ORIGINAL PAPER

Improvement of Protein Bioavailability by Solid-State
Fermentation of Babassu Mesocarp Flour and Cassava Leaves

E. M. Morales1 • R. N. Domingos2 • D. F. Angelis1

Received: 8 June 2016 / Accepted: 28 October 2016 / Published online: 19 November 2016

� Springer Science+Business Media Dordrecht 2016

Abstract

Purpose The worldwide demand of food is continuously

increasing along with its selling price. Higher food costs

affect the access of poor communities to nutritious food, so

become a priority problem to be solved, according to the

United Nations. This study provides an alternative and

viable contribution to the enrichment of food for poor

communities using agricultural byproducts.

Methods Samples of a mixture of cassava leaves and

babassu mesocarp fermented in solid-state way with Rhi-

zopus oligosporus were submitted to evaluation of protein

quantity and quality, by the comparison of the digestibility

before and after the solid-state fermentation. The assess-

ment of essential amino acids, relative nutritional value and

protein digestibility-corrected amino acid score (PDCAAS)

estimated the quality of the protein.

Results The solid-state fermentation increased by 15.2%

the amount of crude protein and improved quality, resulting

in a food with relative nutritional value for 98.18%,

equating to the casein (100%). The PDCAAS of fermented

mixture (BMF ? 32% CLF) showed results similar to

beans and protein textured soybean (PTS) reported, indi-

cating a good source of amino acids (0.6037 to the fer-

mented mixture, 0.6296 for beans and 0.6481 to PTS). The

SSF also reduced by 94.18% the amount of cyanide present

in the mixture, which is a substance derived from the

cassava leaves.

Conclusion By the obtained results, it was observed that

the use of SSF in byproducts from agroindustry can pro-

duce more nutritious food for humans by the transforma-

tion of energetic food in structural food which has a higher

amount of proteins.

Keywords Relative nutritional value � Cassava leaves �
Babassu mesocarp � Rhizopus oligosporus � PDCAAS �
Amino acid

Introduction

Hunger and food wastage are (nowadays) the two biggest

problems that the world faces [1]. According to Gustavsson

et al. [2], there are approximately 25,000 deaths per day

related to starvation, 18,000 of which are children. On the

other hand, about 1.3 billion ton of food is wasted annually.

Nutritional deficiencies during childhood affect the child’s

development and lead to a high infant mortality. Additional

effects of malnutrition during this age cause injury in

psychomotor development, low educational performance

and low productive capacity in adulthood [3, 4].

According to the United Nations [5], the number of

undernourished people increased in developing countries

by 20 million between the years of 2000 and 2008. The UN

still claim that the malnutrition is directly linked to poverty

and social inequalities. This document mentions that, even

with the prospect of increased demand for food by 70%

until 2050, there is a slowdown in the ‘‘green revolution’’

in agriculture. However, the first aim proposed by the UN

to the millennium is eradicating extreme poverty and

& E. M. Morales

emorales@rc.unesp.br

1 Department of Biochemistry and Microbiology, Institute of

Bioscience, Univ. Estadual Paulista - UNESP, Avenida 24-A,

1515, Bela Vista, Rio Claro, SP 13506-900, Brazil

2 Pro-Rectory of Research, Fundação Universidade do

Tocantins, 108 Sul Alameda 11, Lote 3, Centro, Palmas,

TO 77123-360, Brazil

123

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hunger, since 27% of the world population still lives in

extreme poverty [5].

The use of alternative nutritional sources allows low-

income people have access to a balanced diet, with good

amounts of protein and other essential nutrients, without

the need for expansion of planted areas [6, 7]. Alternative

sources of food should present good sensory characteris-

tics, ease of production and desirable functional and

nutritional characteristics [8]. The most foods produced in

the world are of the energetic group, whose nutritional

quality is directly linked to the supply of energy for the

organism. However, the main reason for malnutrition is

associated with low protein intake. Thus, the conversion of

energetic food into another food with high protein levels

can contribute to an improvement in the diet, in relation

with protein intake.

Examples of common energetic foods (great amounts of

carbohydrates) are corn, wheat, rice, manioc, potatoes and

others. Babassu (Orbignya sp.) is an important plant for the

economy of the Northeast of Brazil, with an annual pro-

duction of approximately 20 billion coconuts. About 20%

of the coconut weight corresponds to the mesocarp.

