PRODUCTION OF PEPTIDES AND AMINO ACIDS FROM MICROBIAL BIOMASS IN FOOD AND FEED INDUSTRIES: BIOTECHNOLOGICAL ASPECTS
Рубрики: RESEARCH ARTICLE
Аннотация и ключевые слова
Аннотация (русский):
Introduction. Microbial biomass is a popular source of food ingredients and feed additives. Its high use has made it focus of many relevant studies. Yeast and fungal biomasses proved to be useful substrates that improve the quality and biological value of functional products. They differ in the content and composition of proteins and polysaccharides. The present research dealt with the enzymatic decomposition of proteins found in a novel fungal and yeast biomass. The research objective was to describe the peptide and amino acid composition of their enzymatic hydrolysates. Study objects and methods. The research featured a new fungal and yeast biomass mix. Aspergillus oryzae is a mycelial fungus and a popular industrial producer of hydrolytic enzymes in food industry. As for the yeast, it was the Saccharomyces cerevisiae strain, which is often used in baking. Results and discussion. The total content of identified amino acids in the fungal and yeast biomass was 306.0 mg/g, which was 1.5 times higher than in the fungal biomass alone. The biomass mix demonstrated a higher biological value of proteins than the yeast biomass. A set of experiments made it possible to compile a scheme for the biocatalytic destruction of polymers in the fungal and yeast biomass under the effect of fungal intracellular and endogenous enzymes. The article also contains a thorough description of the obtained enzymatic hydrolysates with various fractional compositions of peptides and free amino acids. Peptides with the molecular weight in the range of up to 29.0 kDa decreased by 2.1 times after 5 h of hydrolysis and by 10.7 times after 18 h. The designed conditions doubled the release of amino acids and increased the content of low-molecular-weight peptides up to 75.3%. Conclusion. The research provided a new algorithm for the biocatalytic conversion of microbial biomass. Regulating the conditions of enzymatic hydrolysis made it possible to obtain enzymatic hydrolysates with a desired degree of protein degradation. They could serve as peptides and amino acids in functional food and feed products.

Ключевые слова:
Microbial biomass, yeast, biocatalytic hydrolysis, enzymes, enzymatic hydrolysates, amino acids, molecular weight, peptide fractions
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INTRODUCTION
The modern concept of healthy diet means that the
range of functional foods keeps expanding to satisfy
various physiological needs of the human organism.
There are many ways to balance the nutritional
and biological value of functional products, e.g.
new formulations, specific raw materials, optimal
technological processes, functional and biologically
active additives, etc. [1–3].
The biotechnological processing of microbial
biomass proved to be a promising direction for the
production of functional food and feed ingredients [4–6].
Microbial biomass is a source of protein substances,
vitamins, polysaccharides, and trace elements. Bacterial
cell walls contain many valuable polysaccharides,
including β-glucans, mannans, aminopolysaccharides,
etc. [7–9]. Fungal biomass is known to produce
chitosanglucan biologics [10–12]. Certain components
of cell walls possess sorption, antioxidant, and other
valuable properties, which makes it possible to use
them in food industry [13–16]. In addition, microbial
cell protoplasm contains a biologically complete protein
with the amino acid score approaching that of animal
protein [4, 6]. However, commercial use of
microorganisms in protein and amino acid production
still requires further research.
The Saccharomyces cerevisiae strain of yeast has
long been focus of scientific attention. Biotechnology
employs it as a substrate for protein food and
feed additives. Enzyme systems can increase the
bioavailability of cellular contents. They catalyze
the hydrolysis of subcellular structures and release
biologically valuable components, e.g. proteins [6,
17, 18]. The functional and biomedical properties
of enzymatic hydrolysates depend on the degree of
biocatalytic decomposition of intracellular proteins.
Proteolytic enzymes owe their regulatory role to their
ability to catalyze the hydrolytic degradation of the
protein by certain peptide bonds. This process results
in physiologically active peptides, which, in turn, can
be bioregulators of certain biological processes [18–21].
The primary structure of the peptides determines their
functions. Biologically active peptides (BAP) have a
low molecular weight, and their amount of amino acid
residues can vary from 3 to 50 [21–24].
