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Fermented meat and digestive tract of snail as
amino acid supplements for functional feed of
native chickens
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The 4th International Conference on Food and Agriculture
IOP Conf. Series: Earth and Environmental Science 980 (2022) 012020
IOP Publishing
doi:10.1088/1755-1315/980/1/012020
1
Fermented meat and digestive tract of snail as amino acid
supplements for functional feed of native chickens
U Suryadi, R T Hertamawati, S Imam*
Department of Animal Science, Politeknik Negeri Jember, Mastrip Street PO BOX
164, Jember 68101, East Java, Indonesia
*Email: shokhirul_imam@polije.ac.id
Abstract. Sources of amino acids and lactic acid bacteria have been developed from the
hydrolysis of snail meat and its digestive tract through a fermentation process using a consortium
of microorganisms present in cow's milk whey, which is fermented by microorganisms from rice
washing water with internal microorganisms from snails. The fermentation process used the
Submerged Fermentation (SmF) method. The fermentation process was carried out using Lactic
Acid Bacteria (LAB) inoculants contained in rice washing water. This product was researched
and developed with the aim of being a feed supplement to reduce the use of fish meal or other
protein sources such as concentrates in the preparation of feed for poultry. Product development
studies were carried out on the LAB content in rice washing water and fermented snail meat and
its digestive tract, as well as their amino acid content. The results of the study showed that at the
dilution of rice washing water, approximately 3 different colonies of LAB were found, the
dilution of 10-1 to 10-4 could not be counted but at the dilution of 10-5, 50 colonies of LAB were
obtained with an amount of 5 x 107 CFU/ml. The fermented snail meat and its digestive tract
contained approximately 4 different LAB colonies and found 3 x 104 CFU/ml at a dilution of 10-
3, and contained 6 types of amino acids, namely; alanine, glycine, cysteine, arginine, lysine, and
proline. The conclusion of this research is that fermented snail meat and digestive tract have
potential as an amino acid supplement for functional feed for native chickens.
1. Introduction
Snails are wild animals as a source of local feed ingredients whose use is often neglected, and their
existence is often exterminated because snails cause huge losses to the agricultural industry. Snails are
natural enemies for farmers that can cause damage to plants. This snail has a high reproductive capacity
so that it has a detrimental impact on the destruction of fields [1].
The population of wild snails is currently decreasing in their natural habitat, and some edible snail
species are overexploited, so that the snails are threatened with extinction. therefore there is an
increasing interest in snail cultivation [2]. However, many difficulties occur in snails because as
agricultural pests, animals that are very vulnerable due to climate change and infection and their
physiology has not been adequately explored [3].
Snails are very important ecologically and economically, and are used as food and medicine. Snails
possess adaptive immunity, snails possess and develop a series of innate defense and immune
mechanisms to respond to invading pathogenic bacteria and more importantly establish and maintain a
beneficial symbiotic microbiota in the gut [4]. Snails physically and chemically externally and internally
The 4th International Conference on Food and Agriculture
IOP Conf. Series: Earth and Environmental Science 980 (2022) 012020
IOP Publishing
doi:10.1088/1755-1315/980/1/012020
2
have defenses such as gastric mucus and gastric acid fluid [5]. lubricating mucus that covers the body
and has a role as antimicrobial activity [6].
Snails are a rich source of protein containing large amounts of essential amino acids, calcium and a
small amount of fat [7]. Snails contain nutrients that can be used as a source of protein, besides that they
also contain microbiota in their intestines. Raw snails contain 81.96% water with crude protein content
of 11.3 g/100 g, ash 2.56 g/100 g, fiber 0.18 g/100 g, and fat 6.78 g/100g on a dry basis, and contains
significant amounts of minerals and amino acids Asp, Ser, Glu, Gly, His, Arg, Thr, Ala, Pro, Tyr, Val,
Lys, Ile, Leu, Phe, Cys, Met, Trp [8].
