1 Recent Progresses on Functionality of Chinese traditional fermented soy products Dr. Yongqiang CHENG College of Food Science & Nutritional Engineering China Agricultural University

2 Outline  Situation of the production of soy products in China  Production way of sufu, douchi and doujiang  Anti-oxidative activity of fermented soy products  ACE inhibitory activity of fermented soy products  Anti-  -glucosidase activity of fermented soy products  other functionalities (AChE inhibitory activity)  Problems to be solved for modernization of traditional fermented soy products

3 Annual output of soybean production in China

4 ■ From the beginning of 21st century, large amount of soybean has been imported; ■ In 2005, about 27 million tons imported; ■ Annual usage for soy foods: about 4 million tons; ■ Soy foods: tofu; soymilk; soymilk powder; fermented soy products (sufu; douchi; doujiang, etc)

5 Consumption of soybean in China (the year of 2000) 68% 6% 23% 3%

6 Appearance of sufu

7 Appearance of soy products

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10 Appearance of soy products

11 Appearance of soy products

12 Appearance of soy products (douchi)

13 Appearance of soy products

14 Research for functionalities of soy products in the world USA and Europe: soy protein, tofu USA and Europe: soy protein, tofu Japan: tofu, soy milk, natto, miso Japan: tofu, soy milk, natto, miso Indonesia: Tempe Indonesia: Tempe Korea: soy paste, soy milk Korea: soy paste, soy milk China: sufu; douchi; doujiang China: sufu; douchi; doujiang USA and Europe: soy protein, tofu USA and Europe: soy protein, tofu Japan: tofu, soy milk, natto, miso Japan: tofu, soy milk, natto, miso Indonesia: Tempe Indonesia: Tempe Korea: soy paste, soy milk Korea: soy paste, soy milk China: sufu; douchi; doujiang China: sufu; douchi; doujiang

15 Main functionalities of soy protein Coronary Heart Disease Coronary Heart Disease Osteoporosis Osteoporosis Menopause Menopause Cancer Cancer Type II Diabetes Type II Diabetes Kidney Function Kidney Function Weight Control & Weight Loss Weight Control & Weight Loss Coronary Heart Disease Coronary Heart Disease Osteoporosis Osteoporosis Menopause Menopause Cancer Cancer Type II Diabetes Type II Diabetes Kidney Function Kidney Function Weight Control & Weight Loss Weight Control & Weight Loss

16 Main functionalities of soy protein

17 Functionalities of soy protein (Isoflavone) OH Equol (isoflavone) HO O Esterogen HO OH

18 In vitro study on functionality of fermented soy products Anti-oxidative activity Anti-oxidative activity Angiotensin I-converting enzyme (ACE) inhibition Angiotensin I-converting enzyme (ACE) inhibition Anti-  -glucosidase activity Anti-  -glucosidase activity Acetyl-chorine esterase (AchE) inhibition Acetyl-chorine esterase (AchE) inhibition Anti-oxidative activity Anti-oxidative activity Angiotensin I-converting enzyme (ACE) inhibition Angiotensin I-converting enzyme (ACE) inhibition Anti-  -glucosidase activity Anti-  -glucosidase activity Acetyl-chorine esterase (AchE) inhibition Acetyl-chorine esterase (AchE) inhibition

19 Production way of sufu

20 Production way of sufu (cont.)

21 Production way of sufu (cont.) Pehtze Salted Pehtze Dressing mixture Salted Pehtze in bottles

22 Production way of douchi For natto, bacteria

23 Production way of douchi (cont.) Fermentation

24 Production way of douchi (cont.) Aging and drying Dressing

25 Production way of doujiang (cont.) Ratio between soybean and rice

26 Production way of doujiang (cont.) (Yamabe et al. Food Chem. 2007) Variety of microorganism

27 In vitro study on functionality of fermented soy products Anti-oxidative activity Anti-oxidative activity Angiotensin converting enzyme (ACE) inhibition Angiotensin converting enzyme (ACE) inhibition Anti-  -glucosidase Anti-  -glucosidase Acetyl-chorine esterase (AchE) inhibition Acetyl-chorine esterase (AchE) inhibition Anti-oxidative activity Anti-oxidative activity Angiotensin converting enzyme (ACE) inhibition Angiotensin converting enzyme (ACE) inhibition Anti-  -glucosidase Anti-  -glucosidase Acetyl-chorine esterase (AchE) inhibition Acetyl-chorine esterase (AchE) inhibition

