INTRODUCTION
Current global trends in food production are aimed
at designing healthy foods to improve public health and
prevent diseases caused by unbalanced nutrition. In the
recent years, the quality and safety of food products has
been a strategic priority in Russia. New laws have been
passed to regulate and encourage the development and
production of a wide range of healthy foods, including
functional products.
Diseases of the musculoskeletal system are
among the most common in modern clinical practice,
especially osteoporosis. According to the World
Health Organization, almost 200 million people suffer
from osteoporosis worldwide, with over 9 million
fractures occurring every year. Women aged 55+
are especially vulnerable to this pathology, which is
presumably associated with estrogen deficiency in the
postmenopausal period.
Postmenopausal osteoporosis is caused by
accelerated bone resorption and systemic calcium
imbalance. Osteoporosis caused by hypoestrogenism
is commonly treated with drugs that prevent bone
resorption or stimulate the formation of bone tissue.
These drugs are mainly based on female sex hormones
or selective estrogen receptor modulators. However,
hormone therapy in postmenopausal women can
be a risk factor for stroke, myocardial infarction,
thromboembolism, and breast cancer. Moreover, these
drugs can cause serious side effects, such as atrial
fibrillation, atypical subcutaneous fracture, delayed
fracture healing, hypersensitivity reactions, hot flashes,
leg cramps, gastrointestinal disorders, etc. Another
cause of osteoporosis is deficiency states due to
insufficient intake of calcium, magnesium, protein, and
vitamin D [1–8].
All these factors determine a need for new ways
of osteoporosis prevention and treatment, namely for
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alimentary correction with functional foods. Such foods
not only meet the intake of essential nutrients, but also
benefit certain bodily functions and prevent the negative
effects of lifestyle and environmental factors. Our diet –
as a whole and its individual components – influences
different physiological processes in our body. Therefore,
food formulators should introduce physiologically
active ingredients with corrective properties, as well
as use technology that preserves the nutritional and
biological value of raw materials and components during
processing and cooking.
Formulation of functional meat products is one
of the current trends in modern meat industry. In
particular, low-value meat-and-bone material can be
used to obtain protein hydrolysates and bone mineral
components [9–12].
Protein hydrolysates are commonly used as an
alternative protein source in commercial products.
They consist of a mixture of proteins and peptides
resulting from the hydrolysis of intact proteins. During
hydrolysis, peptide bonds of intact proteins get broken,
which leads to a range of peptides of different sizes.
Protein hydrolysates are used in various products
depending on their properties [13, 14]. Numerous
studies show that protein hydrolysates can be used in
diets due to their high nutritional and therapeutic value
– low immunological reactivity, bioactive peptides, and
antioxidant activity. Protein hydrolysates are widely
used in the diet for people who cannot digest whole
protein. Protein hydrolysis can be carried out using
enzymes, acids, or alkalis, but enzymatic hydrolysis
is preferable for food purposes since it can produce
hydrolysates with a well-defined peptide profile [15].
Collagen-containing products of meat processing
are the main source of collagen with a unique amino
acid composition. Collagen can be transformed into
active peptides and amino acids to be used as functional
ingredients in food formulations. It is a protein that is
present in large quantities in the connective tissue of
animal materials. Connective tissue is part of cartilage,
tendons, subcutaneous tissue, bone, intercellular
substance of muscles, parenchymal organs, and vascular
walls. It accounts for about 50% of the animal’s body
weight. Connective tissue contains proteoglycans,
whose polysaccharide group includes glucosamine or
galactosamine. One of its main functions is that it takes
part in the formation of organs and their restoration.
Enzymatic hydrolysis increases the bioavailability
of collagen and glycosaminoglycans for the body to
assimilate. It produces peptides and amino acids that are
absorbed into the bloodstream and then enter the cells of
the connective tissue matrix [16].
The content of protein fractions and their amino acid
composition in hydrolysates can be regulated by modes
of hydrolysis, type of enzyme, processing method,
temperature, and other factors.
Pork legs and ears are a valuable source of collagen
hydrolysates. They contain 23.5 and 21.0% of protein,
respectively, with 15.3 and 12.6% in the connective
tissue, respectively. In our previous work, we
substantiated hydrolysis parameters for the production
of active peptides and free amino acids (10–15%) [17].
