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Effectiveness inactivation of trypsin inhibitor from Brazilian cultivars of beans (Phaseolus vulgaris L.)/Eficacia da inativacao do inibidor de tripsina em cultivares brasileiras de feijao (Phaseolus vulgaris L.).

INTRODUCTION

The beans seeds of Phaseolus vulgaris L. are important source of protein, dietary fiber, fat, starch, flavonoids and vitamin for human diet. (8,11,12,15, 21, 23, 26) In spite of these favorable features, the bean also contains antinutritional factors that are not good for human and animal nutrition, such as trypsin inhibitors, lectin, tannins, and phytate. (3,11,12,32) In addition, feed trypsin inhibitor can reduce nutrient uptake, animal growth (14,16,31) and is considered an effective pancreatic hypertrophy agent. (13,16,17,31) On the other hand, it is well established that protease inhibitors can prevent cancer. (9,20,28)

Although trypsin inhibitor activity has been partially or fully inactivated by different treatments (18) and the inactivation process might be attributed to formation of high molecular weight aggregated among trypsin inhibitor and others proteins, (7,8) beans nutritional value have been improved by thermal inactivation treatment, (8,11,18) highpressure, (31) extrusion cooking, (21) natural fermentation, (15) pressure-cooking (121[degrees]C.30 [min.sup.1]) or germination. (12) In addition, it has been shown that purified inhibitor is resistant to inactivation (24, 29) and trypsin inhibitor purified from rice lost its activity after 2 hours of in vitro digestion with pepsin and pancreatin. (27) An important point that remains inconclusive is that if this protein is resistant to inactivation and become active into digestive systems of humans and animals.

Due to the importance of animal and human nutrition, many investigations have been carried out to develop treatments in order to reduce or even remove the content of antinutritional factors and enhance the nutritional quality. These changes differ widely depending on the technology and conditions involved. (15)

Therefore, the study of tripsyn inhibitor reactivation from Phaseolus vulgaris L. submeted to pressure-cooking might provide new information about efficiency of thermal treatment, or necessity of a combination of several treatments.

In this paper we report the trypsin inhibitors concentration in ten cultivars of Phaseolus vulgaris and the study of the trypsin inhibitors reactivation using double-digestive pepsin-pancreatin in vitro of bean grains samples submitted to pressure-cooking. The degree of hydrolysis and amino acid composition were performed in the IACArua cultivar due to higher recovery of inhibitor activity after digestion.

MATERIAL AND METHODS

Bean Cultivars

Ten Brazilian varieties of Phaseolus vulgaris L. namely: IAPAR-14; IAC-Carioca; Ruda; Corrente, IAC-Arua, IAPAR-16, IAPAR-57, IAC-Carioca Pyata, Carioca, Apore were chosen for this study.

Sample Preparation

Bean grains were soaked in distilled water (1:5 p:v) for 12 hours under laboratory conditions at 26[+ or -]5[degrees]C and submitted to pressure-cooking at 121[degrees]C and 141 kPa for 30 min, according established previously by Carvalho et al. (8) After cooling, the erlenmeyer was immersed in crushed ice, dried in an oven with air flow forced circulation at 60[degrees]C for 18 hours and grounded into a fine powder using a grinder. The powder was sieved through a screen and stored at -20[degrees]C. Flour bean without pressured-cooked was included in each experiment to quantify the inactivation degree of trypsin inhibitor in each varieties cultivars.

Crude Extract

The trypsin inhibitors extraction was carried out at 26[+ or -]5[degrees]C Heat treated and untreated samples were homogenized (1:50 w.[v.sup.-1]) with volumes of 0.025M HCl, with starring for 1h filtered in Whatman no. 2 paper and then the filtrate was used in the enzyme inhibition assay.

Assay of Inhibitory Activity

Inhibitory trypsin activity was assayed discontinuously, at 37[degrees]C, in a Hitachi U-1000 spectrophotometer by following the liberation of p-anilide at 410nm. The reaction was initiated by addition of the substrate and stopped with 0.1mL of 30% acetic acid. Standard conditions were 0.1mL the trypsin (0.11mg.[mL.sup.-1]. in 0.001N HCl), after 10 min of incubation 0.7mL 1mM BAPNA (N-benzoyl-DL-arginine p-nitroanilide) previously prepared in 50mM Tris.HCl buffer, pH 8.2, containing 20mM Ca[Cl.sub.2] in a final volume of 1.0mL. (19) The inhibitory activity was measured by the difference in enzyme activity with and without the inhibitor presence. One trypsin unit (TU) was defined as an increase of 0.01 unit of absorbance at 410nm by 100mL of reaction medium. The results were expressed as Units Trypsin Inhibited (UTI) per milligram of sample.

