1

Biodegradation of Gluten waste from bakery industry by Staphylococcus sciuri

I.S. Sony
TKM Institute of Technology, Karuvelil, Kollam-691505, Kerala, India
Email id:sonyfiro@gmail.com

Abstract
The gluten degrading bacterial strain Staphylococcus sciuri was isolated, and gluten degradation into aminoacids by
bacterial culture was investigated in shake flask fermentation as well as RP-HPLC studies. Among the bakery
industries, halwa manufacturing units produce high amounts of proteinaceous gluten waste and the increasing number
of halwa manufacturing units in Kerala, India has formulated a need for innovative solutions to deal with this gluten
waste. In the present study, highly efficient gluten degrading bacterial culture, Staphylococcus sciuri (TKMFT 8) was
isolated from soil and wastewater collected from halwa manufacturing units, and its gluten degrading ability was
studied. Different isolates were screened for protease production, among them, S. sciuri was found to be the most potent
protease producer highest protease activity, 215.66±1.98 U/ml. Further, TLC and RP-HPLC analysis confirmed the
gluten degrading ability of S.sciuri into amino acids. The study concludes that, treatment of gluten waste with the
selected isolate provided the significant amount of amino acids after 48h of fermentation and that the utilization of S.
sciuri to treat gluten waste has future potential in the amino acid industry.

Keywords: Halwa. Gluten waste. Bacterial isolate. Staphylococcus sciuri. Amino acids

Introduction
Halwa is one of the sweet Indian dishes made up of different substrates primarily maida-the finely milled and refined
wheat flour 9. Bakery industry is considered to be one of the sources of food industry wastes, and among bakery
industries, halwa manufacturing units generate lots of residues which can be either disposed of or can be recycled. In
many food processing units, separation of useful food constituents from undesired one creates a huge fraction of solid
wastes in the initial stage of processing 2. During the manufacturing of Halwa, the flour milk is extracted by mixing
maida flour with water in equal proportion and the dough which remains after extracting the milk composed of gluten.
The food industry waste is characterized by a high ratio of product-specific waste, and this waste generation is
unavoidable. Moreover, the waste product primarily consists of the organic residue of the processed raw material. In the
food processing industries, waste disposal and by-product management give rise to problems in the field of
environmental protection and sustainability 15
To discard gluten waste, a range of waste management practices have been adopted by Halwa manufacturer’s viz.,
landfillg, incineration, feeding to livestock and finally disintegration using biogas plants. Disposal of solid waste by
dumping in landfilling is not recommended due to the generation of malodours. In addition to this, landfilling takes up
precious land space and cause soil, air, and water pollution. On the other hand, gluten waste application as animal feed
and disposal of waste by incineration represents a huge loss of valuable by-products and nutrients. Moreover, the
disposal of gluten waste to biogas plant is becoming less favorable because of blockage in biogas plant pipelines due to
the bulky structure and insoluble nature of gluten waste. Hence the development of low-cost technology for the
conversion of gluten waste into amino acids should be seriously taken care of by food scientists and microbiologists.
Nowadays, the significant challenge for the food processing industry is to retrieve and use the beneficial components
contained in the food waste 3. Hence recycling is economically and ecologically beneficial as well as the most practical
method for the disposal of bakery waste.

Gluten is a mixture of proteins present in wheat and similar grains, including barley, rye, oat, and all their species and
hybrids like spelt, Kamut, and triticale 19.Moreover, gluten has viscoelastic properties and gives elasticity to dough,
helping it rise and often gives the final product a chewy texture 11.The amino acid composition of commercial vital
wheat gluten possesses Arginine, Aspartic Acid, Histidine, Alanine, Cystine, Proline, Serine, Glutamic acid, Glycine,
Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tyrosine, and Valine. 4

Since protease production is an inherent capacity of all microorganisms protease producing microbial agents can be
used for gluten degradation into amino acids leading the recycling of food industry waste. It is presumed that soil and
wastewater collected from the food industry may be a good source of proteolytic organisms. In the present study, the
proteolytic bacterial population were isolated and used for the degradation of gluten waste into amino acids. Given the
increasing demand for amino acids, there is a need for economical production of amino acids from novel sources to
meet the local demand.

In the present study, the bacterial strain Staphylococcus sciuri was isolated and tested for its potential to degrade gluten.
Therefore the main objective of the work was to degrade gluten waste into amino acids by the highly efficient protease
producing Staphylococcus sciuri.