In particular, we described a method for obtaining
hydrolysate from pork legs and ears using enzymecontaining
pancreas homogenate (15% of the raw
materials.) at T = 50 ± 2°C, τ = 6 h, and further freezedrying
at –40°C.
Thus, functional foods play a special role in the
prevention and nutritional correction of osteoporosis,
especially products based on hydrolyzed connective
tissue proteins. They contain large amounts of collagen
peptides and amino acids that stimulate the synthesis
of physiological collagen and other substances creating
cartilage and bone matrix. In addition to collagen
peptides, the diet should meet the intake of calcium,
magnesium, copper, zinc, as well as vitamins D, A, E, C,
and group B.
In this regard, we aimed to formulate a functional
product based on collagen fermentolysate for
osteoporosis prevention and to confirm the identified
properties in animal experiments. Our objectives
were to substantiate the formulation in terms of its
composition and component ratios, evaluate its sensory,
microbiological, and toxicological indicators, as well as
assess its restorative effect on bone metabolism impaired
by oophorectomy in experiments on rats.
STUDY OBJECTS AND METHODS
Our study objects were dried collagen fermentolysate
and a functional product based on it.
Collagen fermentolysate was obtained from pork
by-products (ears and legs, 1:1) using raw pancreas
homogenate as an enzyme-containing material. The
resulting hydrolysate was dried under vacuum in an
Alpha 1-2 LD freeze-dryer (Germany) at –40°C. Then,
it was crushed to a particle size < 0.2 mm. The resulting
hydrolysate was a homogeneous fine powder of light
beige color, readily soluble in water.
The molecular weight distribution of protein
fractions in the collagen fermentolysate was studied
by electrophoresis in a 10% polyacrylamide gel with
sodium dodecisulfate (SDS) according to Laemmli. The
amino acid composition was determined on an Agilent
1260 Infinity LC liquid chromatograph in line with State
Standard 34132-2017. The hydroxyproline content was
measured in line with State Standard 23041-2015.
Protein content in the product was determined by
the Kjeldahl method according to State Standard 25011-
2017. Mass fractions of vitamin D3 and calcium were
measured according to State Standard 32307-2013 and
State Standard R 55573-2013, respectively (the latter by
atomic absorption). The method to determine vitamin C
involved the vitamin’s extraction (by sequential acid and
enzymatic hydrolysis), precipitation of proteins, and high
performance liquid chromatography in the ultraviolet
(UV) region at a given wavelength. The resulting peak
in the chromatogram was compared with the peak of a
standard with a known concentration.
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The method to determine vitamin E was based on
alkaline hydrolysis of the sample and extraction with
diethyl ether. The obtained extract was analyzed by high
performance liquid chromatography in the ultraviolet
(UV) region at a given wavelength. The resulting peak
in the chromatogram was compared with the peak
of a standard vitamin solution with a known mass
concentration.
Microbiological indicators were determined using
the following standards:
– State Standard 10444.15-94 for the quantity
of mesophilic aerobic and facultative anaerobic
microorganisms (QMAFAnM);
– State Standard 31747-2012 for coliform bacteria;
– State Standard 31659-2012 for salmonella bacteria; and
– State Standard 10444.12-2013 for mold.
Toxic elements were determined according to the
following standards:
– State Standard 26927-86 for mercury;
– State Standard 26930-86 for arsenic;
– State Standard 26932-86 for lead; and
– State Standard 26933-86 for cadmium.
Biological experiments were carried out on female
Wistar rats (n = 42) weighing 340 ± 20 g. The animals
were kept and studied in a vivarium in strict accordance
with State Standard 33216-2014.
After quarantine (7 days), the rats were randomly
divided into two groups: 1) intact rats (n = 10), who
were fed on a standard diet throughout the experiment,
and 2) rats exposed to experimental osteoporosis
modeling (n = 32).
The standard vivarium diet contained 12% casein
proteins, 72% soluble carbohydrates, 11.5% saturated
and polyunsaturated fatty acids, 1.0% fat-soluble
vitamins, 0.1% water-soluble vitamins, and 4.0%
minerals [18].
The osteoporosis modeling involved complete
oophorectomy under general anesthesia (Zoletil 100,
Virbac S.A., France; Xila, Interchemie, Netherlands).
