Ppt - Lunasin

Processing Methods to Concentrate Bioactives in Soy Products

International Forum Emerging Technologies in Food Processing

September 23-25, 2009 Urbana-Champaign, IL

Elvira Gonzalez de Mejia Associate Professor

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign

Department of Food Science and Human Nutrition Agricultural, Consumer and Environmental

Sciences

http://www.aces.uiuc.edu

OUTLINE   Soybean components and their concentration through

processing

  Lunasin as a model

  Analysis and characterization of lunasin

  Process to isolate lunasin

  Optimization of lunasin concentration in soybean

  Mechanisms of action of lunasin and health benefits

  Conclusions

  Future Studies

To offer an overview of the processing methods to concentrate bioactives in soy

products. Lunasin as a model

Objective �

Distribution in soybean world production

Soybean production

  U.S. soybean production revenue was over 1 trillion dollars in 2006 (3.188 billion bushels) (USDA, 2007).

  Lunasin

  Bowman Birk inhibitor

  Bioac2ve pep2des   Isoflavones

  Saponins

  Lower prevalence of high plasma cholesterol, bowel and kidney cancer, diabetes mellitus, and obesity. Peptides are potential bioactive components of soybean

(Messina et al., 2006).

Bioactive components in soybean

Glycoside Aglycone

Genistin Genistein Daidzin Daidzein

Glycitin Glycitein

Soybean Composition

Soy Isoflavones

Phytochemicals

Saponins Antiviral, antifungic,

diuretic, antiinflammatory

(Berhow, 2006; Dia et al., 2008)

Hipocolesterolemic (Potter, 1995 ; Lee et al., 2005)

Soy is a good source of bioactive peptides�

Wang and de Mejia.2005. CRFSFS 4(4):63-‐78

α-conglycinin, β-subunit�

Soybeans Cleaning Cracking Soy Chips

Hulls

Flaking Fat Extraction

Soybean Oil

Edible Defatted Flakes

Protein Extraction

Protein Precipitates

Drying

Soy Protein

Isolate (SPI)

Sugar Removal

Drying

Soy Protein

Concentrate (SPC)

Grinding and Sizing

Soy Grits Grinding

Soy Flour Blending

Texturing

Drying Screening

Water

TVP Products

Soybean processing flow chart

Commercial soybean products

Food processing affects bioactive components

– Heat •  Heating of soybean Remove trypsin inhibitor (Brandon

et al., 1991)

•  Heating of soymilk Unfolding, dissociation, denaturation, and aggregation of proteins (Kwok and Niranjan, 1995)

– Fermentation •  Soy proteins Smaller peptides, amino acids, and

ammonia (Whitaker, 1978; Shreffler et al., 2001)

Value-‐added soy products

Soybean Lunasin

Predicted secondary structure of lunasin

Amino acid sequence of lunasin

de Lumen, B. O., Nutr Rev, 63, 2005

Wang et al., J AOAC Intl, 91, 2008

Lunasin Facts  Activity is found in processed legumes and

dry cereals. It is heat stable, surviving

temperatures up to 100°C for 10 min.

 Resistant to proteolytic digestion, gets absorbed, and enters target tissues.

 Major component of the Bowman-Birk protease inhibitor, a cancer preventive component from soybeans.

Effect of lunasin on skin tumorigenesis in female Sencar mice

Galvez et al., Cancer Research 61: 7473-7478, 2001

Isolation and Purification of Lunasin

Anion exchange chromatography DEAE, pH 7.5

Size exclusion chromatography

25 kDa

Size exclusion chromatography

7 kDa Lunasin (86%)

Ultrafiltration 3 kDa

SDS-PAGE, Western blot, ELISA,

RP-HPLC, MALDI–TOF, LC-MS-MS

Defatted soybean flour In water (1:5, w/v)

Enzyme Linked Immuno Sorbent Assay

Factors affecting purification of lunasin

pH 8.2 Soy flour (Arcon F from ADM), material/solvent ratio (1:5) suspended in different solvents and extracted at 40 °C for 70 min with sonication

Effect of pH on binding of soy protein and synthetic lunasin on DEAE resin

Wang et al., 2008. Analysis of soybean protein-derived peptides and the effect of cultivar, environmental conditions, and processing on lunasin concentration in soybean and soy products. JAOAC Int. 91 (4): 936-946.