Total bacterial content ranged from 1.00-1.50 x 108 cfu/g, Coliform count ranged from 1.68-2.20 x
107 cfu/g, Salmonella/Shigella count ranged from 5.2-8.2 x 107cfu/g, the number of lactic acid bacteria
ranged from 1.03-1.30 x 108 cfu/g and the number of fungi ranged from 7.3 x 107 - 1.00 x 108 cfu/g.
Microorganisms that can be isolated are Bacillus subtilis, Staphylococcus aureus, Lactobacillus spp.,
Escherichia coli, Micrococcus luteus and Bacillus cereus while the fungal isolates are Aspergillus
terrus, Aspergillus fumigatus, Absidia sp., Fusarium oxysporum, Eurotium sp. and Aspergillus flavus
[9].
In the intestinal tract of snails, Lactobacillus was found which has the potential as a specific probiotic
for itself. Intestinal commensal lactobacilli strains interact with the gastrointestinal mucosa of snails and
enhance cellular immune responses such as: chemotaxis and phagocytosis, as well as the antimicrobial
activity of hemolymph factors [3].
The technological innovation that can be used to increase the nutritional value and digestibility of
feed ingredients is the fermentation process. Fermentation is the oldest technique in food preservation,
besides being low in cost, fermentation can also produce products that have better nutritional value than
the original ingredients [10]. Fermentation is all kinds of metabolic processes with the help of enzymes
from microbes (microorganisms) to carry out oxidation, reduction, hydrolysis and other chemical
reactions, resulting in chemical changes in an organic substrate by producing certain products [11].
Fermented foods are more nutritious than unfermented foods [12], this is due to the enzymatic
processes produced by microorganisms through catabolic and anabolic processes, breaking down
complex compounds, and synthesizing vitamin complexes and other growth factors [13]. Fermentation
is a natural way to increase vitamins, essential amino acids, anti-nutrients, protein, food appearance,
taste and aroma enhanced. Fermentation also helps in reducing the energy required for cooking as well
as making safer products [14,15].
Taking into account the potential for the existence of snails in epidemiology, the content of nutritional
elements, and microbiological elements, then with a touch of biotechnology application of snail meat
fermentation and its digestive tract it is possible to process it into amino acid supplements. This study
aims to examine the potential of snail meat and it’s digestive track as a amino acid supplement for
functional feed of native chickens.
2. Materials and Methods
2.1. Fermentation of snail meat and its digestive tract
Rice washing water is heated to a temperature of 63°C, then cooled and cured for 3 days. Rice washing
water is used to ferment cow's milk for 5 days to form whey (cloudy liquid) and curd (clumps). The
resulting whey is mixed with sugar water and then used to ferment the snail meat and its digestive tract
in a 1:1:1 proportion. Fermentation was carried out for 3 weeks so that the pH of the liquid with an acid
aroma was obtained, the color was cloudy brown, pH 3.71 and a temperature of 28°C. This liquid is
used as a source of internal inoculants of microorganisms.
2.2. Isolation of lactic acid bacteria
Snail samples that have been fermented with several media as much as 25 ml were suspended in 225 ml
of 0.85% NaCl aseptically (considered a dilution of 10-1). Then a series of dilutions were carried out in
stages from 10-2 to 10-5. A total of 1 ml of suspension from each dilution was grown on GYP (Glucose
The 4th International Conference on Food and Agriculture
IOP Conf. Series: Earth and Environmental Science 980 (2022) 012020
IOP Publishing
doi:10.1088/1755-1315/980/1/012020
3
Yeast Extract Peptone) agar media (glucose 10 g, yeast extract 10 g, peptone 5 g, beef extract 2 g,
Sodium Acetate 2 g, 0.5 ml salt solution), 10 g of tween 80 and 12 g of agar in one liter of distilled
water) to which 5 g of CaCO3 was added, then incubated at 37°C for 3 days. The isolation method was
carried out by sprinkling the spread plate method. Lactic acid bacteria was indicated by the formation
of a clear zone around the colony. Colonies considered as lactic acid bacteria were sampled using a
sterile ose needle and inoculated on GYP media. CaCO3 agar to obtain pure colonies. Pure colonies
obtained were re-grown on GYP media. CaCO3 agar slanted as stock for further tests.