After 14 days from the surgery, the ovariectomized
female rats were divided into three groups:
1) control animals (control), which daily received
intragastrically administered distilled water (0.5 ml/
head) for 28 days (n = 10);
2) experimental animals (experiment 1), which daily
received an intragastrically administered glucosaminechondroitin
solution in a dose of 0.014 g per 1 kg of live
weight (Pharmacor Production, Russia) (0.5 ml/head) for
28 days (n = 11); and
3) experimental animals (experiment 2), which daily
received an intragastrically administered functional
product based on collagen fermentolysate and dissolved
in water (12 g/100 mL) in an amount of 0.5 g per 1 kg of
live weight (0.5 mL/head) for 28 days (n = 11).
The rats were kept in IV S cages (Tecniplast, Italy),
5 animals each, under standard vivarium conditions:
temperature 20 ± 3°C, humidity 48 ± 2%, day/night
lighting (from 6.00 to 18.00), as well as free access to
water and feed [18].
Before the study and after administering the
functional product, the animals were weighed every
4 days on a laboratory electronic balance (Adventurer
Pro AV2101, USA). On the 42nd day of the experiment,
the animals were euthanized in a chamber (VetTech,
UK), with blood samples extracted from the heart.
The experiments were conducted in compliance
with Order No. 267 of the Russian Ministry of Health
of June 19, 2003 “On the rules of laboratory practice”
and European Community Directive 86/609EEC. The
study was approved by the bioethical commission of the
V. M. Gorbatov Federal Research Center of Food
Systems (protocol No. 01/2019 of May 09, 2019) [18].
Following autopsy, all the animals underwent a
thorough examination of their body surface, as well as
intracranial, thoracic, and abdominal cavities and their
contents. Their internal organs (liver, kidneys, spleen,
adrenal glands, thymus, and heart) were separated and
wet-weighed immediately after dissection. Femurs were
sampled to determine mass fractions of phosphorus
(State Standard 32009-2013), magnesium (State Standard
33424-2015), and calcium (State Standard R 55573-2013).
The blood cytometric assay involved counts
of lymphocytes (LYM), granulocytes (GRA), and
Figure 1 Molecular weight distribution of protein-peptide fractions in collagen fermentolysate
0
5
10
15
20
25
30
35
40
45
>400-600 230-400 170-230 100-170 40-100 20-40 10-20
Total protein, %
Molecular weight, kDa
6
8
10
12
14
16
acids, g/100 g protein
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monocytes (MON) according to cell size and granularity
on a Guava Easy Cyte flow cytometer (Merck Millipore,
Germany). The content of leukocytes was determined as
a sum of lymphocytes, granulocytes, and lymphocytes.
Relative contents of lymphocytes, granulocytes, and
monocytes were calculated using the formulas: LYM/
WBC×100%, GRA/WBC×100%, MON/WBC×100%.
Biochemical parameters of blood serum were
determined on an automatic BioChem FC-360 analyzer
(HTI, USA) using a set of reagents (HighTechnology,
USA) [18]. Biochemical analysis measured total protein,
albumin, bilirubin (total and direct), urea, creatinine,
triglycerides, aspartate aminotransferase (ASAT),
alanine aminotransferase (ALAT), alkaline phosphatase
(ALP), gamma-glutamyltransferase (GGT), lactate
dehydrogenase (LDH), calcium, cholesterol, glucose,
phosphorus, and magnesium.
Osteocalcin in blood serum was quantified by
enzyme-linked immunosorbent assay (ELISA) using
a set of rat-specific reagents on an Immunochem 2100
analyzer (HTI, USA).
Statistical analysis was performed in STATISTICA 10.
Statistical significance was determined by the Kruskal-
Wallace H-test (P ≤ 0.05).
RESULTS AND DISCUSSION
First, we studied the physicochemical parameters
of collagen fermentolysate. The molecular weight
distribution of its fractions is shown in Fig. 1.
As can be seen in Fig. 1, about 41% of fractions
weighed from 10 to 20 kDa. Peptides with such
a molecular weight should be used as the basis
of a functional beverage, since they ensure high
bioavailability and good taste characteristics.
The amino acid composition of collagen
fermentolysate is shown in Fig. 2.
According Fig. 2, collagen fermentolysate contained
relatively high contents of glutamic acid (14.8%),
aspartic acid (10.8%), glycine (7.3%), alanine (6.9%),
and proline (4.9%). These amino acids are known to
stimulate cartilage and bone cells and restore joint
tissues. Alanine is the main component of connective
tissue, while proline and lysine are precursors of
hydroxylysine and hydroxyproline, which are used by
the body to form collagen, tendons, and heart muscle.