Aspergillus fungal mycelial biomass has also been
a popular subject of scientific studies. Aspergillus
oryzae produces industrially significant metabolites,
e.g. enzymes, organic acids, etc. [4, 10–12, 14, 25–27].
Various studies of microbial biomass as a substrate
for food and feed additives revealed differences in
the amount of proteins and polysaccharides. Their
structure and biochemical composition also vary,
which can affect the functional properties of biological
products. The biomass of A. oryzae fungus contains
almost twice as little protein as the S. cerevisiae yeast.
However, the fungal biomass proved a valuable source
of polysaccharides [25]. A fungal and yeast biomass
mix can improve the quality and biological value of
functional products and is a promising direction in
substrate production.
Protein substances, e.g. polypeptides, low-molecularweight
peptides, and amino acids, are an important
component of any balanced diet. Proteins and amino
acids are responsible for the formation of all tissues in
a living organism. They also play a regulatory role in
metabolic processes. It is the composition and amount
of key amino acids that matters. This fact proves
the relevance of studies aimed at obtaining various
functional ingredients of food and feed products
from microbial biomass as a source of biologically
complete protein.
The research objective was to study the processes
of enzymatic decomposition of proteins in fungal and
yeast biomass. The project also focused on the effect
of peptide and amino acid composition of microbial
biomass enzymatic hydrolysates on the functional
properties of food and feed ingredients.
STUDY OBJECTS AND METHODS
The research was performed on the premises of
the Russian Research Institute of Food Biotechnology
– branch of Federal Research Center of Nutrition,
Biotechnology, and Food Safety (Moscow). It featured
the biomass of the Aspergillus oryzae mycelial fungus,
an industrial producer of hydrolytic enzymes for the
food industry, and the Sаcharomyces cerevisiae strain of
baker’s yeast.
The A. oryzae fungal biomass was obtained by a
10-minute centrifugation at 5000 rpm. The resulting
mycelial biomass was mixed with yeast in a ratio
of 1:2. It served as a substrate for the biocatalytic
decomposition of intracellular polymers. After
centrifugation, the filtrate of the culture fluid was used
to obtain a complex enzyme preparation (CEP), which
served as a source of proteinases and peptidases.
The biocatalytic decomposition of the fungal
and yeast biomass happened because of the autolytic
processes caused by intracellular fungal enzymes. The
exogenous enzymatic systems of proteolytic (CEP) and
β-glucanase (Brewzyme enzyme preparation) action
were introduced to increase the polymer hydrolysis.
The enzymatic activity in the enzyme systems was
measured using standard methods. The mannanase
activity was determined by the degree of mannan
hydrolysis under certain conditions with the formation
of reducing carbohydrates. The chitinase hydrolysis
was assessed according to the chitin hydrolysis. State
Standard R 53974-2010I was used to evaluate the general
proteolytic activity, while State Standard R 53973-2010II
served to measure the β-glucanase activity.
We determined the hydrolysis of the fungal and
yeast biomass mix according to the concentration of
reducing substances, amine nitrogen, and amino acids
during enzyme hydrolysis. The anthrone method made
it possible to measure the concentration of reducing
substances, while the copper method helped to define the
concentration of amine nitrogen [28]III.
We used high-pressure exclusion chromatography
to assess the mass distribution of peptide molecules in
the enzymatic hydrolysates. The superose 12 column
(1.0×30 cm) was calibrated with standard globular watersoluble
proteins provided by SERVA (Germany) [29].
I State Standard 53974-2010. Enzyme preparations for food
industry. Method for determination of proteolitic activity. Moscow:
Standartinform; 2011. 16 p.
II State Standard R 53973-2010. Enzyme preparations for food
industry. Method for determination of β-glucanase activity. Moscow:
Standartinform; 2011. 12 p.
III OFS.1.2.3.0022.15 Opredelenie aminnogo azota metodami
formolʹnogo i yodometricheskogo titrovaniya [General Pharmacopoeia
Article No. 1.2.3.0022.15 Determination of amine nitrogen by
formol and iodometric titration].