In vitro protein digestibility and inhibitor reactivation

In vitro hydrolysis of raw and pressure-cooking cultivar extracts was carried out as described by Akerson & Stahman (2) using a double-digestive pepsin-pancreatin. After hydrolysis, proteins that were not hydrolyzed were precipitate with TCA, centrifuged at 12.000xg for 20 min, at 4[degrees]C, filtrated at Watman no. 2 paper and used for determination of digestibility and assay of inhibitor reactivation.

The protein was measured using the semimicro-Kjeldahl procedure and expressed as percent of the total N, by using the following formula:

Were: dN - nitrogen; aN - auto digestible nitrogen; - tN - sample total nitrogen.

Limited proteolysis with pepsin-pancreatin

Raw and pressure-cooking protein samples of IAC-Arua were incubated using double-digestive pepsinpancreatin. (2) The hydrolyzed protein was analyzed measuring the amount of a-amine groups liberated with TNBS (1) using L-leucine as standard.

Gel filtration chromatography

Raw and pressure-cooking extract of IAC-Arua cultivar were fractionated on a 1.50 x 120cm Sephadex G-75 chromatography column, equilibrated and eluted with 50mM Tris.HCl buffer, pH 7.5. Fraction of 4.2mL was collected and protein 280nm and trypsin inhibitor activity assayed. The fraction with activity were pooled, frozen in liquid nitrogen and stored at -20[degrees]C for a period of one month without appreciable loss of activity.

Amino acid composition

Proteins from IAC-Arua treated and untreated were hydrolyzed with 6N HCl at 110[degrees]C for 24 hours. (22) The amino acid composition was determined in a Dionex DX 300 Ion Chromatography System by using a cation exchange column and a ninhydrin derivation post-column. The amino acid standard was used to determine the samples amino acid concentrations. Yearly average and standard deviations were determined for each amino acid. Alls determination was carried out at Protein Chemistry Center of USP-Ribeirao Preto

RESULTS AND DISCUSSION

Protein digestibility was investigated in ten bean cultivars of treated and untreated Phaseolus vulgaris L. As shown at Table 1, there was a significant difference (p<0.01) among digestible protein that ranged from 40% (Carioca) to 60% (IAC-Arua). These results were accordingly to Carvalho et al. (8) who reported values from 47% for cultivar OC and 59% for Parana. Similar value for protein digestibility (36.3-56%) has also been reported by Salunke & Kadam. (25) On the other hand, these values were lower than those reported (65.6-80.7%) by different cultivars of Phaseolus vulgaris growth in difference countries. (15,26,30) These variations in beans digestibility can be influenced by specie, cultivar and environmental conditions. (10,11,18,26)

When compared to other food proteins, the beans have low digestibility due to their resistance to enzymatic hydrolysis.

The pressure-cooking at 121[degrees]C for 30 min increased digestible protein in vitro (P<0.01) from 11 to 37%. These results are quite similar to that reported by Carvalho et al., (8) in which digestibility at some conditions showed an increase of 32.6% using the heating process.

There was a significant difference of trypsin inhibitor (P<0.001) among cultivars studied. The IAC-Arua (Table 2) shows 36.18UTI.[mg.sup.-1] while IAPAR-16 for 63.33UTI. [mg.sup.-1]. The medium value for trypsin inhibitor widespread in almost varieties is about 46UTI.[mg.sup.-1]. Was found in other cultivars of bean growth in different countries. (5,8,11,15,18,26) The values of trypsin inhibitor harmful to animals are not yet fully known. So, cultivars with trypsin inhibitor with a lower activity and/or more susceptible to inactivation is expected to increase the nutritional quality of grains. Although trypsin inhibitor activity was totally inactivated by pressure-cooking at 121[degrees]C for 30 min, there was a significant difference (P<0.001) in trypsin inhibitors reactivation after pressure-cooking and incubating in vitro using a double digestive pepsin-pancreatin (Table 2). The recovering ranged from 19.24UTI/mg for IACCarioca Pyata to 36.12UTI/mg for IAC-Arua. According to Roychaudhuri et al. (24) the reversibility or not of thermal denaturation of trypsin inhibitor depends on the rate of the renaturation.