We also found high contents of leucine and threonine.
These essential amino acids are important for the
biosynthesis of glycine and serine, which are responsible
for the production of collagen, elastin, and muscle tissue.
Based on collagen fermentolysate, our functional
product with an antiosteoporosis effect also contained
dietary fiber, bioactive substances, as well as macro- and
microelements.
The formulation was in line with the biomedical
requirements for the quality, composition, and safety of
functional products with corrective properties. To have
a real physiological effect, the product should contain at
least 50% of collagen fermentolysate. However, it should
also have good consumer appeal. To neutralize the flavor
of fermentolysate, pumpkin powder was used as dietary
fiber. It has a pleasant taste and, at the same time,
contains various carbohydrate components, including
pectins, cellulose, fiber, calcium, magnesium, iron, B
vitamins, vitamin PP, beta-carotene, and vitamin C.
Our formulation was primarily aimed at normalizing
metabolic processes and preventing diseases of the
musculoskeletal system. The component quantities met
the physiological needs of adult humans.
Increased calcium intake is an integral part of
osteoporosis prevention and treatment. To assimilate
calcium, we added vitamin D3, as well as vitamins E
and C with antioxidant properties. Products based on
collagen hydrolysates in combination with vitamin C
are more effective in stimulating collagen fibrils and
proteoglycans in the cartilage matrix, thus improving
joint mobility [19]. Oxidative stress is an important
factor of aging that also contributes to osteoporosis. It
induces bone resorption due to superoxide production by
osteoclasts, which leads to bone degradation [5, 20, 21].
The dietary fiber included in the formulation is
a prebiotic that ensures normal functioning of the
gastrointestinal tract and has a beneficial effect on lipid
and carbohydrate metabolism. In addition, indigestible
oligosaccharides increase the absorption of various
minerals, contributing to bone mineralization [22].
Figure 2 Amino acid contents in collagen fermentolysate
0
5
10
15
20
25
30
35
40
45
>400-600 230-400 170-230 100-170 40-100 20-40 10-20
Total protein, %
Molecular weight, kDa
0
2
4
6
8
10
12
14
16
Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-Cys Val Met Phe Ile Leu Lys OPro Pro
Amino acids, g/100 g protein
0.8
1.2
1.6
2.0
elements, mg/kg
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Of great importance are chondroprotectors –
glucosamine sulfate and chondroitin sulfate. They have a
positive effect on metabolic processes in cartilage tissue,
slowing down degenerative changes in joints and the
spine.
Our choice of ingredients was determined by two
main objectives. Firstly, we aimed to formulate an
efficient functional product with a high nutritional
value. Secondly, we wanted this product to have good
sensory characteristics. Both objectives could be
achieved with pumpkin powder of the Gribovskaya
variety. The pumpkin was peeled, cut into 5–8 mm
pieces, blanched for 3–5 min, and placed on racks for
10–15 min to remove water. The pieces were then dried
in a convection drying chamber in two stages – first,
at 90 ± 5°С to a moisture content of 40–42% and then,
at 60 ± 5°C to a moisture content of 3.0–5.0%. The dried
pumpkin was crushed to a particle size of 0.2 mm. The
resulting powder had a sweetish taste and yellowish
color.
To determine an optimal ratio between collagen
fermentolysate and dried pumpkin, in both functional
and sensory terms, we carried out a sensory experiment.
The panelists preferred the taste characteristics of a
80:20 ratio between protein hydrolysate and pumpkin
powder. A higher content of hydrolysate gave the
product a pronounced bitter taste, which was considered
unacceptable.
The amounts of functional ingredients had to meet
the standard physiological needs without spoiling
the consumer appeal. These ingredients included
calcium lactate, glucosamine sulfate, chondroitin
sulfate, ascorbic acid (vitamin C), tocopherol acetate
(vitamin E), and cholecalciferol (vitamin D3).
The formulated product is a dry powder for
preparing a functional drink (Table 1).
