J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Review

Volume 22, June 2023, pages 9-16

Trypsin inhibitors, antinutrients or bioactive compounds? a mini review

**Table 1.** Extant technologies for deactivating trypsin inhibitors in legumes
Method	Raw materials	Treatment conditions	Trypsin inhibitor inactivation (%)	Advantages	Disadvantage
Source: Adapted from Avilés-Gaxiola et al. 2018.
Thermal treatment	Peas	Microwaves (2,450 MHz, 4 min)	100	High efficiency.	Energy demand.
Mung bean	Boiling (100 °C for 90 min)	100	Standardized process.	Reduced solubility.
Garbanzo	Soaked cooked seeds (95 °C for 1 h)	88.4	Scalable process.	Decreased lysine, tryptophan, and sulfurous amino acids.
Lentil	Cooking (80 °C for 1 min)	95.6	Increases aromatic amino acids.	Mineral loss (Na, Ca and Mg).
Soy	Baking (200 °C for 20 min).	67.3	Improves gelling capacity.	Reduced B vitamins.
Extrusion	Peas	Prior cooking and humidifying processes at 120 °C, opening nozzle 55 mm, velocity 380 rpm.	58.9	Reduces total phytate and tannin levels	Reduces emulsion activity.
Garbanzo	Prior cooking and humidifying processes at 120 °C, nozzle diameter 55 mm, velocity 380 rpm.	91.8	Raises protein digestibility.	Pre-processing may be needed.
Fava	Prior cooking and humidifying processes at 120 °C, nozzle diameter 55 mm, velocity 380 rpm.	53.7
Soy	Dry extrusion at 150 °C	95
Soy	Wet extrusion at 150 °C	60
Lentil	Wet extrusion between 140–180 °C.	> 93.0
Ultrafiltration	Garbanzo	Filter at 50 kDa	9.47	Selective method, with no toxic residues on the final product.	Energy demand.
Ultrasound	Soy	20 kHz, 3.3 s pulses for 20 min	55	Selective method.	Energy demand.
High hydrostatic pressure	Soy	550 MPa, 65 °C for 15 min (prior treatment with 0.5% bicarbonate of sodium)	76	Prolongs shelf life.	Must be combined with other methods for peak effectiveness.
Common bean	600 MPa and 60 °C for 60 min	84	Conserves and improves organoleptic characteristics.
Peas	600 MPa at 60 °C.	3.1	Reduces phytic acid and lectin content, maintaining thermolabile compounds.
Gamma radiation	Soy	8 kGy	38.7	Improves isoflavone, phenol and anthocyanin content.	Reduced vitamin C.
Soy	5 kGy	63.3
Soaking	Soy	96 h	35,0	Simple and profitable.	Long process
Black-eyed peas	22 h at room temperature, sample: water of (p/v)	18.2	Loss of water-soluble proteins and other components including minerals.
Common bean	2 h at room temperature, sample: water of 1:5 (p/v)	19.4
Peas	2 h at room temperature, sample: water of 1:5 (p/v)	19.8
Acids and bases	Soy	NaOH of 1 % at 74 °C for 15 min	63	Decreases required TI inactivation temperature.	If not processed correctly, chemical substances can remain in the final product.
Germination	Black bean	3 days at 25 °C	88.2	No energy demand.	Long process, low inactivation output
Garbanzo	3 days at 25 °C in darkness	34	Improves natural compound content.
Common bean	5 days at 25 °C in darkness	19.2
Fermentation	Common bean	Lactobacillus fermentum at 37 °C for 3 days	38	Fat content reduced.	Long process
Soy	Aspergillus oryzae for 5 days	89.2	Decreased phytic acid content.
Soy	Lactobacillus plantarum for 5 d	99.2	Scalable process.
Soy	Lactobacillus acidophilus for 2 d	82.6	No excessive energy requirements.

Table 1. Extant technologies for deactivating trypsin inhibitors in legumes

Method

Raw materials

Treatment conditions

Trypsin inhibitor inactivation (%)

Advantages

Disadvantage

Source: Adapted from Avilés-Gaxiola et al. 2018.