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Serba Е.М. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 268–276
0.2 M sodium chloride served as an eluent at elution
rate = 0.4 cm3/min and a wavelength λ = 280 nm
using a UV132 flow-through ultraviolet detector
and a Multichrom 3.1 data processing software. The
chromatograms were integrated by the gravimetric
method. The range of molecular weights varied from
free to full volume of the chromatographic column.
The research employed a KNAUER EUROCHROM
2000 chromatograph to measure the amino acid content
in the microbial biomass and enzymatic hydrolysates.
After that, the components were determined by a
spectrophotometric Smartline UV Detector 2500 at a
wavelength of λ = 570 nm (Germany). The aminograms
were calculated by comparing the areas of the standard
and the sample [30].
RESULTS AND DISCUSSION
The microbial biomass proved to vary in the amino
acid composition (Table 1). The content of essential
amino acids amounted to 53.13% of the total number
in the fungal protein, while it was only 41.30% in the
yeast protein. The level of tryptophan and methionine
in the fungal protein was 2.2 times higher, leucine and
tyrosine – by 1.8 times, and valine – by 1.5 times. As
for the yeast protein, it appeared to contain proline; the
amount of glutamic acid was by 1.4 times higer, lysine
and threonine – by 1.2 times.
The yeast and fungal biomass mix had a total amino
acid content of 306.0 mg/g, which was 1.5 times higher
than that in the fungal biomass (202.8 mg/g). The yeast
and fungal biomass had a slightly higher biological value
of proteins, while the share of essential amino acids was
44.26% (Table 1).
We conducted a comparative analysis of the amino
acid composition of the protein in the yeast and fungal
biomass mix with that of the reference protein approved
by the Food and Agricultural Organization (WHO). The
reference protein shows to what degree a certain protein
satisfies the physiological need of the body for essential
amino acids [31].
The amino acid score (ACS) was calculated
according to the formula:
ACS = А : S × 100% (1)
where ACS – amino acid score;
А – essential amino acid content in a particular
protein;
S – amino acid content in the reference protein.
The yeast and fungal biomass demonstrated a high
biological value of the protein: the total content of
essential amino acids was 1.2 times higher than in the
reference protein. The biomass contained two limiting
amino acids, namely phenylalanine and methionine.
Their amino acid score was 70% and 55% of the
reference protein, respectively (Fig. 1). Tryptophan,
lysine, threonine, and leucine proved to have the highest
amino acid score.
Therefore, the biomass fortified with essential amino
acids obtained from proteins of the S. cerevisiae yeast
strain and the A. oryzae fungus can be a promising
substrate for the production of new biologically active
peptide and amino acid additives with a wide range of
functional properties.
The microbial biomass mix had a higher level of
protein in the substrate, and its biological value also
increased. In addition, it demonstrated a higher content
of chitin-glucan and mannan polysaccharides, as well as
intracellular enzymes.