Materials and Methods

 2

Isolation of protease producing bacteria

Soil and water samples were collected from halwa manufacturing units located in Kollam and Karunagappally region,
Kerala, India. Gluten degrading microorganisms were isolated by serial dilution agar plate technique. 16

Screening of protease production

A total of 87 dissimilar colonies from nutrient agar plates were selected (TKMFT 01 to TKMFT 87) and subjected to
primary screening for the production of protease using gelatin agar medium. The organisms were inoculated into gelatin
agar plates followed by incubation at 37 ºC for 48 h; the clear zone diameter was measured by flooded the plates with
mercuric chloride solution. Based on the zone diameter, TKMFT 8, TKMFT 22, TKMFT 25, TKMFT 39 were found to
be protease producers who are identified by biochemical and molecular techniques and selected for secondary
screening. The selected organism was inoculated into conical flasks containing 100 ml MRS (De Man, Rogosa and
Sharpe) broth (HiMedia Laboratories, Mumbai) as it possesses a low degree of selectivity of lactobacillus. Hence
secondary microflora may grow well. The flasks were incubated over ten days at 37⁰ C in a rotary shaker. MRS broth
without bacterial inoculation kept as control. The contents of the flask were centrifuged at 10000 rpm for 10 minutes at
4⁰ C to get the crude enzyme. The protease assay was carried out from the first day to10 thday to find out the day with
maximum enzyme production for fermentation study according to the method using casein as a substrate. 20 Enzyme
activity was expressed as U/ml. The total protein content of the samples was determined by the Lowry method using
Bovine Serum Albumin (BSA) as standard.12 OD was measured at 660 nm and expressed in milligram per milliliter
(mg/ml).

Biochemical and molecular identification of protease producing bacteria

The selected organisms were identified using VITEK 2 compact-Biomerieux, automatic system (France) in Cashew
Export promotion Council of India (CEPCI), Kollam and the test method was A O A C OMA 2012.02. VITEK-2 system
imparts an automated, computer based technique of species identifications, relies on advanced colorimetry technology,
the measurement of light attenuation associated with each biochemical reactions in VITEK cards (Gram-negative
fermenting and non-fermenting bacilli (GN), Gram-positive cocci and non-spore-forming bacilli (GP), yeasts and yeast-
like organisms (YST), Gram-positive spore-forming bacilli (BCL)). The reagent cards have 64 wells and each well
contain an individual test substrate. Substrates assess various metabolic activities such as alkalinisation, acidification,
enzyme hydrolysis, and growth in the presence of inhibitory compounds. The VITEK-2 compact system combines
several advantages like rapid identification, a simple methodology, a high level of automation and taxonomically
updated databases.
Molecular identification of bacterial isolates using 16S Ribosomal RNA Sequencing

Genomic DNA of bacterial isolates TKMFT 8, TKMFT 22, TKMFT 25, TKMFT 39 and TKMFT 53 was extracted
following the method of Murray and Thompson (1980). Liquid culture (5ml) with the selected bacterial isolate was
inoculated and grown in appropriate conditions for the proper growth of the strain. The culture (1.5ml) was taken in
eppendrof and centrifuged for 2 minutes and discarded the supernatant. Resuspended the pellet in 567 μl TE buffer by
repeated pipetting and 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K were added. The eppendrof was mixed
thoroughly and incubated for 1 hr at 37°C. After incubation, 100 μl of 5M NaCl and 80 μl of CTAB/ NaCl solution
were added and finally incubated for 10 min at 65°C. Later, equal volume (0.7 to 0.8 ml) of chloroform/isoamyl
alcohol, was added, and centrifuged (10000rpm) 4 to 5 min in a microcentrifuge. Transferred the aqueous supernatant to
a fresh microcentrifuge tube and 0.6 vol isopropanol was added. Washed the pellet with 70% alcohol, respun for 5 min
and redissolved the pellet in 100 μl TE buffer.
Ribosomal 16S RNA gene from genomic DNA was amplified with a pair of primers 8-27F, 5‘-
AGAGTTTGATCCTGGCTCAG-3‘and 1495R 5'-ACGGCTACCTTGTTACGA-3’. PCR assay was performed in Bio-
Rad S1000 thermal cycler. The PCR reaction mixture contained 1μl of genomic DNA (100 ng), 2μl of each primer (100
pmol/ μl ), 2 μl 10 mM dNTP, 2.5 μl PCR buffer containing MgCl 2, 2μl of (3U/ μl) Taq DNA Polymerase, and 13.5 fill
of deionized water to make up the total volume to 25μl. The PCR programme was conducted with an initial
denaturation of the template DNA at 94°C for 3min followed by denaturation at 94°C for 1min, the annealing step was
 3