Thermal treatment

Peas

Microwaves (2,450 MHz, 4 min)

100

High efficiency.

Energy demand.

Mung bean

Boiling (100 °C for 90 min)

100

Standardized process.

Reduced solubility.

Garbanzo

Soaked cooked seeds (95 °C for 1 h)

88.4

Scalable process.

Decreased lysine, tryptophan, and sulfurous amino acids.

Lentil

Cooking (80 °C for 1 min)

95.6

Increases aromatic amino acids.

Mineral loss (Na, Ca and Mg).

Soy

Baking (200 °C for 20 min).

67.3

Improves gelling capacity.

Reduced B vitamins.

Extrusion

Peas

Prior cooking and humidifying processes at 120 °C, opening nozzle 55 mm, velocity 380 rpm.

58.9

Reduces total phytate and tannin levels

Reduces emulsion activity.

Garbanzo

Prior cooking and humidifying processes at 120 °C, nozzle diameter 55 mm, velocity 380 rpm.

91.8

Raises protein digestibility.

Pre-processing may be needed.

Fava

Prior cooking and humidifying processes at 120 °C, nozzle diameter 55 mm, velocity 380 rpm.

53.7

Soy

Dry extrusion at 150 °C

Soy

Wet extrusion at 150 °C

Lentil

Wet extrusion between 140–180 °C.

> 93.0

Ultrafiltration

Garbanzo

Filter at 50 kDa

9.47

Selective method, with no toxic residues on the final product.

Energy demand.

Ultrasound

Soy

20 kHz, 3.3 s pulses for 20 min

Selective method.

Energy demand.

High hydrostatic pressure

Soy

550 MPa, 65 °C for 15 min (prior treatment with 0.5% bicarbonate of sodium)

Prolongs shelf life.

Must be combined with other methods for peak effectiveness.

Common bean

600 MPa and 60 °C for 60 min

Conserves and improves organoleptic characteristics.

Peas

600 MPa at 60 °C.

3.1

Reduces phytic acid and lectin content, maintaining thermolabile compounds.

Gamma radiation

Soy

8 kGy

38.7

Improves isoflavone, phenol and anthocyanin content.

Reduced vitamin C.

Soy

5 kGy

63.3

Soaking

Soy

96 h

35,0

Simple and profitable.

Long process

Black-eyed peas

22 h at room temperature, sample: water of (p/v)

18.2

Loss of water-soluble proteins and other components including minerals.

Common bean

2 h at room temperature, sample: water of 1:5 (p/v)

19.4

Peas

2 h at room temperature, sample: water of 1:5 (p/v)

19.8

Acids and bases

Soy

NaOH of 1 % at 74 °C for 15 min

Decreases required TI inactivation temperature.

If not processed correctly, chemical substances can remain in the final product.

Germination

Black bean

3 days at 25 °C

88.2

No energy demand.

Long process, low inactivation output

Garbanzo

3 days at 25 °C in darkness

Improves natural compound content.

Common bean

5 days at 25 °C in darkness

19.2

Fermentation

Common bean

Lactobacillus fermentum at 37 °C for 3 days

Fat content reduced.

Long process

Soy

Aspergillus oryzae for 5 days

89.2

Decreased phytic acid content.

Soy

Lactobacillus plantarum for 5 d

99.2

Scalable process.

Soy

Lactobacillus acidophilus for 2 d

82.6

No excessive energy requirements.