Babassu mesocarp is a food widely used in the region, but

has only energetic characteristics [9–11], which makes it an

important source for use in SSF.

Mesocarp flour is a high-carb and low-protein supply

that is used as supplementary food for students of ele-

mentary school in some low-income communities from

Brazil, especially in the Northeast region [12, 13]. The

composition of babassu mesocarp is approximately 14.0%

of water, 64.7% of total carbohydrates, 2.3% of protein,

2.8% of lipids, 1.3% of ashes and 14.9% of other com-

pounds [14]. Likewise, other crops rich in carbohydrates

from various regions of the world can become a potential

substrate for Solid-State Fermentation (SSF) and thereby

increase the possibility of dietary protein enrichment.

Africa is the largest producer of cassava (Manihot

esculenta Crantz) led by Nigeria accounting for 50.5% of

this production, followed by Asia (33.7%) and South

America (15.2%). In the same period (2008), Brazil con-

tributed with 11.4% of world production, corresponding to

26.7 million tons [15]. It is estimated that about 2250 kg of

cassava leaves dry weight are produced per hectare [16].

Although cassava leaves present low digestibility index

caused by the large amount of fiber [17], they have high

protein, vitamin and mineral concentration but this poten-

tial is unexplored [18]. According to Oni et al. [19], cas-

sava leaves present 88.4–90.1% of moisture, 19.7–24.0%

of crude protein, 6.0–7.3% of lipids, 6.5–16.1% of ashes

and 54.0–65.7% of total carbohydrates. The same authors

report that 59.6–66.2% of the total carbohydrates are

related to fibers.

Materials of low digestibility may be modified by

microbial fermentation making them more suitable for

nutrition [20]. Solid-state fermentation is presented as a

simple procedures and low infrastructure cost that may be

successfully applied in cassava leaves to increase the

amount of digestible proteins derived from the mycelial

growth on plant fiber [20, 21]. During the fermentation,

cyanogenic compounds may be metabolized and at the

same time new organoleptic characteristics can be added to

the final product [22, 23]. The possible decrease of anti-

nutrients by fermentation is important, since the leaves

present some of them as hydrocyanic acid (113–292 ppm),

polyphenols (5.2–9.2 g 100 g-1), trypsin inhibitor

(1.9–2.8 ITU mg-1), saponin (1.7–3.6 g 100 g-1), oxalate

(2.2–2.9 g 100 g-1) and nitrate (&75.0 g 100 g-1) [24].

The fungus Rhizopus oligosporus is important eco-

nomically for the treatment of waste, the production of

enzyme and foods (such as tempeh) and inhibition of

Gram-positive bacteria [25–27].

The experiences of using R. oligosporus suggest that its

use is recommended for obtaining food with low fat con-

tent and high protein, high antioxidant activity and espe-

cially food producing free of toxins [28–30].

The aim of this study was to evaluate the efficiency of

SSF with fungus R. oligosporus to produce a food with

high nutritional value using mesocarp babassu and cassava

leaves.

Materials and Methods

Organism

Rhizopus microsporus var. oligosporus CCT 3762 (ATCC

22959) was acquired from Tropical Research and Tech-

nology Foundation ‘‘André Tosello’’, Campinas—SP,

Brazil. Cultures grown in Petri plates at 30 ± 2 �C for 72 h

in malt extract agar 5% were transferred to the 250 mL

Erlenmeyer flasks containing the substrate.

Substrates

Babassu Mesocarp Flour (BMF)

The Babassu Mesocarp Flour (BMF) was provided by the

Superintendency of Food and Life Quality (PROVIDA),

from State Secretariat of Labor and Social Assistance

(Secretaria Estadual do Trabalho e de Assistência Social—

SETAS) from the municipality of São Miguel do Tocan-

tins—TO, Brazil. The flour was packaged in PVC packs

and kept under refrigeration until ready to use.

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Cassava Leaves Flour (CLF)

Cassava leaves (CL)with 1–2 years oldwere harvested at the

UNESP, Campus Rio Claro, and SP, Brazil. Leaves were

sun-dried until to achieve the minimum of 70% dehydration,

according to Silva et al. [31]. After they were triturated in a

blender and stored in PVC packs under refrigeration.