2.3. Counting Total Bacteria
Total plate count is all colonies growing on NA media. The number of bacterial colonies counted on
petri dishes is between 25-250 colonies. After that the amount obtained is multiplied by the dilution.
The method of calculating TPC is: The number of bacteria in a petri dish x 1/ dilution factor.
2.4. Amino acid analysis
Amino acids were analyzed using High Performance Liquid Chromatography (HPLC). The principle of
this amino acid analysis is that amino acids from proteins are liberated by hydrolysis with 6N HCl. The
hydrolyzate was dissolved in sodium citrate buffer and each amino acid was separated using HPLC.
Prior to the hydrolysis process, protein extraction was carried out using the Kjeldahl method.
3. Results and Discussion
Isolation and characterization of bacteria in food products is important to determine and differentiate
the beneficial or detrimental effects of the microbiota in a particular sample. Isolation in the form of
natural microbial communities can be exploited in one of the media with various bioprocesses to
encourage the conversion process by selecting certain microbial species. One of the potential media that
can be used as a source of inoculants is rice washing water. Rice water washing contains starch as a
source of microbial food, especially Lactic Acid Bacteria (LAB).
The results of observations of LAB colonies from rice washing water were found about 3 different
colonies. The morphological and physiological characteristics of the three LAB colonies used in this
experiment are presented in Table 1. The existence of 3 types of LAB colonies contained in rice washing
water, this may consist of 3 different types, according to [16], microorganisms grow together, both in
natural communities and in artificial or synthetic cultures. The results of the 10-1 to 10-4 dilutions cannot
be calculated, but the 10-5 dilutions obtained the number of 50 colonies with a total of 5 x 107 CFU/ml
(Table 2.).
Table 1. Morphological and physiological characteristics of Lactic Acid Bacteria colonies from
rice washing water.
Colony
Characteristics
Form colony
Edge colony
Colony surface
Top color
Bottom color
A1
Circular
Entire
Convex
Milky white
Creamy
white
A2
Circular
Entire
Convex
Yellow
Yellow
A3
Circular
Entire
Convex
Creamy white
Cream
LAB is a group of bacteria that produce lactic acid as a result of carbohydrate fermentation in the
substrate [17]. LAB is a large group of bacteria with the ability to ferment sugars into lactic acid. Lactic
acid bacteria (LAB) is a type of gram-positive bacteria that uses carbohydrates as the only or main
carbon source [18]. Fermentation microorganisms mainly involve LAB. such as Enterococcus,
Streptococcus, Leuconostoc, Lactobacillus, and Pediococcus [19]. The fermentation process also
increases the activity of microbial enzymes because it provides an acidic environment at a temperature
of 22-25ºC [19]. Lactic acid fermentation is an intracellular anaerobic enzymatic process to convert
The 4th International Conference on Food and Agriculture
IOP Conf. Series: Earth and Environmental Science 980 (2022) 012020
IOP Publishing
doi:10.1088/1755-1315/980/1/012020
4
sugar into lactic acid, which is one approach to obtain the energy needed for cell life processes under
anaerobic conditions [19–22].
Table 2. Results of Isolation of LAB and TPC from rice washing water.
Sample
Different
types of
LAB
TPC results
Results CFU/ml
Dilution
10-1
10-2
10-3
10-4
10-5
10-1
10-2
10-3
10-4
10-5
Rice
washing
water
3
CNC
CNC
CNC
CNC
50
CNC
CNC
CNC
CNC
5 x
107
Note: CNC = Cannot be calculated
The presence of LAB content from the soaking of rice washing water is possible that these bacteria
can degrade snail meat through fermentation. The presence of microbial diversity implies that
qualitatively there is an opportunity to convert substrates and and produce naturally produced secondary
metabolites [16]. The results of the isolation of lactic acid bakeria colonies from meat and digestive tract
of fermented snails found approximately 4 different LAB colonies and at a dilution of 10-3 found 3 x
104 CFU/ml.
Table 3. Observations of LAB colonies from meat and digestive tract fermented snail.