performed at 58°C for 1 min and elongation at 72°C for 1 min. The cycle was then followed by 34 cycles of
denaturation, annealing and elongation followed by a final extension step at 72°C for 10 min. The amplified PCR
products were resolved in 1.2% agarose gel composed of 0.5 mg/ml ethidium bromide -and visualized under UV (Gel-
Doc IT TM).

Sequencing of the 16S rRNA was performed using ABI Prism 3500 genetic analyzer using the big dye Terminator kit
version 3.1” (Applied Biosystems), at Chromous Biotech Pvt. Ltd, Bangalore, India. All the sequences were analyzed
against nucleotide sequences present in GenBank using the BLAST software (Altschul et al., 1980). The 16S rRNA
sequences of selected bacterial strains have been deposited in the GenBank data base and accession numbers have been
obtained.

Gluten fermentation by Staphylococcus sciuri -Shake flask fermentation

Gluten samples for fermentation study were collected from different halwa manufacturing units in and around Kollam
and Karunagappally region. Staphylococcus sciuri (TKMFT8) was grown in MRS broth, and for the fermentation study,
the bacterial isolate was cultivated until the late exponential phase of growth was reached (12 h growth cultures) .14 The
flask was added with 40 g of gluten mass, 160 ml of tap water and 10 ml culture of each organism and incubated for ten
days at 37 oC with stirring (ca.200rpm). The filtrate was extracted in diethyl ether from the first day to10 thday whereas
uninoculated samples were kept as control.

Extraction of amino acids from fermented Gluten

The extraction was performed using diethyl ether in a separating funnel fed with 200 ml fermented broth and 100 ml
diethyl ether and the separated ether layer was collected and used for further experimental studies.

Amino acids separation by Thin Layer Chromatography (TLC)

Qualitative analysis of amino acids using TLC was performed to detect the gluten degrading ability and subsequent
production of amino acids by selected bacterial isolates. Presence of amino acids were determined every 24 hrs till the
end of fermentation period (10th day) and the effect of fermentation day on amino acids production was determined.
Thin Layer Chromatography is a sensitive technique generally used for rapid and efficient analysis and separation of
components of a mixture. Separation is based on the liquid–liquid partition of the compounds between two immiscible
phases. Materials used for TLC were Silica Gel plate (Stationary phase); 1-butanol, Glacial Acetic acid and water
(mobile phase) in the ratio 4:1:1 ; known solutions of amino acids, unknown solutions of amino acids, micropipette,
developing tank, 2% ninhydrin solution, heat gun, pencil and gloves. TLC plates were prepared by mixing 25 g of the
adsorbent such as silica gel with 50 ml water (2:1 ratio). The mixture was spreaded as thick slurry on glass plate which
was air dried for 15 minutes and activation of the plate was carried out by heating in an oven at 120 oC for 30 minutes.
Silica gel plates were carefully held by the sides to prevent disturbing the silica gel layer. A pencil line was drawn about
1.5 cm from the bottom of the plate. Points were marked on the line for each one of the known and unknown amino
acids. Leaved margins on both sides of at least 1.5cm. Known amino acid standards were marked as S1, S2 and S3
which composed of mixture of amino acids and unknown amino acid sample was marked as S.
Each plate consists of 6 spots, of which 3 spots for known amino acid standards (S1, S2 and S3) and another 3 spots for
unknown amino acids in sample (S). At the point S1, a very small drop of the known amino acid standard was added.
After the evaporation of the liquid (only a few seconds) a second drop of another known amino acid was added to the
same spot. This procedure was repeated for the remaining four known amino acid standards for the spot S1. Like this,
remaining amino acid standards were added to the spots S2 and S3. The amino acid extract in diethyl ether from the
gluten fermentation broth were used as the sample (S). Fermentation was carried out using 5 bacterial cultures for a
period of 10 days. For each organism, the amino acid analysis was performed for 10 times which represented 10 days of
analysis. After the addition of standards and samples to the respective spots, all spots were allowed to dry completely.
 4

