase, maltase, sucrase, aminopeptidase N (ApN), aminopeptidase A (ApA) and dipeptidyl-peptidase IV (DPPIV) in the homogenates were analysed by spectrophotometry using corresponding sugars and peptides as substrates.

(102) Intestinal absorptive functions measured by galactose test on day 3 tended to be higher in Bio group relative to the Con group (P=0.11, FIG. 12), whereas the function measure by lactose test on day 4 did not differ between two groups (FIG. 12).

(103) Intestinal permeability measured by lactulose/mannitol test tended to be lower in the Bio group then that in the Con group (P=0.07, FIG. 13).

(104) When it comes to ex vivo brush border enzyme activities, only lactase in Prox differed between two groups with higher values in the BioWPC group (P<0.05, FIG. 14).

(105) The activity of lactase in Mid and Dist and of other brush boarder enzymes in any of the three regions did not differ between groups (data not shown).

Example 9Intestinal Morphology and Goblet Cell Density

(106) Paraformaldehyde-fixated tissues from Prox and Dist were embedded in paraffin, sectioned (3 m), mounted on slides and stained with hematoxylin and eosin. Digital histology images were obtained by use of a light microscope (Ortho-plane, Leitz, Germany) and an attached camera. Mean villus height (m), and crypt depth (m) were measured on the digital images in ten representative vertically well-oriented villus-crypt axes in each region. (Image J 1.44p, National Institutes of Health, Bethesda, MD, USA). An average of 10 measurements in each region was used as the representative villus height or crypt depth for each pig. Clarke's fixated Dist intestine as well as colon samples were embedded in paraffin, sectioned (2 m), mounted on slides and stained with Alcian Blue-Periodic Acid Shiff (AB-PAS) for the evaluation of goblet cell density. Slides were visualized using a light microscope (20 lens, Olympus BX45TF, Tokyo, Japan) equipped with a camera (Olympus). STEPanizer (version 1.0, company, city, country) was used to visualize the tunica mucosa including the goblet cells, and the goblet cell density was calculated as the area fraction of the total tunica mucosa that was covered by goblet cells (lamina muscularis mucosa excluded).

(107) In the Prox region, pigs in the Bio group had increased villus height and decreased crypt depth compared with the Con group (both P<0.05, FIG. 15).

(108) Villus height and crypt depth did not differ between two groups in the Mid and Dist regions (data not shown).

(109) Goblet cell density did not differ between two groups neither in the Dist small intestine nor in the colon (FIG. 16).

(110) However, when comparing pigs with Dist NEC score 1 with those with score 2, pigs scored 2 had decreased goblet cell density relative to pigs scored 1 (P<0.01, FIGS. 17 and 18).

(111) No comparison was made for other scores due to lack of samples (only 3 pigs scored higher than 2 in the Dist small intestine).

Example 10Effects of WPCs on Cytotoxicity and Proliferation of Porcine Immature IECs In Vitro

(112) Porcine IPEC-J2 cell line (DSMZ, Braunschweig, Germany) derived from the jejunum of a newborn pig, were used to test the effects of WPCs in vitro. Cells were cultured between passage 5-25 in advanced DMEM/F12 medium supplemented with 2% heat-inactivated fetal bovine serum, 40 U/ml penicillin, 40 g/ml streptomycin and 2 mM Glutamax (all from Life Technologies, Naerum, Denmark), at 37 C. and 5% CO2.

(113) Centrifuged BioWPC and HWPC solutions at 10 g/L prepared as mentioned above were sterile filtered at 0.2 m, and diluted in serum-free culture medium to reach protein concentrations of 1, 0.1 and 0.01 g/L.

(114) Cell cytotoxicity: cells were seeded in 96 well plates at 2104 cells/well, and allowed to attach for 24 h. BioWPC and HWPC at 1, 0.1 and 0.01 g/L were mixed with Sytox green 5 M (Life Technology), a non-membrane permeable dye binding to extracellular DNA, and stimulated with IECs for 6 h.