Colony
Characteristics
Colony form
Colony edge
Colony surface
Top color
Bottom color
AB1
Circular
Entire
Convex
Milky white
Cream
AB2
Circular
Entire
Flat
Clear white
Cream
AB3
Circular
Entire
Raised with
concave beveled
edge
Milky white
Cream
AB4
Circular
Entire
Convex
Cream
Yellow
cream
Table 4. Results of isolation of bacteria and TPC LAB from meat and digestive tract of snails
fermented.
Sample
Different
types of
LAB
TPC results
CFU/ml results
Dilution
10-1
10-2
10-3
10-4
10-5
10-1
10-2
10-3
10-4
10-5
Rice
washing
water +
Snail
4
65
16
3
-
-
65 x 102
16 x 103
3 x 104
-
-
Note: - = Cannot be calculated
All fermented foods and beverages were identified to contain LAB which is the most important
dominant microbiota that contributes to beneficial effects in fermented foods/drinks [19]. Lactobacillus,
Lactococcus, Streptococcus, Pediococcus, Leuconostoc, and Enterococcus are some of the
facultative/obligate anaerobic gram-positive and acidogenic (lactic acid) bacteria used to metabolize
saccharides with different degrees of efficiency into alcohols, lactic acid, lipids, amino acids, and
compounds. Aliphatic [23]. Enzymes such as amylase, proteinase, mannase, catalase, cellulose, are
generally produced from the fermentation of microorganisms, especially Bacillus [24].
The 4th International Conference on Food and Agriculture
IOP Conf. Series: Earth and Environmental Science 980 (2022) 012020
IOP Publishing
doi:10.1088/1755-1315/980/1/012020
5
The presence of LAB from the soaking of rice washing water, it is possible that these bacteria can
degrade snail meat through fermentation. Snail meat fermentation process can cause snail meat protein
to be hydrolyzed into amino acids through the services of microorganisms that produce protease
enzymes. The results of the analysis of the amino acid content of fermented snail meat and its digestive
tract obtained 6 types of amino acids (Table 5).
Table 5. Amino acid content of fermented snail meat and its
digestive tract.
No
Types of amino acids
Amino acid level (%)
1
Glycine
0.226
2
Alanine
0.189
3
Lysine
0.176
4
Arginine
0.121
5
Cystein
0.203
6
Proline
0.185
During the fermentation process, enzymes produced by microorganisms initially degrade the
macronutrients of the substrate [15], The three main pathways by which LAB are involved in the
manufacture of fermented foods and the development of their taste, are (a) glycolysis (fermentation of
sugars), (b) lipolysis (fat degradation) and (c) proteolysis (protein degradation) [23]. Protein degradation
will result in increased protein solubility and the availability of several micronutrients and essential
amino acids [25]. Sharma et al., [26] microorganism activity plays an important role in food fermentation
by showing changes in chemical and physical properties of food. As a fermentation strain, LAB must
have several important metabolic characteristics, such as the ability to produce acid and aroma, the
ability to hydrolyze protein, the ability to produce viscous exopolysaccharides and the ability to inhibit
bacteria [27].
Fermentation techniques are used either to produce large numbers of microbial cells or to produce
extracellular microbial products (e.g. lactic acid), enzymes, amino acids, vitamins and other
pharmaceutical compounds [28]. There are various roles of LAB in fermented foods such as to produce
better taste, aroma, and texture, increase nutritional value, safety, and some functional compounds [29].
Fermentation processes in foods often cause changes in nutritional and biochemical qualities relative
to the starting material. Fermented foods consist of a very complex ecosystem consisting of: enzymes
from raw materials that interact with the metabolic activity of fermenting microorganisms. Fermented
microorganisms provide a unique approach to food stability through physical and biochemical changes
in fermented foods. These fermented foods can benefit consumers compared to simple foods in terms of
antioxidants, peptide production, organoleptic and probiotic properties, and antimicrobial activity. It
also helps in anti-nutrient levels and toxin levels. The quality and number of microbial communities in
fermented foods varies based on the manufacturing process and storage/durability conditions. This
review contributes to current research on biochemical changes during food fermentation. The focus is
on changes in biochemical compounds that determine the characteristics of the final fermented food
product from the original food source [26].