The reactivation studies showed an average recovering percentage of 68.22%. The highest value observed was for IAC-Arua (100%) following by the IAPAR-14, with efficiency of 85% as well as by Jourdan et al. (18) for inactivation of trypsin inhibitor activity from Brazilian varieties of beans. This observation is in agreement with Carvalho et al. (7) and has been interpreted as non specific interactions among inhibitor with other grain components, but not due to inhibitor thermal inactivation. Taken together, these results show that inhibitors of digestive enzymes are highly resistant to denaturation by thermal treatment and its activity is restored during the digestion process. Since it remains to be clarified if the reactivation of trypsin inhibitor alter the nutritional value and shows an adverse toxic effect on human, treatment to reduce or even remove the antinutritional factors of bean have been looking forward. (15,21,31)

The amino acids composition of raw and pressurecooking IAC-Arua (Table 3) was similar to those reported for different cultivars of Phaseolus vulgaris. (8, 23) This cultivar showed high levels of aspartic acid, glutamic acid, lysine, proline, leucine but is limiting in sulfur amino acids. The values obtained for methionine (0.62), cystein (0.37) and for lysine (7.43) of raw and autoclaved samples were not significantly modified by treatments. Despite its low content in sulphur amino acids, the presence of high level of lysine suggest that bean can be used as a good complement of those cereals deficient in lysine. (4)

The amino acid content, expressed as mile moles of amino acids.[gram.sup.-1] of proteins (htot) was not significantly modified by treatment which is 8.31 in the raw and 8.27mM.[g.sup.-1] protein to the treated samples, respectively (Table 3), and are in accordance to those reported by Candido (6) who found htot of 8.0mM.[g.sup.-1] protein.

In spite of both raw and pressure-cooking IAC-Arua beans have showed similar hydrolysis degree by pepsin at least for 180 minutes (Figure 1), the pressure-cooking at 121[degrees]C for 30 min increased the susceptible to proteolysis by pancreatin and the digestibility increases from 59% to 63%. It should be stressed that trypsin inhibitor activity was not modified by autoclaving at 121[degrees]C for 30 min, and was quite similar to those reported for crude extracts. However, these results do not exclude unequivocally the possibility that the inhibitor might be structurally modified without loss of its activity. Since it activity depend of native configuration, only after structural studies is possible determine whether there are changes in the native configuration of the inhibitor.

Chromatography on Sephadex G-75 column (Figure 2) of raw extracts and of the samples pressure-cooking (121[degrees]C and 141kPa) for 30 min and treated with a double-digestive pepsin-pancreatin showed that inhibitor of crude extract was eluted at fraction of 127mL. On the other hand, when the treated sample was chromatographed under the same conditions, the inhibitor was eluted at 176mL volume. Our results shows that the native inhibitor is resistant to proteolysis with double-digestive pepsinpan-creatin, whereas pressure-cooking at 121[degrees]C for 30 min results in a non-native conformation that is significantly susceptible to proteolysis by both enzymes, improving the digestibility and inactivate differentially the activity of trypsin inhibitors. The results of the thermal treatment of the beans show inactivation of the inhibitors, which may be due to formation of high molecular weight aggregates with other substances of the grain. The pepsin-pancreatin digestion of the inactivated inhibitor restores the activity, probably due to its retention by the digested fragments.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

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Recebido em: 13/10/2010

Aprovado em: 21/06/2011

Kelli Cristina PAIVA **

Maria Regina Barbieri de CARVALHO ***

Joao Martins PIZAURO JUNIOR ***

* Financial support by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP.

** Post-graduated in Food & Nutrition - College of Pharmaceutical Sciences - Sao Paulo State University - UNESP - 14801-902 - Araraquara - SP - Brazil.

*** Department of Technology - College of Agricultural and Veterinarian Science - Sao Paulo State University - UNESP - 14884-900 - Jaboticabal - SP - Brazil. E-mail: mrbcar@fcav.unesp.br.
Table 1--Digestible protein in vitro (%) of raw and autoclaved beans
of ten cultivars of Phaseolus vulgaris.

    Cultivars              Raw beans                Autoclaved
                                                       beans