Table 1 Amino acids in the microbial biomass mix
(Aspergillus oryzae and Sаcharomyces cerevisiae)
Amino acids Amino acid content in microbial biomass
Yeast Fungal Mix
mg/g % mg/g % mg/g %
Aspartic acid 37.86 10.01 20.05 9.89 30.06 9.82
Serine 22.15 5.86 11.04 5.44 19.25 6.29
Threonine 18.57 4.91 8.40 4.14 15.63 5.11
Glutamic acid 64.83 17.15 25.54 12.59 51.02 16.67
Proline 36.05 9.54 – – 14.06 4.60
Glycine 17.21 4.55 10.03 4.95 14.46 4.73
Alanine 25.71 6.80 11.00 5.42 18.37 6.00
Valine 14.85 3.93 11.85 5.84 14.38 4.70
Methionine 5.51 1.46 6.62 3.26 5.92 1.94
Isoleucine 13.00 3.44 6.31 3.11 11.02 3.60
Leucine 23.66 6.26 22.92 11.30 23.25 7.60
Tyrosine 6.45 1.71 6.09 3.00 6.02 1.97
Phenylalanine 14.87 3.93 8.46 4.17 12.75 4.17
Histidine 11.66 3.08 5.47 2.70 8.21 2.68
Lysine 27.96 7.40 12.74 6.28 23.95 7.83
Tryptophan 25.57 6.76 30.45 15.02 28.62 9.35
Arginine 12.16 3.22 5.83 2.87 9.03 2.95
Total amount
of amino
acids
378.07 100 202.80 100 306.00 100
Essential
amino acids
156.15 41.30 107.75 53.13 135.42 44.26
Figure1 Essential amino acids in the protein of the microbial
biomass mix (Aspergillus oryzae and Sаcharomyces
cerevisiae) vs. reference protein
0 2 4 6 8
Threonine
Valine
Methionine
Isoleucine
Leucine
Phenylalanine
Lysine
Tryptophan
Microbial protein Reference protein
5
10
15
20
25
30
Reducing substances, NH2,
free amino acids, %
1
3
2
Stage I
Biocatalysis of the biomass mix
by fungal endo-enzymes
(τ = 2 h, t = 50°C)
Biomass mix
(1:2)
hydrolysate I
Stage II
Biocatalysis of the biomass mix by fungal
endo-enzymes and exogenous β-glucanase
Brewzyme 50 units
of β-glucanase per g
biomass Saccharomyces cerevisiae biomass
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Serba Е.М. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 268–276
The biocatalytic conversion made it possible to
increase the bioavailability of polymers in the microbial
biomass mix and to obtain easily digestible peptides
and amino acids. The biocatalytic conversion included
three stages (Fig. 1). Stage I featured the fungal biomass,
which contained residual proteolytic and β-glucanase
enzymes (Table 2). The autolytic decomposition of
the microbial biomass polymers lasted 2 h at 50°C.
Enzymatic hydrolysate I of the biomass mix appeared
after 2 h of autolysis under the effect of fungal
intracellular enzymes.
The Brewzyme BGX enzyme preparation is known
as a source of β-glucanases and other hydrolases
(Table 2). The Brewzyme enzyme made it possible to
increase the decomposition rate of cell walls during
Stage II. Mannans and β-glucans, as well as protein-
Table 2 Enzymatic activity of enzyme preparations used for biocatalysis of the microbial biomass mix (Aspergillus oryzae and
Sаcharomyces cerevisiae)
Source of enzymes Enzyme activity, unit/g (cm3)
Protease β-glucanase Mannanase Chitinase
Fungal biomass 5.10 1.44 0.12 0.02
Brewzyme BGX enzyme 0 600.00 78.00 0.76
Complex enzyme preparation (CEP) 450.00 113.00 48.00 1.98
Figure 2 Biocatalytic conversion of the microbial biomass mix (Aspergillus oryzae and Sаcharomyces cerevisiae)
0 2 Threonine
Valine
Methionine
Isoleucine
Leucine
Phenylalanine
Lysine
Tryptophan
Microbial protein 0
5
10
15
20
25
30
0 2 4 6 Reducing substances, NH2,
free amino acids, %
Enzymolisis 1 – reducing substances, % 3 – free amino acids, %
0
10
20
30
40
50
2 Molecular weight distribution
of peptide fractions, %
Proteolyses 72.9–29.0 29.0–8.0–4.1 4.1–Stage I
Biocatalysis of the biomass mix
by fungal endo-enzymes
(τ = 2 h, t = 50°C)
Biomass mix
(1:2)
Enzymatic hydrolysate I
Stage II
Biocatalysis of the biomass mix by fungal
endo-enzymes and exogenous β-glucanase
(τ = 3 h, t = 40°C)
Brewzyme 50 units
of β-glucanase per g
Aspergillus oryzae biomass Saccharomyces cerevisiae biomass
Stage III
Biocatalysis of the biomass mix