Sample Preparation for Fermentation and Substrate

Moisture

To determine the best babassu mesocarp fermentation

conditions used in this study, different culture medium (4%

BMF, 4% of the mixture BMF ? CLF (9:1) and 4% CLF)

were mixed in Petri dishes containing agar. The mycelial

growth halo of this culture medium was measured after 48

and 96 h of incubation at 30 ± 2 �C.
The ideal moisture of the substrate was calculated by the

visualization of the growth of mycelia on these different

blends (BMF, BMF ? CLF and CLF) in different contents

of water. Samples with 40, 50, 55 and 60% of moisture

were analyzed to verify the proper condition to use during

the fermentation process.

Solid-state Fermentation (SSF)

The solid-state fermentation was performed by the prepa-

ration of five sets of five PVC packs (quintuples) con-

taining 30 g of each sample (substrate) as following

information: (1) BMF (pH 5.13); (2) 92% BMF ? 8% CLF

(pH 5.08); (3) 84% BMF ? 16% CLF (pH 5.07); (4) 68%

BMF ? 32% CLF (5.03); and (5) CLF (pH 4.86). No

corrections on pH of the substrates were made. The plastic

bags were stopped with a cotton stopper to allow the

change between the atmospheric gases. The substrates were

humidified to 57% and sterilized at 121 �C/5 min (1 atm)

followed by the inoculation with R. oligosporus

(7.59 9 107 spores/30 g of sample). The packs were

maintained at 30 ± 2 �C/3 days before all analyses of

fermented material.

Analyzed Parameters

Determination of Total Crude Protein

Quantification of total nitrogen was carried out (in tripli-

cate) by the Kjeldahl method [32], using the factor 6.25 to

convert to total crude protein.

Digestion

According Akeson and Stahman [33], the digestion was

performed in Erlenmeyer flasks 50 mL, adding 1 g of the

tested sample (BMF or BMF ? CLF or CLF) and 7.5 mL of

pepsin solution (2 mg mL-1 in HCl 0.1 M), followed by

incubation at 37 �C during 3 h. The samples were alkalin-

ized with 7.5 mL of NaOH 0.2 M, followed by addition of

3.75 mL of pancreatin solution (2 mg mL-1 in Phosphate

Buffer 0.1 M, pH 8) and incubated at 37 �C for 24 h.

Thereafter, the reaction was stopped with addition of 5 mL

of trichloroacetic acid (TCA) 30% (w/v) and the volume

adjusted to 25 mL with addition of TCA 5% (w/v). The

samples were centrifuged at 10,000 RPM at 4 �C for 30 min.

In Vitro Digestibility

The digestibility of each sample was calculated as the

difference between total protein measured by the amount in

the precipitate (non digested material) obtained after

digestion with pepsin and pancreatin, both determined by

the Kjeldahl method [32].

The precipitated material from each sample was sub-

mitted to drying in a similar method to the fermented

samples (54 ± 1 �C), in which the amount of protein was

quantified. The digestibility of the samples was expressed

as a percentage of solubilized proteins.

Determining the Relative Nutritional Value (RNV)

All the procedures were performed in this study according

Ford [34], as following. The assays to calculate the RNV

were performed in quadruplicate. The samples were previ-

ously degreased using n-hexane in a Soxhlet apparatus [32].

After, the samples and the casein were hydrolyzed under

stirring in 4 mL of pronase Sigma P-5147 (1 mg mL-1) and

40 mL of sodium glycerophosphate buffer (Sigma

G-5422–20 g L-1), adjusted to pH 8.0 (with NaOH 0.1 Mor

H3PO4 0.033 M) at 48 �C for 3 h. After hydrolysis, the pH

and the volume of the samples were adjusted to 7.2 and

100 mL respectively. In the sequence, the samples were

sterilized in filter 0.22 lm(cold sterilization). After, 2 mLof

the basal medium, 2 mL of the hydrolyzed sample, 7 mL of

sterilized water and 100 lL of Enterococcus zymogenes

inoculum were incubated in test tube at 37 �C for 48 h.

Subsequently, the samples were sterilized by autoclaving

(121 �C/15 min) and the growth measurements performed

by acidimetry. The titrating of each tube was performedwith

NaOH 0.01 M or H3PO4 0.0033 M. The value of the total

volume used in the test was calculated discounting the vol-

ume used for the titration of the blank (carried out with

inactive inoculum). The data obtained in the test with E.

zymogenes are compared with value of standard protein

(casein–SIGMA C-7078), considered 100%.