**Table 2.** Trypsin inhibitors’ effects as anti-nutrients
Sample	Trypsin inhibitor	Study type	Time	Dose	Result	Reference
TIA: Trypsin inhibition activity, U: Units of trypsin inhibition activity, TI: trypsin inhibitor, BBI: Bowman-Birk Inhibitor.
Unheated soy flour extract (1); TI-free soy flour extract (2)	AIT 1 = 125.1 U/mg protein; AIT 2 = 12.9 U/mg protein	In vivo: Male rats, 21 days old	20 days	Around 10% of the diet was soy flour extract	Less growth in the group of rats fed with unheated soy flour extract.	Kakade et al. (1973)
Pea flour 1 (H1); Pea flour 2 (H2); Pea flour 3 (H3); Pea flour 4 (H4)	AIT H1 = 1.5 U/mg dry material; AIT H2 = 8.7 U/mg dry material; AIT H3 = 1.8 U/mg dry material; AIT H4 = 7.4 U/mg dry material	In vivo: Rats	11 days	10 g dry matter + 150 mg of N	Direct relation between inhibiting activity, decreased protein digestibility, and biological value of protein.	Hedemann et al. (1999)
Raw soy flour	Not indicated	In vivo: Male Wistar rats	36 weeks	–	No symptoms present at 24 weeks, but pancreatic cancer emerged at 36 weeks.	McGuinness et al. (1987)
Fat-free soy flour: 1. Raw; 2. Toasted; 3. Overtoasted; Isolated soy protein; 1. Raw, high in TI; 2. Raw, high in TI; 3. Raw, medium TI; 4. Raw, low in TI; 5. Heated, low in IT; 6. Heated, low in IT	Fat-free soy flour: 1. 57.7 mg/g protein; 2. 16.0 mg/g protein; 3. 9.3 mg/g protein; Isolated soy protein; 1. 35.8 mg/g protein; 2. 29.4 mg/g protein; 3. 20.4 mg/g protein; 4. 7.0 mg/g protein; 5. 6.1 mg/g protein; 6. 3.2 mg/g protein	In vivo: Male Wistar rats, 21 days old	28 days	10 and 30% of protein in diet	Diets with higher TI concentrations increased pancreatic pathology rates.	Gumbmann et al. (1986)
Soy trypsin inhibitor	Not indicated	In vivo: Male Sprague-Dawley rats, 14 days old	14 weeks	1.0 g/100 g food + 9 or 30 µg Zinc	Regardless of dietary zinc level, diets with soy TI caused pancreatic hyperplasia and/or hypertrophy.	Ellwood et al. (1994)
Raw and toasted soy	Not indicated	In vivo: Golden hamster, 6 weeks old	28 days	Diet with 55% raw or toasted soy flour	Short-term trophic effects appeared in the pancreas (significant pancreatic weight increases). Raw soy did not favor pancreatic cancer development in treated hamsters.	Herrington (1994)
Bowman-Birk Inhibitor	Not indicated	In vivo: Humans, 15 men and 6 women, ages between 19 and 37 years	55 min	4 mg of BBI/mL of pancreatic juice	Inhibition of around 95% of trypsin and chymotrypsin activities. BBI doubled or tripled production of trypsin, chymotrypsin, elastase and amylase enzymes.	Liener et al. (1988)

Table 2. Trypsin inhibitors’ effects as anti-nutrients

Sample

Trypsin inhibitor

Study type

Time

Dose

Result

Reference

TIA: Trypsin inhibition activity, U: Units of trypsin inhibition activity, TI: trypsin inhibitor, BBI: Bowman-Birk Inhibitor.

Unheated soy flour extract (1); TI-free soy flour extract (2)

AIT 1 = 125.1 U/mg protein; AIT 2 = 12.9 U/mg protein

In vivo: Male rats, 21 days old

20 days

Around 10% of the diet was soy flour extract

Less growth in the group of rats fed with unheated soy flour extract.

Kakade et al. (1973)

Pea flour 1 (H1); Pea flour 2 (H2); Pea flour 3 (H3); Pea flour 4 (H4)

AIT H1 = 1.5 U/mg dry material; AIT H2 = 8.7 U/mg dry material; AIT H3 = 1.8 U/mg dry material; AIT H4 = 7.4 U/mg dry material

In vivo: Rats

11 days

10 g dry matter + 150 mg of N

Direct relation between inhibiting activity, decreased protein digestibility, and biological value of protein.

Hedemann et al. (1999)

Raw soy flour

Not indicated

In vivo: Male Wistar rats

36 weeks

–

No symptoms present at 24 weeks, but pancreatic cancer emerged at 36 weeks.