28 Anti-oxidative activity of tofuyo and sufu (Wang et al, JARQ, 2003) Sample 1 to sample 4, various types of tofuyo from Okinawa, Japan; sample 5, sufu from China.

29 DPPH radical-scavenging activity of 17 kinds of douchi Anti-oxidative ability of douchi

30 Anti-oxidative ability of douchi ABTS radical-scavenging activity of 17 kinds of douchi

31 Anti-oxidative ability of douchi during fermentation Fermentation time

32 Anti-oxidative activity Anti-oxidative activity Angiotensin-I converting enzyme (ACE) inhibition activity Angiotensin-I converting enzyme (ACE) inhibition activity Anti-  -glucosidase activity Anti-  -glucosidase activity Acetyl-chorine esterase (AchE) inhibition Acetyl-chorine esterase (AchE) inhibition Anti-oxidative activity Anti-oxidative activity Angiotensin-I converting enzyme (ACE) inhibition activity Angiotensin-I converting enzyme (ACE) inhibition activity Anti-  -glucosidase activity Anti-  -glucosidase activity Acetyl-chorine esterase (AchE) inhibition Acetyl-chorine esterase (AchE) inhibition

33 ACE inhibitory activities of tofuyo and sufu extracts (Wang et al, JARQ, 2003) Sample 1 to sample 4, various types of tofuyo from Okinawa, Japan; sample 5, sufu from China.

34 SDS-PAGE patterns of tofuyo and sufu extracts (Wang et al, JARQ, 2003) Sample 1 to sample 4, various types of tofuyo from Okinawa, Japan; sample 5, sufu from China.

35 Zhang et al. Food Chem ACE inhibitory activities as a function of fermentation time (douchi) Changes in ACE inhibitory activity after pre-incubation with ACE

36 Zhang et al. Food Chem Elution profile of ACE inhibitor extracted from douchi qu １ ２ ３ ４

37 Zhang et al. Food Chem HPLC profile of Peak 1-4 Peak ２ Peak ３ Peak 1 Peak 4

38 ACE inhibitory activities of douchi were improved following the fermentation The ACE inhibitors in 48 h-fermented douchi qu were fractionated into four, major peaks by gel filtration chromatography on Sephadex G-25. Peak 2, which had the highest activity, had only one peptide

39 Anti-oxidative ability Anti-oxidative ability Angiotensin converting enzyme (ACE) inhibition Angiotensin converting enzyme (ACE) inhibition Anti-  -glucosidase ability Anti-  -glucosidase ability Acetyl-chorine esterase (AChE) inhibition Acetyl-chorine esterase (AChE) inhibition Anti-oxidative ability Anti-oxidative ability Angiotensin converting enzyme (ACE) inhibition Angiotensin converting enzyme (ACE) inhibition Anti-  -glucosidase ability Anti-  -glucosidase ability Acetyl-chorine esterase (AChE) inhibition Acetyl-chorine esterase (AChE) inhibition

40 Mechanism of the funchiton of  - glucosidase inhibitor starch amylas e glucose inhibit  -glucosidase inhibitor Oligosaccharide  -glucosidase

41 Method for assay of anti-α- glucosidase activity Inhibition gradient: slope value of the inhibitory activity vs. dilution; higher, stronger inhibition activity (Yamaki et al, Nippon Shokuhin Kagaku Kogaku Kaishi (In Japanese), 2006, 53 (4), ) IC50:

42 Anti-α- glucosidase activity of water-extract from commercial douchi samples sample con. 10%(w/v), enzyme con. 25mg/ml, PNPG con mg/ml, incubation time 50min.