Pre-mixtures based on the compatibility and fineness
of ingredients were introduced into a drum-type mixer
in two stages to ensure uniformity. The first premixture
included fine ingredients in smaller quantities
(vitamins D3, E, and C, chondroitin sulfate, and
glucosamine sulfate). At the second stage, they were
combined with the remaining components (protein
hydrolysate, calcium lactate, dried pumpkin, and dietary
fiber). A drink can be prepared by mixing 12 g of the
concentrate with 100 mL of water. Three servings per
day are needed to provide a good preventative effect.
Next, we studied the sensory, physicochemical,
microbiological, and toxicological indicators of
the developed product. The nutritional value of the
powdered product is shown in Table 2.
When formulating a functional product based on
hydrolysates, it is important to crease a characteristic
sensory profile. The sensory indicators of our functional
product, both in powdered and ready-to-use form, are
presented in Table 3.
The microbiological properties and contents of toxic
substances (lead, arsenic, cadmium, and mercury) in the
Table 1 Product formulation
Components Mass fraction, %
Collagen fermentolysate 56.00
Dried pumpkin 20.00
Inulin 11.00
Calcium lactate 7.00
Glucosamine sulfate 3.00
Chondroitin sulfate 1.00
Ascorbic acid (vitamin C) 0.41
Tocopherol acetate (vitamin E) 0.09
Cholecalciferol (Vitamin D3) 0.13×10–4
Table 2 Nutritional value of the powdered product
Component Content
per 100 g
powder
Content per
serving (% of
daily intake)
Proteins, g 50.0 6.0
Carbohydrates, g 14.0 2.0
Dietary fiber, g 12.0 1.4 (7%)
Ascorbic acid, mg 410.0 49.0 (80%)
Tocopherol, mg 90.0 10.8 (108%)
Cholecalciferol, μg 13.0 1.6 (31%)
Calcium, mg 1100.0 140.0 (15%)
Glucosamine sulfate, mg 3000.0 360.0 (51%)
Chondroitin sulfate, mg 1000.0 120.0 (20%)
Table 3 Sensory characteristics of the functional product
Indicator Product characteristics
Powder Drink
Appearance Fine, light yellow powder consisting
of single agglomerated particles
Transparent light yellow liquid without sediment
Consistency Loose, agglomerated particles disintegrate
under light mechanical impact
Liquid, homogeneous, without settling
Color Light yellow Light yellow, intense, with no gloss
Aroma Mild, with a pumpkin note Mild, with a light pumpkin note
Taste – Pleasant, slightly sweet, with a pumpkin
flavor, and slight sourness
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functional product were analyzed against the Technical
Regulations of the Customs Union 021/2011 “On food
safety” (Table 4 and Fig. 3).
We found that the concentrations of lead and arsenic
were significantly below the permissible values, and
the contents of cadmium and mercury were within the
norms established by TR CU 021/2011. This means that
our functional product met the safety requirements of
TR CU 021/2011.
Thus, we developed a powdered functional
product based on collagen fermentolysate to prevent
osteoporosis. Mixed with water, the drink can be used
as an additional source of connective tissue protein,
calcium, vitamins C, E, and D3, as well as dietary fiber
and chondroprotectors.
Its effectiveness was confirmed in the experiment
on laboratory animals with modelled osteoporosis
(ovariectomized female rats).
The weight of the intact animals was mostly stable
throughout the experiment, with a slight increase from
the 9th to the 15th day and from the 19th to the 26th
day. The control animals, which received distilled water,
gained weight during the entire experiment, especially
from the 1st to the 12th day and from the 19th to the
26th day. Two groups of experimental animals, which
received glucosamine + chondroitin and the drink based
on collagen fermentolysate, also gained weight from the
1st to the 12th day and from the 15th to the 26th day.
By the end of the experiment, the weight gain
in the ovariectomized rats treated with distilled
water, glucosamine + chondroitin, and the collagen
fermentolysate drink was 16.0, 12.5, and 14.3%,
respectively. The weight gain in the intact group was
5.3% (Fig. 4). Our data were consistent with the results
of other studies [23]. Our findings were associated with
a deficiency of estrogen that decreases the secretion of
leptin (a hormone with anorexigenic effect) from adipose
tissue, thus leading to hyperphagia.