      IAPAR         55,0 [+ or -] 1,4 (ab)    61,0 [+ or -] 2,7 (bcd)
   IAC-Carioca      49,0 [+ or -] 4,2b (cd)   62,0 [+ or -] 1,4 (bcd)
      Ruda          53,0 [+ or -] 2,8 (abc)   58,0 [+ or -] 4,0 (cd)
    Corrente        53,0 [+ or -] 2,8 (abc)   60,0 [+ or -] 1,6 (cd)
    IAC-Arua         60,0 [+ or -] 4,2 (a)     73,0 [+ or -] 2,8 (a)
    IAPAR-16        53,0 [+ or -] 2,8 (abc)   60,0 [+ or -] 1,6 (cd)
    IAPAR-57        48,0 [+ or -] 4,2 (bcd)   63,0 [+ or -] 2,5 (bc)
IAC-Carioca Pyata   56,0 [+ or -] 1,4 (ab)    68,0 [+ or -] 2,4 (ab)
     Carioca         40,0 [+ or -] 3,0 (d)     55,0 [+ or -] 3,0 (d)
      Apore         45,0 [+ or -] 4,0 (cd)    56,0 [+ or -] 1,4 (cd)
        F                  10,17 **                   14,51**
       CV                    6,16                      4,02
       DMS                   9,12                      7,15

    Cultivars       % of digestibility
                       autoclavated

      IAPAR                11,0
   IAC-Carioca             27,0
      Ruda                 13,0
    Corrente               13,0
    IAC-Arua               22,0
    IAPAR-16               13,0
    IAPAR-57               31,0
IAC-Carioca Pyata          21,0
     Carioca               37,0
      Apore                24,0
        F                   -
       CV                   -
       DMS                  -

All values are means of triplicate [+ or -] standard deviation.

**- means with different superscript within a column indicate
statistically significant differences (P<0.01).

Table 2--Trypsin inhibitor activity (UTI.[mg.sup.-1]) contente of
raw and autoclaved and subject to proteolysis double-digestive
pepsin-pancreatin of ten cultivars of Phaseolus vulgaris.

    Cultivars          Raw          ASP       Recovery (%)

      IAPAR         38,18 (de)   32.42 (b)         85
   IAC-Carioca      44,00 (d)    33,08 (b)         76
      Ruda          44,08 (cd)   26,00 (c)         59
    Corrente        41,31 (de)   34,00 (ab)        82
    IAC-Arua        36,18 (e)    36,12 (a)        100
    IAPAR-16        63,33 (b)    32,49 (b)         51
    IAPAR-57        55,12 (b)    32,03 (b)         58
IAC-Carioca Pyata   38,32 (b)    28,74 (d)         75
     Carioca        56,47 (de)   19,24 (bc)        34
      Apore         50,30 (bc)   23,91 (cd)        47
        F            54,12 **     25,43 **         -
       CV              2,16         1,95           -
       DMS             6,24         5,63           -

**- means with different superscript within a column indicate
statistically significant differences (P<0.01).

ASP = autoclaved and subject to proteolysis.

Table 3--Amino acids composition of raw and autoclaved beans of
IAC-Arua.

  Amino acids                      Raw beans

                  g.100[g.sup.-1]prot.   m mM.[g.sup.-1] prot.

      ASP                14,74                   1,11
      TRE                 4,62                   0,39
      SER                 7,18                   0,68
      GLU                23,36                   1,59
      PRO                 7,83                   0,68
      GLY                 4,62                   0,62
      ALA                 5,00                   0,56
      CYS                 0,37                   0,03
      VAL                 3,62                   0,31
      MET                 0,62                   0,04
      ILE                 2,44                   0,19
      LEU                 7,25                   0,55
      TYR                 3,37                   0,19
      PHE                 5,56                   0,34
      LYS                 7,43                   0,51
      HIS                 2,69                   0,17
      ARG                 5,68                   0,33
      TRP                 0,37                   0,02
[H.sub.tot] (1)            -                     8,31

  Amino acids                 Autoclaved beans

                  g.100[g.sup.-1] prot.   mM.[g.sup.-1] prot.

      ASP                 13,52                  1,02
      TRE                 4,39                   0,37
      SER                 6,55                   0,62
      GLU                 22,66                  1,54
      PRO                 7,51                   0,65
      GLY                 6,91                   0,92
      ALA                 4,45                   0,50
      CYS                 0,84                   0,07
      VAL                 3,61                   0,31
      MET                 0,60                   0,04
      ILE                 2,41                   0,18
      LEU                 6,97                   0,53
      TYR                 3,79                   0,21
      PHE                 5,29                   0,32
      LYS                 7,09                   0,49
      HIS                 2,58                   0,17
      ARG                 5,41                   0,31
      TRP                 0,36                   0,02
[H.sub.tot] (1)             -                    8,27

(1) mile moles of amino acids.[gram.sup.-1] of proteins.
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Author:Paiva, Kelli Cristina; de Carvalho, Maria Regina Barbieri; Pizauro, Joao Martins, Jr.
Publication:Alimentos e Nutricao (Brazilian Journal of Food and Nutrition)
Date:Jul 1, 2011
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