43 Anti-α-glucosidase activity of sufu water-extract sample con. 10%(w/v), enzyme con. 25mg/ml, PNPG con mg/ml, incubation time 50min.

44 Aspergillus oryzae Materials ： black bean Incu. Temp. and time ： 28 ℃， 3 d ； Inoculation ： 10 6 spores/g bean ； Actinomucor elegans 3.118

45 Rhizopus arrhizus Products Material ： Black bean ； Aging temp. & time: 45 ℃， 4 months; salt ： 10% （ w/w)

46 Changes of anti-  -glucosidase activity of douchi during prefermentation type Time (h) 0h12h24h36h48h60h Asper.0.116± ± ± ± ± ±0.02 Mucor0.166± ± ± ± ± ±0.06 Rhiz.0.301± ± ± ± ± ±0.03 For all the 3 types of moulds ， the activity gradually increased with the prefermentation time, however, not so much

47 Changes during aging to 4 weeks (Aspergillus) Changes during aging to 4 weeks (Rhizopus) Changes during aging to 4 weeks (Actino.)

48 Some commercial douchi showed fairly high anti-  -glucosidase activity For all the 3 types of moulds ， the activity gradually increased with the prefermentation time, however, not so much During post fermentation (aging), Aspergillus- douchi showed higher inhibitory activity than the other two.

49 Anti-oxidative ability Anti-oxidative ability Angiotensin converting enzyme (ACE) inhibition Angiotensin converting enzyme (ACE) inhibition Anti-  -glucosidase ability Anti-  -glucosidase ability Acetyl-chorine esterase (AChE) inhibition Acetyl-chorine esterase (AChE) inhibition Anti-oxidative ability Anti-oxidative ability Angiotensin converting enzyme (ACE) inhibition Angiotensin converting enzyme (ACE) inhibition Anti-  -glucosidase ability Anti-  -glucosidase ability Acetyl-chorine esterase (AChE) inhibition Acetyl-chorine esterase (AChE) inhibition

50 Mechanism Alzheimer`s disease (AD) Concern with the lack of acetylchorine (ACh) in the brain AChE catalyzes the hydrolysis of acetylchorine Inhibition of AChE can prevent the loss of acetylcholine, and is one of the treatment method for AD

51 Method for measuring AChE inhibition (Yamaki, et al) Substrate: Acetylcholine iodide Enzyme: Acetylcholinesterase, AChE Using Dithiobisnitrobenzoic acid to generate color Absorbance measurement at 405 nm Absorbance ↑ chorine ↑, inhibition ↓

52 AChE inhibition ability of commercial douchi (IC 50 )IC 50 Sample No.

53 Commercial samples IC50 of different type of douchi Aspergillus Mucor Bacteria

54 Comparison of IC50 for different foods

55 Some varieties of commercial douchi showed AChE inhibitory activity, especially Aspergillus-type. AChE inhibitory activity of douchi is similar with that of green tea

56 Change of AChE inhibition ability during fermentation of sufu (Actinomucor elegans 3.118) tofuPre Fer. Salt Add 1 mon. 2 mon.

57 Tofu did not show any AChE inhibition activity During prefermentation of sufu, anti-AChE activity increased comparing with start point, the addition of salt decreased it

58 Problems to be solved for traditional soy products in vivo experiments for functionality Isolation and identification of some unknown active components systemization of traditional production technologies decrease of the salt content of products provision of healthy fermented soy products

59 WHEN CAN I EAT MORE CHINESE FERMENTED SOY FOODS EVERYDAY?

60 What a way to go!

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64 Welcome to our college !!!!!! Thank you !

65 Calculation of IC50 IC50 = concentration (in mg/mL) of extract required to inhibit enzyme activity by 50%. Lower IC50 value, stronger inhibition activity

66  -glucosidase inhibitor Vegetable originated oligosaccharides …… alkaloid amine, derivatives of carbohydrates Flavonoids, Isoflavone, Saponion