Blood analysis showed a 29.6–60.5% increase in
leukocytes in the ovariectomized animals, compared
to the intact group. However, statistical significance
was only registered in the group that received distilled
water. A statistically significant (P < 0.05) increase of
Table 4 Microbiological indicators of the functional product
Indicator Content
in the powder
Standard content,
as in TR CU 021/2011
Mesophilic aerobic and facultative anaerobic microorganisms, CFU/g, max. 8×104 1×105
Mass of the product in which coliforms are not allowed, g not detected 0.1
Mass of the product in which pathogenic bacteria, including salmonella, are not allowed, g not detected 25.0
Mold, CFU/g, max. 30 200
Figure 3 Contents of toxic elements in the functional product
Figure 4 Weight changes in animals throughout
the experiment
Molecular weight, kDa
0
2
4
6
8
10
12
14
16
Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-Cys Val Met Phe Ile Leu Lys OPro Amino acids, g/100 g protein
0.0
0.4
0.8
1.2
1.6
2.0
lead arsenic cadmium mercury
Toxic elements, mg/kg
Permissible level (TR CU 021/2011)
Content of toxic elements in the functional product
4
3
2
1
1 – rats on the standard diet (intact), 2 – rats on distilled water
(control), 3 – rats on glucosamine + chondroitin (experiment 1),
4 – rats on the collagen fermentolysate drink (experiment 2)
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46.5% in lymphocytes was observed in the rats treated
with glucosamine + chondroitin (reference sample),
compared to the intact animals. All the ovariectomized
animals had an increased content of granulocytes (up
to 86.4%), compared to the intact group, although we
found significant variation between the experimental
rats within the group. Yet, the increased concentrations
of lymphocytes and granulocytes did not significantly
affect their relative content.
The control rats treated with distilled water had
a statistically insignificant increase in monocytes of
27.8% in relation to the intact group. This growth was
more pronounced in both experimental groups (1 and 2),
averaging 2.3 times (P < 0.05) in absolute terms and up
to 47.8% (P < 0.05) in relative terms (Table 5).
The cytometric analysis showed that both
supplements to the diet (glucosamine + chondroitin
and the functional drink) contributed to increased
contents of leukocytes, granulocytes, lymphocytes, and
monocytes, although to a different extent. This meant
that they activated the blood immunity.
According to the biochemical blood analysis
(Table 6), the control rats had a significant increase,
compared to the intact rats, in alkaline phosphatase,
phosphorus, and calcium by 36.0, 2.8, and 15.6%,
respectively. They also had a significant decrease
in magnesium by 15.3%. We know that increased
concentrations of these parameters in the blood are
among the diagnostic criteria for osteoporosis. However,
the supplements of glucosamine + chondroitin and
Table 5 Cytometric blood analysis at the end of the experiment
Parameter Standard diet
(intact)
Supplements to the diet
Distilled water
(control)
glucosamine + chondroitin
(experiment 1)
drink based on collagen
fermentolisate (experiment 2)
Lymphocytes, 109/L 4.82 ± 1.47 5.48 ± 1.89 7.06 ± 1.47* 7.09 ± 2.73
Leukocytes, 109/L 6.68 ± 2.14 8.66 ± 3.56 10.72 ± 2.95* 10.25 ± 4.14
Mixture of monocytes, eosinophils,
basophils and immature cells, 109/L
0.46 ± 0.22 0.75 ± 0.35 1.04 ± 0.45* 1.01 ± 0.46*
Monocytes, % 6.68 ± 1.20 8.54 ± 1.15 9.34 ± 1.89* 9.89 ± 2.22*
Granulocytes, 109/L 1.40 ± 0.59 2.43 ± 2.15 2.61 ± 1.54 2.15 ± 1.22
Lymphocytes, % 72.50 ± 5.23 64.93 ± 11.88 67.85 ± 11.03 69.93 ± 6.78
Relative content of granulocytes, % 20.82 ± 4.73 26.53 ± 11.83 22.81 ± 9.68 20.17 ± 5.97
* – significant difference from the intact group (Р < 0.05); ** – significant difference from the control group (Р < 0.05); + – significant difference
between the experimental groups (Р < 0.05)