The determination of RNV was taken from the growth

of E. zymogenes, which occurs according to the quality of

the protein present in the sample [35]. The growth response

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is related to the absolute nutritional necessity of essential

amino acids (aa) by the microorganism as arginine, glu-

tamic acid, histidine, isoleucine, leucine, methionine,

tryptophan, valine and the lysine. Even the lysine not being

considered an essential aa to E. zymogenes, its deficiency

can impair the growth of the microorganism [34, 35]. As

the needs of aa of the microorganism are very similar to

human needs (histidine, isoleucine, leucine, lysine,

methionine, phenylalanine, threonine, tryptophan and

valine), this indirect method of evaluation of RNV presents

a good match between these both organisms, because the

present enzymes in the human gastrointestinal tract are the

same enzymes used in the hydrolysis of the sample.

Essential Amino Acids

Total amino acids quantification was performed in the

laboratory of the Institute of Food Technology, Campinas-

SP, according to the methods described by White et al. [36]

and Hagen et al. [37], excepted the tryptophan that was

determined by the method of Spies [38].

Protein Digestibility-Corrected Amino Acid Score

(PDCAAS)

The PDCAAS was obtained by the amino acid profile from

samples [39] corrected by the digestibility observed in vitro [35].

From the total crude protein values obtained by the

Kjeldahl method, it was possible to estimate the amount of

sample (BMF ? 32% CLF) containing 1 g of protein.

With this value, it was calculated the amount of each

essential amino acid for this amount of sample.

The amino acid ratio was individually calculated to His,

Thr, Val Met ? Cys, Ile, Leu, Phe ? Tyr, Lys, and Trp, by

dividing the amount of each amino acid of the sample

(corresponding to 1 g of protein) by the reference value of

the same amino acid for children 2–5 years, as defined by

FAO/WHO [40].

The PDCAAS value of BMF ? 32% CLF was calcu-

lated by multiplying the value of the amino acid found in

less quantity in the sample by the value of in vitro

digestibility of protein [41].

Cyanide Content

Total cyanide content was evaluated by Center of Tropical

Roots and Starches (CERAT—Centro de Raı́zes e Amidos

Tropicais), Botucatu, SP, Brazil.

Statistical Analyzes

The statistical significance was verified by one-way ana-

lyzes of variance (ANOVA) and subsequent application of

the t test at SigmaPlot 11.0, comparing the differences

between the samples (fermented and unfermented mate-

rial). Significant differences were understood at P\ 0.05.

Results

Preliminary experiments were performed to verify if the

presence of cassava leaf (CLF) in the substrate containing

just babassu mesocarp (BMF) would improve the devel-

opment of the fungus. For this test, was observed that the

inclusion of cassava leaf in the fermentation substrate

(BMF) provided more favorable conditions for fungal

growth. In the substrate made only with BMF, the fungus

growth was virtually zero during the first 48 h. After 96 h

only few hyphae were observed. In the plates with

BMF ? CLF and with CLF, halos sizes was from 4 cm in

diameter after 48 h and 7 cm after 96 h. In the plates

containing only CLF there was formation of a higher

density mycelial.

Furthermore, the relationship between the humidity of

the substrate and the fungus growth was also considered.

Tests for four different percentages of moisture (45, 50, 55

and 60%) were performed and the best growth response

was visually observed in tests carried out with 55–60%

humidity.

Considering that the addition of CLF in the substrate

with mesocarp promoted a higher growth of the R. oli-

gosporus, tests to evaluate the best concentration of CLF to

be incorporated into the BMF substrate were performed.

Five sets of different substrates with different quantities

of BMF and CLF were performed with the intent to eval-

uate which mixture would present the ideal results in

3 days. It was found that the addition of 32% CLF in BMF

resulted in a significant increase (P[ 0.05) in crude pro-

tein concentration (12.5%), after fermentation of 72 h long,

while samples containing only the BMF did not show

significant increase difference of crude protein (Table 1).

The samples BMF ? 8% CLF, BMF ? 32% CLF and

100% CLF that showed significant results (p\ 0.05) after

fermentation were submitted to biological assay for esti-

mating the Relative Nutritional Value (RNV) of their

proteins. The digestibility and RNV results before and after

fermentation, presented in Table 2, indicated that when

incorporated 32% of CLF in the BMF there was only

improvement in nutritional quality (RNV) when the mix-

ture was submitted to the solid-state fermentation process

(from 19.40 to 98.67). The digestibility percentual of the

fermented mixture was 77.4%. The RNV value post-fer-

mentation was very similar to the values obtained to casein,

which is a complete protein used as standard.