(115) Fluorescence intensity was then measured at 485/520 nm (excitation/emission). Cell cytotoxicity was calculated by the fluorescence intensity of treatments subtracted by that of serum-free medium alone.

(116) Cell proliferation: cells were seeded in 96 well plates at 2104 cells/well, allowed to attach for 24 h prior to treatments with BioWPC and HWPC at 1, 0.1 and 0.01 g/L for 48 h. Cell proliferation was quantified by Celltiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Nacka, Sweden) according to the manufacturer's instructions.

Example 11Physical Activity

(117) Physical activity was recorded by continuous video surveillance using infrared cameras installed over each incubator and connected to an HD recorder with built-in motion detection. The digital output for each camera allowed recording of the status of the individual piglets as being either active or resting.

(118) With the PIGLWin application (Ellegaard Systems, Faaborg Denmark), the proportion of active time was automatically registered for every hour. The cameras were turned off during any handling and contact with the pigs.

(119) Activity recording was performed from total enteral feeding commenced on day 3 at 9:00 am and ended just before pigs were euthanized on day 5 at 9:00 am.

(120) The proportion of active time was analyzed from means of recordings covering the day and night time, respectively.

(121) On day 4, spontaneous motor activity was evaluated in an open field arena (1.201.20 m), with a video camera mounted from the ceiling (bird's eye view) during a 3-min recording period. From these recordings, piglet movements were tracked and analyzed using a commercially available software (EthoVision XT10, Noldus Information Technology, Wageningen, The Netherlands) providing information on distance travelled inside the arena. Pigs that were clinically ill on day 4 were excluded from the test.

(122) Initially, home cage activity was similar between groups, but from late day 3 and onwards, the activity level was higher in the Bio group compared to the Con group (P=0.09), and significantly increased during the last half day prior to the euthanasia (P<0.05). Further, in the open field test on day 4, Bio pigs walked almost twice the distance as that of the Con pigs (P<0.05, FIG. 4), overall supporting an increased motor activity in Bio pigs.

(123) Result

(124) BioWPC did not affect body and intestinal weights but significantly improved feeding tolerance compared with control pigs (P<0.01). BioWPC pigs also tended to show higher hexose absorptive capacity (P=0.09) and lower gut permeability (P=0.07).

(125) BioWPC increased the villus height to crypt depth ratio, and proximal intestinal lactase activity (P<0.05). The time for acquisition of basic motor skills was similar between groups, and the home cage activity was initially similar between groups, but from late day 3 and onwards, the activity level was higher in the Bio group compared to the Con group (P=0.09), and significantly increased during the last half day prior to the euthanasia (P<0.05), but the BioWPC pigs tended to have more activity bouts (p=0.09), and longer activity time (p=0.10), compared with controls. The distance travelled in the open field arena was consistently longer in the BioWPC group, relative to controls (n=8, p<0.05).

CONCLUSION

(126) The BioWPC intervention increased physical activity and locomotion in preterm pigs. This may be explained by a combination of metabolic effects, improved gut maturation or direct beneficial effects on early brain development.

REFERENCES

(127) Anders D. Andersen, Per T. Sangild, Sara L. Munch, Eline M. van der Beek, Ingrid B. Renes, Chris van Ginneken, Gorm O. Greisen, and Thomas ThymannDelayed growth, motor function and learning in preterm pigs during early postnatal lifeAm J Physiol Regul Integr Comp Physiol 310: R481-R492, 2016. Yanqi Li, Mette V. stergaard, Pingping Jiang, Dereck E. W. Chatterton, Thomas Thymann, Anne S. Kvistgaard, and Per T. SangildWhey Protein Processing Influences Formula-Induced Gut Maturation in Preterm PigsThe Journal of Nutrition 2013/09/18/jn.113.182931