McGuinness et al. (1987)

Fat-free soy flour: 1. Raw; 2. Toasted; 3. Overtoasted; Isolated soy protein; 1. Raw, high in TI; 2. Raw, high in TI; 3. Raw, medium TI; 4. Raw, low in TI; 5. Heated, low in IT; 6. Heated, low in IT

Fat-free soy flour: 1. 57.7 mg/g protein; 2. 16.0 mg/g protein; 3. 9.3 mg/g protein; Isolated soy protein; 1. 35.8 mg/g protein; 2. 29.4 mg/g protein; 3. 20.4 mg/g protein; 4. 7.0 mg/g protein; 5. 6.1 mg/g protein; 6. 3.2 mg/g protein

In vivo: Male Wistar rats, 21 days old

28 days

10 and 30% of protein in diet

Diets with higher TI concentrations increased pancreatic pathology rates.

Gumbmann et al. (1986)

Soy trypsin inhibitor

Not indicated

In vivo: Male Sprague-Dawley rats, 14 days old

14 weeks

1.0 g/100 g food + 9 or 30 µg Zinc

Regardless of dietary zinc level, diets with soy TI caused pancreatic hyperplasia and/or hypertrophy.

Ellwood et al. (1994)

Raw and toasted soy

Not indicated

In vivo: Golden hamster, 6 weeks old

28 days

Diet with 55% raw or toasted soy flour

Short-term trophic effects appeared in the pancreas (significant pancreatic weight increases). Raw soy did not favor pancreatic cancer development in treated hamsters.

Herrington (1994)

Bowman-Birk Inhibitor

Not indicated

In vivo: Humans, 15 men and 6 women, ages between 19 and 37 years

55 min

4 mg of BBI/mL of pancreatic juice

Inhibition of around 95% of trypsin and chymotrypsin activities. BBI doubled or tripled production of trypsin, chymotrypsin, elastase and amylase enzymes.

Liener et al. (1988)

**Table 3.** Trypsin inhibitors’ effects as bioactive compounds
Sample	Study type	Time	Dose	Result	Reference
KTI: Kunitz-type trypsin inhibitor, BBI: Bowman-Birk inhibitor, uPA: urokinase plasminogen activator, UIC: Units of chymotrypsin activity.
Purified Kunitz and Bowman-Birk type soy inhibitors	Cell culture (human ovarian cancer cell line HRA)	24 h	0 - 10 µM	KTI, but not BBI, could inhibit cell impassivity, at least via suppression of the signal cascade from uPA.	Kobayashi et al. (2004a)
Bowman-Birk black soy inhibitor	Cell culture (human nasopharyngeal carcinoma cell lines CNE-2 and HNE-2)	-	0.71 µg/mL	KTI presented inhibiting activity against the inverse transcriptase from HIV-1, with immunostimulant activity and inhibition of tumor cell growth.	Fang et al. (2010)
Kunitz-type black soy inhibitor	Cell culture (Breast cancer MCF-7 cells and hepatoma HepG2 cells)	-	35 µM	BBI from black soy presented anti-proliferation activity against breast cancer cells and hepatoma cells, as well as inhibiting HIV-1 inverse transcriptase.	Ho and Ng (2008)
BBI	In vivo: Humans (men and women over 21 yrs)	16 weeks	8000 UIC/day	Soy extract could be associated with disease regression in ulcerative colitis patients without apparent toxicity or adverse side effects.	Lichtenstein et al. (2008)
Interalpha inhibitor (KTI)	In vitro using serum	-	3.5 % of protein in serum	Possible anti-inflammatory activity, relevant in local inflammation sites	Okroj et al. (2012)
Inter-α-trypsin inhibitor	In vivo: Mice	-	100 µg/mL	Decreases tissue inflammation in a murine pulmonary lesion model	Garantziotis et al. (2007)
Urinastatin	In vivo: Humans	14 days	150 000 U	Protects against cisplatin-induced nephropathy.	Umeki et al. (1989)
Soy trypsin inhibitors	In vivo: Humans (women)	60 min	60 mg IBB or 180 mg KTI	Protects the pancreas and the pancreatic conduct from premature trypsinogen activation.	Reseland et al. (1996)
Aprotinin (KTI)	In vivo: Sheep	3 h	280 mg	No reduction of peri-operational bleeding measured by drainage or hemoglobin loss	Ohri et al. (2001)
Bikunin	In vivo: 7-week rats	7 days	30 mg/kg	Possible anti-metastatic activity in humans	Kobayashi et al. (2004b)
Trypsin inhibitor of soy + genistein	In vivo: 7-week rats	2 h	100 mg/kg	Anti-inflammatory activity	Sadeghalvad et al. (2019)
Bowman-Birk soy inhibitor	Cell culture (Normal human prostate epithelial cells (PrEC), 267B1, BRF-55T, 267B1/Ki-ras, LNCaP, and PC-3 cells)	8 days	100 µg/mL	May be a useful agent for treating prostate ailments.	Kennedy and Wan (2002)