Table 3 shows the total and digestible essential amino

acid present in the unfermented BMF (used as

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supplementary foods in low-income communities) and in

the fermented mixture of BMF ? 32% CLF. The fer-

mented mixture of BMF ? 32% CLF shows high amounts

of essential amino acids when compared to the unfer-

mented BMF (Table 3). In this table it was also possible to

observe that the incorporation of 32% of the CLF in BMF

increased significantly the values of the essential amino

acids in the mixture, except tryptophan.

The necessary amounts of each essential amino acid for

a balanced diet [42] and the results obtained are listed in

Tables 3 and 4. The comparison between the data present

in both tables become possible to observe that a daily

intake of 100 g of mixture BMF ? 32% CLF fermented

can supply a good daily requirements of essential amino

acids, as recommended by WHO [42], mainly during

childhood (Table 4).

The PCDAAS was presented in Table 5.

During the solid-state fermentation process a decrease of

94.18% (from 10.14 to 0.59 mg kg-1) in cyanide concen-

tration was also observed.

Discussion

Countries whose economy is based in agriculture end up

generating large amounts of agricultural waste, which can

be used in bioprocesses to obtaining nutritional higher

value products [43, 44]. The solid-state fermentation (SSF),

which is a biotechnological process where microorganisms

grow on solid matrix and act as a processing agent, also is a

promising method for the use of agricultural residues in

food production with more nutricional value [45]. The high

nutricional value of the fermented material is related to

both components: from waste used (substrate) and the

proteins present in the fermenter microorganism, since

according Sargantanis et al. [46], 27.58% by weight of R.

oligosporus cells are constituted of crude protein.

Associating the need to minimize environmental

impacts caused by the disposal of agricultural waste with

the potential usage of these residues in food production, the

application of SSF can be an effective technique to increase

the nutritional value of protein, vitamins, phenolic con-

tents, flavonoids, lipids (unsaturated fatty acids) and other

important compounds for foods made from such waste,

which characterize in an important strategy for solving

problems related to the limitation and wastage of the food

[47, 48].

The results of this study showed that the presence of

CLF in BMF exerted a favorable condition in the growth of

fungi. This was probably due to the presence of protein

compounds in the cassava leaves, which are essential

nutrients for the metabolism and growth of fungi. Campos

et al. [47] obtained similar results by SSF when used

cashew peduncle bagasse. The authors reported a value of

20.3% of protein post fermentation, which corresponded a

3 times higher increase of that the reported in the beginning

Table 1 Crude protein quantity

before and after fermentation by

Rhizopus oligosporus and the

relative improvement of crude

protein

Crude protein (%)

Before fermentation After fermentation Improvement % P

BMF 1.53 ± 0.156 1.75 ± 0.158 14.38 0.301

BMF ? 8% CLF 3.38 ± 0.146 3.82 ± 0.149 13.02 0.058

BMF ? 16% CLF 5.86 ± 0.399 6.07 ± 0.335 3.58 0.645

BMF ? 32% CLF 9.74 ± 0.277 11.22 ± 0.184 15.20 0.005*

CLF 26.93 ± 0.284 29.85 ± 0.179 10.84 \0.001*

* Results for P\ 0.05 presents significance between the samples (a = 0.05)

Table 2 Relative nutritional

value (RNV) calculated by

Enterococcus zymogenes

growth in samples of the BMF

and CLF before and after

fermentation compared with

standard protein casein

Samples RNV (%)a Digestibility (%) Real RNV (%)

Casein (Sigma C-7078) 100.00 100.00 100.00

BMF ? 8% CLF 23.26 ± 4.13 66.9 15.56

BMF ? 8% CLF—fermented 29.98 ± 1.45 64.5 19.34

BMF ? 32% CLF 23.46 ± 1.39 82.7 19.40

BMF ? 32% CLF—fermented* 127.48 ± 3.08 77.4 98.67

CLF 81.00 ± 1.02 75.2 60.91

CLF—fermented 38.06 ± 1.43 87.3 33.23

a Calculated before the application of samples’ digestibility (in vitro)

* Results for P\ 0.05 presents significance between the samples (a = 0.05)

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of the fermentation. The data of Domingos [12] also point

to the enrichment of BM by rice bran, whose inclusion of

the rice bran increased the protein concentration of the

medium, providing greater development of the fungus.