Table 6 Biochemical analysis of blood serum at the end of the experiment
Parameter Standard diet
(intact)
Supplements to the diet
Distilled water
(control)
Glucosamine +
chondroitin
(experiment 1)
Drink based on
collagen fermentolisate
(experiment 2)
Proteins Total protein, g/L 76.50 ± 1.26 76.68 ± 2.62 80.52 ± 5.88 78.65 ± 4.70
Albumin, g/L 42.18 ± 1.34 42.16 ± 1.91 42.11 ± 2.15 41.95 ± 1.03
Low-molecularweight
nitrogencontaining
substances
Creatinine, μmol/L 65.09 ± 4.45 60.41 ± 3.55 60.13 ± 4.68 60.14 ± 2.99
Urea, mmol/L 8.62 ± 1.60 9.65 ± 1.36 8.80 ± 1.16 8.89 ± 1.52
Pigments Bilirubin (total), μmol/L 3.02 ± 0.42 2.63 ± 0.54 2.98 ± 0.45 2.77 ± 0.49
Bilirubin (direct), μmol/L 2.12 ± 0.27 2.22 ± 0.45 2.18 ± 0.36 2.08 ± 0.31
Enzymes ASAT, U/L 90.63 ± 9.70 95.80 ± 18.74 86.77 ± 9.79 94.86 ± 15.77
ALAT, U/L 51.06 ± 16.58 48.11 ± 9.19 45.25 ± 6.54 44.48 ± 8.71
Alkaline phosphatase, U/L 218.0 ± 41.0 339.2 ± 43.4* 194.9 ± 62.4** 208.4 ± 31.5**
GGT, U/L 3.53 ± 1.30 3.55 ± 0.81 3.51 ± 0.88 3.15 ± 0.56
LDH, U/L 189.8 ± 114.3 180.2 ± 87.8 186.7 ± 49.9 191.6 ± 85.0
Lipids Cholesterol, mmol/L 2.18 ± 0.23 2.43 ± 0.58 2.73 ± 0.43* 2.60 ± 0.70
Triglycerides, mmol/L 1.73 ± 1.23 1.45 ± 0.82 1.71 ± 1.21 1.22 ± 0.37
Carbohydrates Glucose, mmol/L 11.33 ± 3.81 9.13 ± 2.32 8.66 ± 0.66 8.65 ± 1.61
Inorganic compounds Calcium, mmol/L 2.82 ± 0.17 3.34 ± 0.19* 2.87 ± 0.17 2.85 ± 0.17
Magnesium, mmol/L 1.18 ± 0.08 1.00 ± 0.05* 1.02 ± 0.08 1.11 ± 0.10**
Phosphorus, mmol/L 2.85 ± 0.22 2.93 ± 0.24* 2.90 ± 0.29 2.86 ± 0.32
* – significant difference from the intact group (Р < 0.05); ** – significant difference from the control group (Р < 0.05); + – significant difference
between the experimental groups (Р < 0.05)
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the functional drink decreased the activity of alkaline
phosphatase to the intact level. This parameter in the
experimental groups was 35.5% (P < 0.05) and 31.0%
(P < 0.05) lower than in the control group. It might
indicate a compensatory activation of the collagensynthetic
function of osteoblastic cells in response to
increased activity of osteoclasts.
The results of the biochemical analysis were
confirmed by the determination of osteocalcin.
Osteocalcin is the main non-collagenous protein of
the bone matrix that is synthesized by osteoblasts. Its
concentration in the blood reflects the metabolic activity
of osteoblasts in bone tissue, since blood osteocalcin
is synthesized, rather than released during bone
resorption [24].
There were no statistically significant changes in
osteocalcin concentrations between the groups (Table 7).
However, we found its increase of 15.0% (statistically
insignificant) in experimental group 2, which received
the functional product based on collagen fermentolysate,
compared to the control.
The contents of calcium, phosphorus, and
magnesium in the bone tissue of the animals under study
are shown in Table 8. As we can see, the control group of
ovariectomized rats had decreased contents of calcium
and magnesium (by 32.0 and 19.3%, respectively),
compared to the intact group. They also had a significant
increase in phosphorus levels (by 22.0%).
The experimental rats that received the functional
drink had increased amounts of calcium and magnesium
(by 25 and 3.0%, respectively), compared to the control
group. Although we also found a 7.0% decrease in
phosphorus, it was not statistically significant. The ratio
between calcium and phosphorus in experimental group
2 was restored to 2.4:1.0 (2.5:1.0 in the intact group).
According to our daily examinations, the general
condition of all the animals was satisfactory in
terms of appearance, coat quality, and behavior. Тhe
experimental animals looked identical to the control
group. Their coat was thick, tight, and glossy, with no
signs of fur loss. They were physically strong and had
no discharge from their natural orifices. Their limbs,
motor functions, and behavioral reactions were normal.