Effect of elution salt concentration on recovery of soy protein and synthetic lunasin from DEAE resin


Purification of lunasin from soy proteins using ion exchange chromatography, XK 50/30 column

packed with DEAE resin

Wang et al., 2008. JAOAC Int. 91 (4): 936-946.

DEAE fractions show similar chromatographic peaks than synthetic lunasin in RP-HPLC using

Vydac C4 column B

C

A


Lunasin purification with gel filtration chromatography Superdex 75 (XK 26/70) and eluted with 20 mM Tris-HCl, pH 7.5 containing 0.15 M NaCl at 4 ml/min


6

8

11 14

16

Lunasin concentration of size exclusion fractions using 25 kDa molecular weight

cut-off


5 kDa

Gonzalez de Mejia and Dia. Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kB pathway in the macrophage. Peptides, 2009. doi:10.1016/j.peptides.2009.08.005

Dose response cytotoxicity of lunasin enriched flour on L1210 cells

IC50 = 0.34 mg/ml C

ell v

iabi

lity

(%)

Lunasin enriched flour (mg/ml)

P < 0.05

Wang et al., JAFC, 2008

Cytotoxicity of lunasin and Bowman Birk inhibitor on L1210 cells �

Lunasin (98% purity) and BBI (95% purity) inhibit prolifera>on of leukemia cells in a dose-‐dependent manner

Wang et al., JAFC, 2008

Lunasin induces apoptosis of leukemia cells

Hoechst stain Caspase stain 50 µM �

Untreated �

Treated 1 mg LEF/mL 24 h �

Gonzalez de Mejia, et al., Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210 leukemia cells by activation of caspase-3. Mol. Nutr. Food Res. 2009. In Press. mnfr.200900073

Lunasin induces apoptosis of leukemia cells 1: Control 2: 5 µM. 3: 15 µM. 4: 50 µM. 5: 250 µM lunasin

4% 3.5% 7.2% 23.2% 43.2%


Comparison of the anti-inflammatory properties of the lunasin-like peptides and their mixture IC30 (µM)

Peptide COX-2 PGE2 iNOS NO 5 kDa 13.0a 17.5a 15.7a 11.8b

8 kDa 33.9b 38.8b 32.7b 32.9c

14 kDa 15.1a 33.6b 33.6b 23.6c

Mixture 114.8c 223.9c 56.3c 1.8a

Gonzalez de Mejia and Dia. Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kB pathway in the macrophage. Peptides, 2009. doi:10.1016/j.peptides.2009.08.005

Industrial Samples

–  Samples from different parts of the soybean process, including:

–  Soy Flour - Soy Powder

–  Soy Protein Concentrate - Hydrolyzed Soy Protein

–  Soy Protein Isolate - Isoflavone Concentrate

–  Soy Flakes - Saponin Concentrate

–  Samples from selected industrial waste effluents of the soy protein process studied for the isolation and purification of lunasin.

Lunasin concentration among different soy products

Lunasin in Chinese germplasm

Gonzalez de Mejia, et al. J Agric. Food Chem., 2004.

Lunasin content in commercially available isoflavone products (mg/g solid material)

Now Extra Strength Soy Isoflavones, 60 mg (soy isoflavone extract, rice flour)

1.0

Vitamin World Soy Isoflavones 23 mg (soy extract)

1.4

PhytoNutramins Isoflavones 11 mg (isoflavones from soybean flour)

3.1

TwinLab Soy Germ Isoflavone Caps 10 mg 4.8

Soy Care for Menopause 25 mg (soy extract, soy protein concentrate)

11.6

Low Isoflavone 15.1 Gonzalez de Mejia, et al. J Agric. Food Chem., 2004.

Lunasin in waste streams

Current research on methods to purify lunasin from soy molasses

Gonzalez de Mejia, et al. J Agric. Food Chem., 2004.

Germination as a process to improve bioactive compounds

•  Increase nutritive value

•  Reduce antinutritional factors

•  Increases methionine value

(Barcelos et al., 2002).

Independent Variables

Levels

-α -1 0 +1 +α

x1 Time (h) 12 21 42 63 72

x2

Temperature (ºC) 18 20 25 30 32

±⏐α⏐=1,41

Experimental design to obtain germinated soy flour

Soybean

Germination

Integral germinated soy flour

Sanitation Grinding

12, 21, 42, 63, 72 h 18, 20, 25, 30, 32 ºC

Drying

Homogenization

Paucar-Menacho, et al. Optimization of germination time and temperature on the concentration of bioactive compounds in Brazilian soybean cultivar BRS 133 using response surface methodology. Food Chem. 118 (3), 2010. 10.1016/j.foodchem. 2009.07.011.