However, the data obtained for different concentrations

of CLF added to the mixtures studied showed an increase

in 15.2% (BMF ? 32% CLF) protein levels after 3 days of

fermentation. These results indicate a higher efficiency of

incorporation of protein per CLF than by rice bran used by

Domingos [12]. According to the results of this author

using the same fungus R. oligosporus for SSF, there was an

increase in the amount of protein after 6 days from the start

of the fermentation process, while in our study it was

necessary only 3 days to increase the same protein amount.

Munhoz [49] observed an increase of 151.6% protein to

sugar cane bagasse fermented by filamentous fungus, after

45 days. The study of Darwish et al. [50] indicates a sharp

increase in the amount in 75% protein level after 7 days

and 175% after 21 days of SSF, when the maize stalks was

used as a substrate and having Pleurotus ostreatus as fer-

menting agent. All these data corroborate the data from this

study, demonstrating the efficient use of microorganisms to

incorporate proteins in vegetable residues.

By the amount of total protein obtained in this study, it

was observed that the inclusion of 32% of CLF in BMF

induced, after the SSF process, a greater increase of protein

Table 3 Improvement of essential amino acid by the addiction of cassava leaves and solid-state fermentation by Rhizopus oligosporus

Essential amino acids BMF—unfermented (mg 100 g-1) BMF ? 32% CLF—fermented (mg 100 g-1) Improvement (%)

Total Applied digestibilitya Total Applied digestibilityb

His 40.00 25.45 240.00 185.76 729.83

Thr 70.00 44.54 630.00 487.61 1094.75

Val 70.00 44.54 680.00 526.31 1181.63

Met ? Cys 0.11 0.07 220.00 170.28 243276.78

Ile 50.00 31.82 540.00 417.95 1313.70

Leu 110.00 69.99 930.00 719.81 1028.40

Phe ? Tyr 90.00 57.27 1220.00 944.26 1648.88

Lys 80.00 50.90 550.00 425.69 836.26

Trp ND 0.00 ND 0.00 0.00

ND Not detected, His histidine, Thr threonine, Val valine, Met methionine, Cys cysteine, Ile isoleucine, Leu leucine, Phe phenylalanine, Tyr

tyrosine, Lys lysine, Trp tryptophan
a Digestibility applied by 63.63% according to REIS [60]
b Digestibility applied by 77.4% according Table 2

Table 4 Daily requirement for essential amino acids recommended by WHO calculated from the average body weight by age and supply rate of

the recommended daily amount of essential amino acids

Amino acids Necessity by age (mg day-1)a Supply rate by age (%)b

0.5 1–2 3–10 11–14 15–18 [18 0.5 1–2 3–10 11–14 15–18 [18

His 165.00 163.50 255.60 546.60 675.95 700.00 112.58 113.61 72.67 33.98 27.48 26.54

Thr 255.00 250.70 383.40 819.90 1044.65 1050.00 191.22 194.50 127.18 59.47 46.68 46.44

Val 367.50 392.40 617.70 1320.95 1720.60 1820.00 143.21 134.13 85.20 39.84 30.59 28.92

Met ? Cys 232.50 239.80 383.40 774.35 983.20 1050.00 73.24 71.01 44.41 21.99 17.32 16.22

Ile 270.00 294.30 489.90 1002.10 1290.45 1400.00 154.80 142.02 85.31 41.71 32.39 29.85

Leu 547.50 588.60 937.20 2004.20 2580.90 2730.00 131.47 122.29 76.80 35.91 27.89 26.37

Phe ? Tyr 442.50 436.00 639.00 1366.50 1720.60 1750.00 213.39 216.57 147.77 69.10 54.88 53.96

Lys 480.00 490.50 745.50 1594.25 2027.85 2100.00 88.69 86.79 57.10 26.70 20.99 20.27

Trp 71.25 69.76 102.24 218.64 276.53 280.00 0.00 0.00 0.00 0.00 0.00 0.00

His Histidine, Thr threonine, Val valine, Met methionine, Cys cysteine, Ile isoleucine, Leu leucine, Phe phenylalanine, Tyr tyrosine, Lys lysine,

Trp tryptophan
a Adapted from WHO [52]
b Percentage of necessity supplied by the consumption of 100 g of BMF ? 32% CLF fermented by Rhizopus oligosporus

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in the mixture. This increase was, according to the

descriptions of Campos et al. [47], probably due to bio-

conversion (by fungicidal activity) of the inorganic nitro-

gen of the substrate (in this case cassava leaf) into organic

nitrogen in the mixture. The data of the present study

corroborate with those obtained by Campos et al. [47] and

Darwish et al. [50], in experiments carried out with cashew

peduncle bagasse and maize stalk, respectively.