Their teeth were white, without plaque but with signs
of abrasion. Their mucous membranes were pale, shiny,
and smooth. The results of necropsy and macroscopic
examination did not reveal any hypofunction or
displacement in internal organs (lungs, liver, spleen,
stomach, kidneys, and pancreas). Their pulmonary
pleurae, as well as pericardial and abdominal layers,
were thin, shiny, and smooth. The hearts and aortas were
unchanged, and the vessels were moderately injected.
Some animals in the control and experimental groups
had an enlarged uterus and mucus in the fallopian tubes.
This might be associated with the involutional processes
in their reproductive organs after surgery.
We weighed the animals’ internal organs and
determined their percentage in relation to the body
weight. The results revealed no significant differences
from the physiological norms for the animals of this
species and age group.
CONCLUSION
We examined the quality characteristics of dried
collagen fermentolysate obtained from low-value byproducts
of the meat industry (pork legs and ears, 1:1).
Collagen fermentolysate contained 41% of peptide
fractions with a molecular weight of 10 to 20 kDa. It
also had high contents of glutamic acid, aspartic acid,
glycine, alanine, proline, leucine, and threonine. These
amino acids stimulate cartilage and bone cells, restore
joint tissue, and are responsible for the production of
collagen, elastin, and muscle tissue.
Table 7 Osteocalcin concentrations in the blood serum of experimental rats
Parameter Standard
diet (intact)
Supplements to the diet
Distilled
water (control)
Glucosamine + chondroitin
(experiment 1)
Drink based on collagen
fermentolisate (experiment 2)
Osteocalcin, ng/mL 2.812 ± 0.569 2.618 ± 0.441 2.761 ± 0.522 3.049 ± 0.585
Table 8 Bone mineral metabolism in rats
Parameter Standard diet
(intact)
Supplements to the diet
Distilled water
(control)
Glucosamine + chondroitin
(experiment 1)
Drink based on collagen
fermentolisate (experiment 2)
Calcium, mg% 15.3 ± 3.5 10.5 ± 2.6 14.0 ± 3.5 13.7 ± 3.4
Phosphorus, mg% 5.70 ± 0.45 7.30 ± 0.58* 6.80 ± 0.54 5.60 ± 0.44
Magnesium, mg% 7.80 ± 1.56 6.30 ± 1.26 6.80 ± 1.36 6.5 ± 1.3
Ca:P ratio 2.5:1.0 1.5:1.0 2.0:1.0 2.4:1.0
* – significant difference from the intact group (Р < 0.05); ** – significant difference from the control group (Р < 0.05); + – significant difference
between the experimental groups (Р < 0.05)
We developed a technology for a functional
product to prevent osteoporosis. Based on collagen
fermentolysate, the formulation contained pumpkin
powder, dietary fiber, calcium, chondroprotectors, and
vitamins E, C, and D3. The 80:20 ratio between protein
hydrolysate and pumpkin powder and the contents of
calcium and vitamin D3 meeting 15 and 30% of the daily
intake, respectively, ensured a high nutritional value,
functional effects, and good sensory characteristics of
the product. The product is a powdered drink designed
to mix with water. The microbiological and toxicological
analyses confirmed that the product complied with the
requirements of TR CU 021/2011.
The experiments on laboratory animals showed that
the formulated product had an osteoprotective effect
on the ovariectomized female rats. We found that those
rats which received the functional product had increased
contents of calcium and magnesium in the bone tissue
(by 25.0 and 3.0%, respectively) and a decreased content
of phosphorus (by 7.0%), compared to the control group.
In addition, their calcium to phosphorus ratio was
restored to 2.4:1.0 and the concentration of osteocalcin in
the blood serum increased by 15%.
Our study makes a theoretical contribution to
the concept of safe bone homeostasis correction and
proves that a functional drink based on connective
tissue protein can be used to prevent postmenopausal
osteoporosis associated with hypoestrogenism.
CONTRIBUTION
All the authors were equally involved in, and
therefore are equally responsible for, developing the
study concept, collecting and analyzing data, writing
and editing the manuscript, and approving its final
version.
CONFLICTS OF INTEREST
The authors declare that there is no conflict of
interest.

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