by fungal endo-enzymes and exogenous proteases
(τ = 13 h, t = 30°C)
Enzymatic hydrolysate II
CEP – a source of a complex
of proteinases
20 of protease per g
Enzymatic hydrolysate III
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Serba Е.М. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 268–276
Table 3 Composition of free amino acids in enzymatic hydrolysates of the microbial biomass mix
(Aspergillus oryzae and Sаcharomyces cerevisiae)
Amino acids Amino acid content, mg/g
Enzymatic hydrolysate I Enzymatic hydrolysate II Enzymatic hydrolysate III
Aspartic acid 3.567 5.097 12.866
Serine 4.246 5.560 9.824
Threonine 6.960 10.476 10.968
Glutamic acid 11.233 14.442 14.487
Proline 2.611 3.439 7.810
Glycine 1.712 2.850 7.264
Alanine 6.304 7.517 9.130
Valine 5.125 6.540 8.509
Methionine 1.088 2.009 2.570
Isoleucine 4.189 5.744 6.558
Leucine 5.916 8.404 11.549
Tyrosine 3.040 4.627 5.497
Phenylalanine 3.575 5.346 6.648
Histidine 3.696 10.053 10.803
Lysine 6.111 9.240 9.527
Tryptophan 6.435 8.044 11.295
Arginine 4.829 7.249 7.714
Total amount of amino acids, where
essential amino acids
80.637
39.398
116.637
55.803
152.019
67.624
Content of free amino acids, % of total 26.4 38.1 49.7
0 2 4 6 8
Threonine
Valine
Microbial protein Reference protein
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
Reducing substances, NH2,
free amino acids, %
Enzymolisis time, h
1 – reducing substances, % 2 – amino nitrogen, %
3 – free amino acids, %
1
3
2
0
10
20
30
40
50
2 5 18
Molecular weight distribution
of peptide fractions, %
Proteolyses time, h
72.9–29.0 29.0–14.6 14.6–8.0
8.0–4.1 4.1–1.6 менее 1.6 кДа
Stage I
Biocatalysis of the biomass mix
by fungal endo-enzymes
(τ = 2 h, t = 50°C)
Stage II
Biocatalysis of the biomass mix by fungal
endo-enzymes and exogenous β-glucanase
(τ = 3 h, t = 40°C)
Brewzyme 50 units
of β-glucanase per g
Stage III
Biocatalysis of the biomass mix
by fungal endo-enzymes and exogenous proteases
(τ = 13 h, t = 30°C)
II
CEP – a source of a complex
of proteinases
20 of protease per g
Enzymatic hydrolysate III
Figure 3 Biochemical parameters of the enzymatic
hydrolysates during hydrolysis of the microbial biomass
mix (Aspergillus oryzae and Sаcharomyces cerevisiae)
mannan and chitin-glucan complexes, were the main
structural polymers [8, 9]. The proportion was 50 units
of β-glucanase per 1 g of biomass dry matters. Stage II
lasted 3 h at 40°С and produced enzymatic hydrolysate
II after 5 h of hydrolysis (Fig. 2).
Complex enzyme preparation CEP was introduced
during Stage III. It provided a deeper enzymatic
hydrolysis of the main subcellular polymers of the
microbial biomass, including protein substances. The
hydrolysis resulted in the formation of easily digestible
biologically active products. The CEP served as a
source of a complex of proteinases and peptidases.
The proportion was 20 units of protease per 1 g of
biomass solids (Fig. 2). Fungal proteolytic enzymes are
thermolabile, so the temperature was reduced to 30°C.
Stage III lasted 13 h; the total biocatalysis time was 18 h.
Stage III produced enzymatic hydrolysate III.
The enzyme system of the A. oryzae fungus
and exogenous enzymes made it possible to obtain
enzymatic hydrolysates from the yeast and fungal
microbial biomass mix. The enzymatic hydrolysates
varied in the degree of decomposition of intracellular
polymers (Fig. 2).
The most intense formation of hydrolysis products of
protein and carbohydrate polymers took place during the
first 5 h. After 5 and 14 h, the concentration of soluble
reducing carbohydrates increased by 9.3 and 12.1 times
(from 2.1% to 25.5%), respectively. The concentration
of amine nitrogen (NH2
+) increased by 6.4 times and
9.6 times (from 0.5% to 4.8%). The concentration of free
amino acids increased by 8.0 times and 12.2 times, from
1.3% to 15.9% (Fig. 3).