About the real quality of the protein, Tavano et al. [35],

by using the Relative Nutritional Value (RNV) technique

to evaluate the nutritional quality of protein by the growth

of E. zymogenes, could distinguish the different nutritional

aspects of protein fractions of chickpea beans. The results,

expressed in Table 2, show that the fermentation increased

protein profile of the substrate, as can be observed in the

sample BMF ? 32% CLF and conferred a significant RNV

increase (about 5 times) in this substrate when it was

compared to the same unfermented sample. The obtained

value was similar to standard casein (Sigma C-7078, a

protein whose RNV pattern is considered 100%).

The analysis of the amino acids results indicates the

improvement in quantity and quality in fermented sample,

suggesting that the action of R. oligosporus increased sig-

nificantly the profile of amino acids available except for

tryptophan. These results corroborate the data obtained by

Suhet and Fioreze [51], on fermented pineapple wastes by

R. oligosporus that showed best result in incorporating

amino acids except for tryptophan as well.

The amount of some essential amino acids supplied by

the consumption of 100 g of the studied material reaches

higher values than those recommended by the WHO [52],

mainly to the demand for children of preschool age. For

adults, the use of fermented mixture (BMF ? 32%

CLF—fermented) may increase about 30% the supply of

essential amino acids and provide to consumers a

healthier diet [53].

The PCDAAS was obtained by the amino acid profile

from samples [39] corrected by the digestibility observed

in vitro value [35]. The PDCAAS value registered for the

BMF ? 32% CLF was 0.6037 and the digestibility of this

mixture was 77.4%, results similar to those described by

Pires et al. [41] for beans (PDCAAS = 0.6296 and

digestibility = 78.7%) and protein texturized soybean

(soybean PTS = PDCAAS = 0.6481 and digestibil-

ity = 86.41%). The PDCAAS value of the mixture was

higher than that found for corn (PDCAAS = 0.3707), wheat

(PDCAAS = 0.4025) and soybeans (PDCAAS = 0.5382),

described by the same authors. These results show that solid-

state fermentation carried out on themixture ofBMF ? 32%

CLF led to increased levels of amino acids and better

digestibility of the final product, comparable to levels found

in grains which are recognized as important sources of pro-

teins, used in basic food (beans and soy).

Since the nineteenth century, there are reports of poi-

soning and death arising from ingestion of cassava, due to

the presence of cyanogenic glycosides. The acidic stomach

pH turns it into hydrocyanic acid, a volatile compound

highly toxic to animals, including man [54]. An unfavor-

able aspect of the consumption of raw CL is the high

concentration of these glycosides. However, processes such

as fermentation and cooking reduce significantly the cya-

nide content of this material [55–57].

Table 5 Protein digestibility-corrected amino acid scores (PDCAAS), Babassu Mesocarp Flour ? 32% Cassava Leaves Flour, casein standard

and daily necessity in mg of amino acid/g of protein for children of 2–5 years old

Essential

amino acids

BMF ? 32% CLF

mg aa/g protein

Casein mg

aa/g protein

Amino acid ratio Standard FAO/

WHO 2–5 years
BMF ? 32% CLFa Casein

His 21.4 18.99 1.13 1.00 19

Thr 56.2 43.22 1.65 1.27 34

Val 60.6 54.95 1.73 1.57 35

Met ? Cys 19.6 30.14 0.78 1.21 25

Ile 48.1 46.91 1.47 1.68 28

Leu 82.9 93.05 1.26 1.41 66

Phe ? Tyr 108.7 109.71 1.73 1.74 63

Lys 49.0 78.66 0.85 1.36 58

Trp ND ND ND ND 11

PDCAAS – – 0.6037 1.0000 –

ND Not detected; BMF Babassu Mesocarp Flour; CLF Cassava Leaves Flour; Data of casein according to Pires et al. [41]; Standard protein

2–5 years, according FAO/WHO (1985); His (histidine); Thr threonine, Val valine, Met methionine, Cys cysteine, Ile isoleucine, Leu leucine,