The TLC plate with the spots was placed in the developing tank with the solvent with the spots towards the bottom and
closed with the lid. The solvent was allowed to ascend through the silica gel plate until the solvent has risen to the
pencil mark at the top of the plate. The plate was removed from the tank and marked the farthest advance of solvent
front. The plate was dried in an oven at 50 0C. In order to locate the amino acids, the plates were sprayed with 2 %
ninhydrin solution and heated. As a result of the reaction of ninhydrin with amino acids, coloured products were
developed and marked. The chromatograms were observed and photographed. The Rf values of standard amino acids
and test samples were calculated and compared. Rf value is defined as the ratio of the distance travelled by the amino
acids to the distance moved by the solvent front from the original spot on the plate and it is the characteristic of the
compound.
Identification of unidentified amino acids in the sample by comparing the Rf value of the spots obtained in the TLC
chromatogram with the Rf value of the standard and spots may be tentatively identified. The identification of spots
within the sample chromatogram using Rf value alone can be unreliable. Hence chromatographic separation of amino
acids and their subsequent detection and quantification were performed by Reversed-Phase High-Performance Liquid
Chromatography (RP-HPLC) at CMFRI, Kochi, Kerala, India.

Amino acid profiling of the samples by Reversed-Phase High- Performance Liquid Chromatography (RP-
HPLC)
TLC results confirmed the ability of selected bacterial isolates to degrade into amino acids. According to the results
obtained in TLC, samples for RP-HPLC were selected based on the greatest separation of amino acids. Moreover, a
comparative study was conducted for the detection of the ability of the selected bacterial isolates for the degradation of
gluten using sample gluten with commercially purchased vital wheat gluten from Biolaxi Corporation, Mumbai. The
Crude protein content of the samples was determined by Kjeldahl method (AOAC, 2002). The amino acids were
identified and quantified by comparing the retention times and peak areas of standards (WAT088122,Waters) at the
Central Laboratory of Central Marine Fisheries Research Institute (CMFRI), Cochin, Kerala, India . Samples were
injected in triplicates into high performance liquid chromatography (HPLC) system from Waters, USA with 2487 dual λ
absorbance detector 1525, binary pumps, Pico-Tag column and the output was analyzed using Waters Breeze GPC
software (Waters™, Milford, MA, USA).

Amino acid profiling of the samples were according to Heinrikson and Meredith (1984) by digesting powdered samples
(0.1 g) with 10 ml 6 N HCl at 110 o C in sealed glass tubes filled with nitrogen for 24 h. Amino acid analysis was
performed by RP-HPLC after pre-column derivatization by phenyl isothiocyanate (PITC). PITC derivatives were
prepared by adding PITC reagent (ethanol-TEA-water-PITC at a ratio of 7:1:1:1), mixed well and incubated at room
temperature for 30 minutes, and the samples allowed to dry under vacuum. Diluent was added in each sample, which
was then processed for filtration (0.45 μm syringe filter) and then 20 μl was injected. RP-HPLC was performed using a
Waters 1525 Binary HPLC pump and Waters 2487 Dual Absorbance Detector. Data was processed and analyzed using
Waters Breeze software. Operating conditions were: column temperature - 380C, column - picotag (Waters, pico tag
system); absorbance - 254 μm; pump pressure - 1500-1700 psi.

Statistical analysis of the data

Mean score and standard deviation of the three triplicates for each test was calculated. The data were subjected to two-
way analysis of variance (ANOVA) using SPSS (Chicago, IL) statistical software package (SPSS for Windows, ver. 16,
2008). To determne whether there were any differences among the means, the Tukey- Kramer HSD test were applied to
the result at 0.05 level of significance (p<0.05) for statistical evaluation of enzyme activities and protein content to find
the interaction effect of microorganism and incubation time on the dependant variables (Protein content and protease
activity).
 5