Table 3 illustrates the composition of the free amino
acids in the obtained enzymatic hydrolysates and
their amount. 26.4% of free amino acids were released
during the hydrolysis of the microbial biomass mix by
intracellular fungal enzymes (enzymatic hydrolysate I).
After exogenous enzymes (β-glucanase and proteolytic
effects) were introduced and the process time was
prolonged, the release of amino acids increased by
1.5–2.0 times. It reached 38.1% in enzymatic hydrolysate
II and 49.7% in enzymatic hydrolysate III. The content
of free essential amino acids also increased (Table 3).
The amount of essential free amino acids increased
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Serba Е.М. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 268–276
by 1.7 times in enzymatic hydrolysate III, rising from
39.398 mg/g to 67.624 mg/g, compared to enzymatic
hydrolysate I.
Thus, controlling the enzymatic hydrolysis of the
combined microbial biomass allowed us to obtain
enzymatic hydrolysates with the desired degree of
decomposition of microbial cell proteins.
We also measured the effect of process time on
the molecular weight of the peptide fractions in the
enzymatic hydrolysates of the biomass mix. Average
yeast proteins consist of 466 amino acid residues and
have a molecular weight of 53 kDa. Fungal proteases
reduced the molecular weight of proteins after 2 h of
autolysis (Fig. 4).
The molecular weight distribution of protein
fractions during the hydrolysis of the biomass mix
confirmed the effectiveness of the decomposition
processes that produced lower-molecular-weight
peptides (Figs. 4 and 5). A longer enzymatic hydrolysis
lowered the content of high-molecular-weight peptides
and increased the number of low-molecular-weight
peptides.
In enzymatic hydrolysate I, peptides in the range
over 4.1 kDa accounted for 38.2% of the total amount
of protein substances. Peptides in the range from 4.1 to
1.6 kDa constituted 26.7%, while those under
1.6 kDa made up 35.1%. The content of highmolecular-
weight peptides decreased significantly
during the hydrolysis of protein polymers. After
5 hours of hydrolysis, the amount of peptides over
29.0 kDa fell by 2.1 times, after 18 hours – by
10.7 times (Fig. 5). In enzymatic hydrolysate II, the
fraction of low-molecular-weight peptides reached
45.4%. As for enzymatic hydrolysate III, the content
of low-molecular-weight peptides in the range up to
4.1 kDa was 75.3%, while the share of those under
1.6 kDa accounted for 52.4%.
CONCLUSION
The present research revealed the composition of
peptides and amino acids in the enzymatic hydrolysates
of a new biomass mix that combined the Saccharomyces
cerevisiae yeast strain and the Aspergillus oryzae
fungus. A set of experiments confirmed that the
enzymatic hydrolysates could be used to fortify food and
feed products.
The new biomass mix demonstrated a higher content
of proteins and essential amino acids, as well as other
(a) Enzymatic hydrolysate I (2 h) (b) Enzymatic hydrolysate II (5 h) (c) Enzymatic hydrolysate III (18 h)
Figure 4 Molecular weight distribution of bioconversion products of protein polymers in the enzymatic hydrolysates of the
microbial biomass mix (Aspergillus oryzae and Sаcharomyces cerevisiae)
Figure 5 Molecular weight distribution of peptides during
the enzymatic hydrolysis of the microbial biomass mix
(Aspergillus oryzae and Sаcharomyces cerevisiae)
0
10
2 5 Molecular of Proteolyses time, 72.9–29.0 29.0–14.6 8.0–4.1 4.1–1.6 Enzymatic hydrolysate III
III.Microbial biomass mix
Enzyme hydrolysis – 2 h
Superose 12 (1, 6 x 50 cm)
Eluent – 0.2 M
NaCl+azide
Elution rate – 2.0 mL/min
UV detector (280 nm)
At the x-axes – molecular
weight, kDa
Enzymolisis – 5 h
Superose 12 (1, 6 x 50 cm)
Eluent – 0.2 M
NaCl+azide
Elution rate – 2.0 mL/min
UV detector (280 nm)
At the x-axes – molecular
weight, kDa
At the y-axes – optical
At the y-axes – optical density at 280 nm, RU
density at 280 nm, RU
0
2 5 Molecular Proteolyses time, 72.9–29.0 29.0–14.6 8.0–4.1 4.1–1.6 Enzymatic hydrolysate III
Enzymolisis – 18 h
Superose 12 (1, 6 x 50 cm)
Eluent – 0.2 M
NaCl+azide
Elution rate – 2.0 mL/min
UV detector (280 nm)