Several strains of Lactaseibacillus casei, Lacticaseibacillus rhamnosus, and Streptococcus
thermophilus contribute to the production of branched-chain amino acid derivatives and aromatic amino
acid derivatives [30]. The final product of hydrolysis is free amino acids, which are rich sources of
nitrogen. Enzymatic breakdown of protein is very important for LAB, because it provides free amino
acids needed for protein synthesis and LAB growth itself, and allows a significant reduction in energy
expenditure in amino acid synthesis [31]. The proteolytic system of lactic acid bacteria consists of
proteinases, peptidases, and specific transport proteins. Proteinases break down casein into peptides,
then peptidases (intracellularly) degrade the peptides into amino acids and smaller peptides [31].
The 4th International Conference on Food and Agriculture
IOP Conf. Series: Earth and Environmental Science 980 (2022) 012020
IOP Publishing
doi:10.1088/1755-1315/980/1/012020
6
Under the influence of proteases in meat, some muscle protein macromolecules; disruption of
disulfide bonds; and an increase in the reactivity of the sulfhydryl, hydroxyl, carboxyl, and amino
groups. During this process, sarcoplasmic proteins are hydrolyzed to a significant degree, and depending
on enzyme specificity, myofibrillar and connective tissue proteins are partially hydrolyzed. Meat under
the influence of proteases, the degree of hydration is increased, and the digestibility of protein is
increased; that is, more meat is available for proteolytic digestive enzymes. Due to this process, the
content of solutes and free amino acids and small peptides is increased, which improves the palatability
of the meat. Proteolytic enzymes contribute to flavor enhancement and are protective factors against
many unfavorable microorganisms [31].
The degradation of meat proteins into peptides is catalyzed by proteolytic enzymes present in LAB,
and the peptides are then further hydrolyzed by exopeptidase and endopeptidase into small peptides and
amino acids. LAB has only a weak proteolytic action on fermented meat myofibrillar proteins [32,33].
However, some Lb. casei, Lb. plantarum, Lb. curvatus and Lb. active sakei strains contribute to the
hydrolysis of sarcoplasmic proteins [34] and the subsequent decomposition of peptides into amino acids.
Some of the activities of Lb. sake, Lb. curvatus and Lb. plantarum strain has leucine and valine
amino-peptidase, which contribute to protein and peptide catabolism yielding free amino acids,
precursors of flavor compounds in the final product [35]. The production of high-quality fermented milk
products depends on the proteolytic system of the starter bacteria, because the peptidases and amino
acids formed have a direct impact on taste or serve as flavor precursors in these products [36].
The proteolysis process can not only be carried out chemically and or the addition of commercial
enzymes to the substrate can also use certain microbial strains that secrete proteases to hydrolyze
substrate proteins [37]. Bacillus species are involved in the process of enzymatic hydrolysis of proteins
as producers of peptides and amino acids, which are claimed to have health benefits [38]. The
physicochemical properties of protein hydrolysates depend on the protein substrate, the specificity of
the enzymes used for proteolysis, and the hydrolysis conditions [39]. Amino acid profile is the most
important nutritional parameter (protein quality and digestibility) of a protein source food.
4. Conclusion
Fermentation of snail meat and its digestive tract produced 4 different LAB colonies with 6 types of
amino acid content, namely; Alanine, Glycine, Cysteine, Arginine, Lysine, and Proline. Fermented snail
meat and its digestive tract have potential as an amino acid supplement for functional chicken feed.
5. Acknowledgments
The author would like to express his gratitude to the Directorate of Research and Community Service,
Deputy for Research and Development Strengthening, Ministry of Research and Technology / National
Research and Innovation Agency, who has funded this research with contract number
753/PL17.4/PG/2021.
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