Results and discussion

Isolation, screening, and identification of protease producing bacteria

The isolated bacterial colonies were purified by subculturing technique and were stored as nutrient agar slants at 4 oC.
The proteolysis ability of bacterial cultures was evaluated using a gelatin agar medium. Highest zone diameter was
observed for TKMFT 8 (26mm) followed by TKMFT 22 (25mm) TKMFT 39 (23mm) and TKMFT 25 (20mm) as
shown in fig 1. Gelatin agar medium was best than skim milk agar medium for the qualitative test for detecting protease
production because the zone of hydrolysis was obtained with more clarity in gelatin agar plates. 17During secondary
screening, the maximum protease activity (215.19±0.90 U/ml) was exhibited by TKMFT 8 at 48h. It was clear from the
results shown in table 1 that increased protease activity decreased the protein content in the inoculated media. Protease
helps to catalyze the hydrolysis of protein into peptides and amino acids.16 In the present study, TKMFT 8 was used as a
cell factory for proteolytic enzymes for the degradation of gluten protein as their proteolytic activity has determined
earlier. A similar study has been demonstrated the use of proteases from lactobacilli, and fungal proteases from
Aspergillus spp as cell factories for multiple and complementary proteolytic enzymes to degrade the gluten of wheat
flour.14

TKMFT8 TKMFT22 TKMFT39

TKMFT39 CONTROL

Fig.1: Zone of hydrolysis of bacterial isolates on Gelatin agar medium

Table 1
Effect of incubation period on protease activity and protein content

Incubation S.sciuri (TKMFT8)

day Protease activity(U/ml) Protein(mg/ml)
1 121.49±0.54 1.34±0.02
2 215.19±0.90 0.77±0.02
3 157.64±1.31 1.10±0.10
4 137.12±1.28 0.75±0.01
5 108.5±1.87 0.73±0.02
6 89.73±1.39 0.71±0.02
7 82.2±1.52 0.68±0.01
8 70.16±1.59 0.65±0.01
9 59.43±1.8 0.66±0.01
10 46.09±1.53 0.53±0.01

Values are mean ± Standard deviation.

 6

The interaction of the incubation period and bacterial isolates showed a significant effect on protease activity (p=0.000).
R squared value (1.000) showed a high degree of interaction between the two variables. Similarly, The interaction of
incubation period and bacterial isolates showed a significant effect on protein levels (p=0.000). R squared value (0.993)
showed a high degree of interaction between the two variables.

The most potent protease producers were biochemically identified as Staphylococcus sciuri (TKMFT 8, TKMFT 22,
TKMFT 25 and TKMFT 39). A study has been reported the performances of VITEK 2 colorimetric cards for
identification of gram-positive and gram-negative bacteria. 21 Earlier findings have proved the efficiency of VITEK -2
system with 85.5% probability of accurate identification of strains. 6 In the present study, it was found to achieve a 99%
probability of identification for Staphylococcus sciuri (TKMFT 8, TKMFT 22, TKMFT 25 and TKMFT 39).18
Table 4.17 Biochemical details of organisms identified using BIOMERIEUX VITEK/GP Cards
Result
Well Test Mnemonic TKMFT 8, 22, 25,
39
2 D-AMYGDALIN AMY +
PHOSPHATIDYLINOSITOL
4 PIPLC -
PHOSPHOLIPASE C
5 D-XYLOSE dXYL -
8 ARGININE DIHYDROLASE 1 ADH1 +
9 BETA-GALACTOSIDASE BGAL -
11 ALPHA-GLUCOSIDASE AGLU +
13 Ala-Phe-Pro ARYLAMIDASE APPA -
14 CYCLODEXTRIN CDEX -
15 L-Aspartate ARYLAMIDASE AspA -
16 BETA GALACTOPYRANOSIDASE BGAR -
17 ALPHA-MANNOSIDASE AMAN -
19 PHOSPHATASE PHOS +
20 Leucine ARYLAMIDASE LeuA -
23 L-Proline ARYLAMIDASE ProA -
24 BETA GLUCURONIDASE BGURr -
25 ALPHA-GALACTOSIDASE AGAL -
26 L-Pyrrolidonyl-ARYLAMIDASE PyrA -
27 BETA-GLUCURONIDASE BGUR +
28 Alanine ARYLAMIDASE AlaA -
29 Tyrosine ARYLAMIDASE TyrA -
30 D-SORBITOL dSOR -
31 UREASE URE -
32 POLYMIXIN B RESISTANCE POLYB -
37 D-GALACTOSE dGAL +
38 D-RIBOSE dRIB +
39 L-LACTATE alkalinisation ILATk +
42 LACTOSE LAC -
44 N-ACETYL-D-GLUCOSAMINE NAG +
45 D-MALTOSE dMAL +
46 BACITRACIN RESISTANCE BACl +
47 NOVOBIOCIN RESISTANCE NOVO +
50 GROWTH IN 6.5% NaCl NC6.5 +
52 D-MANNITOL dMAN +
53 D-MANNOSE dMNE +
54 METHYL-B-D-GLUCOPYRANOSIDE MBdG +
56 PULLULAN PUL -
57 D-FAFFINOSE dRAF -
58 O/129 RESISTANCE (comp. vibrio.) O129R +
59 SALICIN SAL +
60 SACCHAROSE/SUCROSE SAC +
62 D-TREHALOSE dTRE +
63 ARGININE DIHYDROLASE 2 ADH2s -
 7