Phe phenylalanine, Tyr tyrosine, Lys lysine, Trp tryptophan
a True protein digestibility obtained in vitro -0.774 (77.4%)

Waste Biomass Valor (2018) 9:581–590 587

123



The data show that the dehydrated CLF, used in this

work, presented initially 29.5 mg kg-1 of cyanide, which

would make unfeasible consumption. The BMF, in natura,

showed a concentration of 1.02 mg kg-1, a value within

the acceptable limits of consumption. A mixture of

BMF ? 32% CLF, before fermentation, showed interme-

diate values recorded for the 2 raw materials used

(10.13 mg kg-1). The toxic potential of the extracted HCN

cassava was quantitated for the LD50 (lethal dose for the

occurrence of 50%) in mice for 35 mg kg-1 (body weight),

while HCN LD50 accepted by the WHO is 10 mg kg-1

[58]. The same author states that the cyanide consumption

in amounts lower than lethal dose allows a detoxification

mechanism to convert the cyanide in thiocyanate, a non-

toxic substance that can be eliminate through urination.

After fermentation, the sample presented values within

the limit of edible ingestion according the legislation

(0.59 mg kg-1), suggesting the possibility of its use in

dietary supplementation. These data about the cyanide

reduction prove the effectiveness of the combination of

heat treatment (sterilization process) followed by fermen-

tation to reduce cyanide to allowed levels for the human

consumption, according to Brazilian food legislation that

establishes the maximum concentration of hydrocyanic

acid equivalent to 4.0 mg kg-1 of product [53].

Studies by Helbig et al. [59] showed that the concen-

tration of hydrocyanic acid in food supplements (multim-

ixtures) that receive CLF not reached levels hazardous to

human health. The authors added only 5% of CLF to not

raise the limit of the cyanide content in the multimixture.

However, our results demonstrated that the fermentation

process increased the protein content of the final product,

using 32% of CLF, without increasing toxicity of produced

food.

Conclusions

The solid-state fermentation of BMF mixture supple-

mented with 32% of CLF (w/w) promoted an increase of

the essential amino acids levels, leading to a gain in total

crude protein concentration of the final product.

The fermentation efficiency to increase Relative Nutri-

tional Value (RNV) of protein and to decrease of cyanide

levels in the mixture fermented by R. oligosporus indicates

that this mixture is a potential food to be used in human

diet.

The production of food from inexpensive sources can be

an interesting strategy to achieve food security and

improved nutrition and promote sustainable agriculture.

This kind of food can be obtained using byproducts from

agriculture, as CLF, to generate more sources of rich pro-

tein food.

In this study, the results showed that the inclusion of

32% of CLF in the BMF mixture led to significant

increases in protein levels after the process of solid-state

fermentation that raised the essential amino acid levels of

the tested mixture.

Essential amino acid levels observed for the fermented

mixture (BMF ? 32% CLF), exceeded the amounts

recorded for corn, wheat and soybeans and became similar

to the levels found in beans and textured soy protein

besides the decrease in the levels of cyanide in the mixture

to acceptable values for consumption.

Based on the results obtained in this work, it can be

inferred that the use of a fermented mixture containing

BMF ? 32% CLF can improve nutritional conditions and

therefore be successfully applied as a food supplement in

communities with limited access to protein food.

Acknowledgements This work was supported by the Coordenação

de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES), Brazil.

Compliance with Ethical Standard

Conflict of interest The authors declare that there are no conflicts of

interest.

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	Improvement of Protein Bioavailability by Solid-State Fermentation of Babassu Mesocarp Flour and Cassava Leaves
	Abstract
	Purpose
	Methods
	Results
	Conclusion

	Introduction
	Materials and Methods
	Organism
	Substrates
	Babassu Mesocarp Flour (BMF)
	Cassava Leaves Flour (CLF)

	Sample Preparation for Fermentation and Substrate Moisture
	Solid-state Fermentation (SSF)
	Analyzed Parameters
	Determination of Total Crude Protein
	Digestion
	In Vitro Digestibility
	Determining the Relative Nutritional Value (RNV)
	Essential Amino Acids
	Protein Digestibility-Corrected Amino Acid Score (PDCAAS)
	Cyanide Content
	Statistical Analyzes


	Results
	Discussion
	Conclusions
	Acknowledgements
	References