Table 3. Trypsin inhibitors’ effects as bioactive compounds

Sample

Study type

Time

Dose

Result

Reference

KTI: Kunitz-type trypsin inhibitor, BBI: Bowman-Birk inhibitor, uPA: urokinase plasminogen activator, UIC: Units of chymotrypsin activity.

Purified Kunitz and Bowman-Birk type soy inhibitors

Cell culture (human ovarian cancer cell line HRA)

24 h

0 - 10 µM

KTI, but not BBI, could inhibit cell impassivity, at least via suppression of the signal cascade from uPA.

Kobayashi et al. (2004a)

Bowman-Birk black soy inhibitor

Cell culture (human nasopharyngeal carcinoma cell lines CNE-2 and HNE-2)

0.71 µg/mL

KTI presented inhibiting activity against the inverse transcriptase from HIV-1, with immunostimulant activity and inhibition of tumor cell growth.

Fang et al. (2010)

Kunitz-type black soy inhibitor

Cell culture (Breast cancer MCF-7 cells and hepatoma HepG2 cells)

35 µM

BBI from black soy presented anti-proliferation activity against breast cancer cells and hepatoma cells, as well as inhibiting HIV-1 inverse transcriptase.

Ho and Ng (2008)

BBI

In vivo: Humans (men and women over 21 yrs)

16 weeks

8000 UIC/day

Soy extract could be associated with disease regression in ulcerative colitis patients without apparent toxicity or adverse side effects.

Lichtenstein et al. (2008)

Interalpha inhibitor (KTI)

In vitro using serum

3.5 % of protein in serum

Possible anti-inflammatory activity, relevant in local inflammation sites

Okroj et al. (2012)

Inter-α-trypsin inhibitor

In vivo: Mice

100 µg/mL

Decreases tissue inflammation in a murine pulmonary lesion model

Garantziotis et al. (2007)

Urinastatin

In vivo: Humans

14 days

150 000 U

Protects against cisplatin-induced nephropathy.

Umeki et al. (1989)

Soy trypsin inhibitors

In vivo: Humans (women)

60 min

60 mg IBB or 180 mg KTI

Protects the pancreas and the pancreatic conduct from premature trypsinogen activation.

Reseland et al. (1996)

Aprotinin (KTI)

In vivo: Sheep

3 h

280 mg

No reduction of peri-operational bleeding measured by drainage or hemoglobin loss

Ohri et al. (2001)

Bikunin

In vivo: 7-week rats

7 days

30 mg/kg

Possible anti-metastatic activity in humans

Kobayashi et al. (2004b)

Trypsin inhibitor of soy + genistein

In vivo: 7-week rats

2 h

100 mg/kg

Anti-inflammatory activity

Sadeghalvad et al. (2019)

Bowman-Birk soy inhibitor

Cell culture (Normal human prostate epithelial cells (PrEC), 267B1, BRF-55T, 267B1/Ki-ras, LNCaP, and PC-3 cells)

8 days

100 µg/mL

May be a useful agent for treating prostate ailments.

Kennedy and Wan (2002)