At the x-axes – molecular
weight, kDa
At the y-axes – optical
density at 280 nm, RU
0 2 4 6 8
Threonine
Valine
Methionine
Isoleucine
Leucine
Phenylalanine
Lysine
Tryptophan
Microbial protein Reference protein
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
Reducing substances, NH2,
free amino acids, %
Enzymolisis time, h
1 – reducing substances, % 2 – amino nitrogen, %
3 – free amino acids, %
1
3
2
0
10
20
30
40
50
2 5 18
Molecular weight distribution
of peptide fractions, %
Proteolyses time, h
72.9–29.0 29.0–14.6 14.6–8.0
8.0–4.1 4.1–1.6 менее 1.6 кДа
units
per g
biomass
of a complex
proteinases
protease per g
less 1.6 kDa
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Serba Е.М. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 268–276
valuable components. The yeast increased the amount
of proteins, while the fungus raised the content of
essential amino acids. The fungus also increased the
amount of intracellular enzymes, which are used during
enzymolisis. As a result of the mutual fortification, the
total amino acid content increased by 1.5 times due to
the higher protein content in the yeast. In addition, the
biological value of the proteins in the new biomass mix
proved to be higher than that in the traditional yeast
biomass. This fact means that the ingredients obtained
from the biomass mix could contribute to a wider range
of functional properties.
The comparative analysis showed the high biological
value of the protein in the yeast and fungal biomass
mix. The total content of essential amino acids was
1.2 times higher than in the reference protein. The
biomass appeared to contain two limiting amino acids
– phenylalanine and methionine. Their amino acid
score accounted for 70% and 55% of their content in
the reference protein, respectively. Tryptophan, lysine,
threonine, and leucine demonstrated the highest score.
A significant amount of tryptophan, typical for fungal
biomass, might add extra functional properties to
ingredients obtained from their peptides and amino
acids. Tryptophan is known as an immunologically
active amino acid. It is a dipeptide with a wide range
of immunomodulatory effects [32, 33]. Tryptophancontaining
drugs have an antidepressant effect and
stimulate the production of vitamin B3 (niacin). In
addition, tryptophan hydroxylation produces serotonin,
an important brain neurotransmitter [34].
The biomass mix fortified with essential amino acids
of proteins obtained from the S. cerevisiae yeast strain
and the A. oryzae fungus could be used as a commercial
substrate. It was found capable of facilitating the
production of new biologically active peptide and amino
acid additives with a wide range of functional properties.
We developed a new algorithm for biocatalytic
polymer conversion in the new microbial biomass
mix. The algorithm made it possible to obtain easily
digestible peptide and amino acid ingredients using
fungal intracellular enzymes, as well as β-glucanase
and proteolytic enzymatic preparations. The conditions
of enzymatic hydrolysis proved to affect the fractional
composition of the enzymatic hydrolysates. A fivehour
hydrolysis lowered the amount of peptides in the
range over 29.0 kDa by 2.1 times, and 18-h hydrolysis –
by 10.7 times. Intracellular proteinases and peptidases
are known to catalyze the decomposition of proteins.
As a result, the enzymatic system with proteinases and
peptidases could provide food and feed ingredients that
contained 75.3% of low-molecular-weight peptides and
up to 50% of free amino acids that are responsible for
biologically active factors with functional properties.
The low-molecular-weight peptides, free amino
acids, and essential amino acids are involved into
various biological processes. They improved the
digestibility of the enzymatic hydrolysates obtained
from the microbial biomass mix, which can be used as
peptide and amino acid components of functional food
and feed products.
CONTRIBUTION
Authors are equally related to the writing of the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict of
interests related to the publication of this article.

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