Optimization of lunasin concentration by germination

Paucar-Menacho, et al. Effect of time and temperature on bioactive compounds of germinated Brazilian soybean cultivar BRS 258. Food Res. Int. 2009. http://dx.doi.org/10.1016/j.foodres.2009.09.016

High concentration lunasin and low concentration of lectin and lipoxygenase

High concentration isoflavones aglycones and

saponins

Optimization of lunasin concentration by germination

Paucar-Menacho, et al. A high-protein soybean cultivar contains lower isoflavones and saponins but higher minerals and bioactive peptides than a low-protein cultivar. Food Chem., , 2009. 10.1016/j.foodchem.2009.09.062.

Complete Model : R2 = 0,95 Ajusted Model : R2 = 0,91

Lunasin

Lunasin (mg/g PS) = 21,08 – 2,45x12 – 4,38 x2

2 – 4,03 x1x2

Paucar-Menacho, et al. Optimization of germination time and temperature on the concentration of bioactive compounds in Brazilian soybean cultivar BRS 133 using response surface methodology. Food Chem. 118 (3), 2010. 10.1016/j.foodchem. 2009.07.011.

 High-protein soybean isolated powder (90% protein)  Whole soybean chili dish prepared at the National Research Soybean Laboratory (University of Illinois)

Lunasin bioavailability

Dia et al. J. Agric Food Chem. (2009)

0 10 20 30 45 60 90 120 150 180

In vitro diges2on experiment

Time (min) 190 200 210 220 235 250 280 310 340 370

pH 7.5

Pancrea2n Pepsin pH 2

 SDS-‐PAGE

 Degree of hydrolysis


Lunasin during simulated gastro-intestinal digestion

Pepsin Pancrea2n

n=6, P< 0.05


Quantification of lunasin in blood of men fed soy protein

Pre-loading of beads

Equilibration of beads

Adsorption of proteins/peptides (~ 50 µg/0.5 mg beads)

Desorption (Using different concentrations of NaCl)

Determination of lunasin in eluate: ELISA, MALDI-TOF, LC/MS-MS

Dia et al. J. Agric Food Chem. (2009)

Lunasin in plasma of men after soy protein consumption

Dia et al. J. Agric Food Chem. 57: 1260 (2009)

Commercial peptide products

Manufacturing of protein hydrolysates/bioactive peptides

•  Batch versus con2nuous process

•  Hydrolysis with/without preserva2ves

•  Hydrolysis by: Acid, Alkali, versus Enzymes and Fermenta2on Processes

•  Downstream Processing: Filter press versus Membrane separa2ons Centrifuge versus con2nuous centrifuge Chromatography, Ion exchange resin columns

Manufacturing of protein hydrolysates/bioactive peptides •  Downstream processing:

•  Pasteuriza2on: Keale hea2ng versus Plate & Frame Pasteurizer and UHT

•  Evapora2on: Hea2ng under vacuum versus mul2ple effect falling film

•  Drying: Drum drying versus spray drying and fluid bed drying for agglomerated products.

Conclusions

•  Lunasin is present in most commercial soy products in proportion to the amount of protein (range 13 to 44 mg/g dry product)

•  Soy processing waste effluents contain 10.4 mg lunasin/g solid material

Conclusions Lunasin can be found in human circulation,

a key requirement for its biological activity.

The average concentration of lunasin 1 h after ingestion was 71.0 ng/mL equivalent to 0.21 mg of lunasin in a person containing 3 L of plasma. This represents an average of 4.5% absorption (range of 2.2-7.8%).

Conclusions

•  Lunasin and lunasin-like peptides inhibited inflammation through suppression of NF-κB activation

•  Lunasin has potential as a unique and novel agent for human health and wellness.

 The consumption of 25 grams per day of soy protein recommended by the FDA for reducing coronary heart disease risk supplies 250 mg of lunasin. Whether this is physiologically relevant remains to be determined in clinical trials.

  Identification of new bioactive soy peptides and their in vivo biological functions

  New commercial production techniques

 Effect of industrial processing on bioactive peptide generation

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Ppt - Lunasin