64 OPTOCHIN RESISTANCE OPTO +

The identity was confirmed by carrying out the partial sequencing of the 16S rRNA gene. Based on % identity the
isolates may be identified as follows: Staphylococcus sciuri (TKMFT 8, TKMFT 22, TKMFT 25 and TKMFT 39). The
BLAST result is presented in table 2. The sequences were deposited in NCBI GenBank with Accession numbers:
KY418027, KY418028, KY418029, and KY418030 for TKMFT 8, TKMFT 22, TKMFT25 and TKMFT 39
respectively.

Table 4.20 Partial sequence of PCR product of 16S rRNA gene sequence of selected Bacterial species (TKMFT 8,

TKMFT 22, TKMFT 25 and TKMFT 39)

Bacteria Sequences
l isolates

CGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAATTT
GTTAGTAACTGAACAAGTCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGC
CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG
CGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCAT
TKMFT
TGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGA
8 AATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGA
CGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCG
TAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATT
AAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGA
CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAA
TCTTGACATCCTTTGACCGCTCTAGAGATAGAGTCTTCCCCTTCGGGGGACAAAGTGACA
GGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA
GCGCAACCCTTAAGCTTATTTGCCATCATTAAGTTGGGCACTCTAGTTGACTGCCGGTGA
CAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGA

TTAGTAACTGAACAAGTCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCC
AGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGC
GCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATT
GGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAA
TKMFT ATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGAC
22 GCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGT
AAACGAT
GAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAATGCTGCATCTAACGCATTAATCACTC
CGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACA
AGCGGTGGAACATGTGGTTTAATTCTAAGCAACGCGAAGAACCTTACCAAATCTTGACA
TCCTTTGAGCGCTCTAGAGATAGAGTCTTCCCCTTCAGGGGACAAAGTGACAGGTGGTG
CATGGTTGTCCTCATCTCGTGTCGTGAGATGTTGGGTTAACTCCCGCAACGAGCGCAACC
CTTAAGCTTATTTGCCATCATTAAGTTGGGCACTCTATGTTGACTGCCGGTGACAAACCG
GAGG
AAGGTGGGGATGACCTCAATCATCATGCCCCTTATG

GGAGCACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGGGAAG
AACAAATTTGTTAGTAACTGAACAAGTCTTGACGGTACCTAACCAGAAAGCCACGGCTA
ACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGG
CGTAAAGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTG
GAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTG
TAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGT
TKMFT CTGTAACT
25 GACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGC
CGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGC
ATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG
GACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCA
AATCTTGACATCCTTTGACCGCTCTAGAGATAGAGTCTTCCCCTTCGGGGGACAAAGTGA
CAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG
AGCG
 8

CAACCCTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAGGTTGACTGCCGGTGACA
AACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACAC
ACGTGCTACAATGGATAATACAAAGGGCAGCGAATCCGCGAGGCCAAGCAAATCCCAT
AAAATTATTCTCAGTTCGGATT

ACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAA
ATTTGTTAGTAACTGAACAAGTCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTAC
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAA
AGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGG
TCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGG
TKMFT TGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAAC
39 TGACGC
TGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAA
ACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAA
GCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACC
CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATC
TTGACATCCTTTGACCGCTCTAGAGATAGAGTCTTCCCCTTCGGGGGACAAAGTGACAG
GTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC
GCAACCCTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTATGTTGACTGCCGGTGAC
AAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACA
CACGTGCTACAATGGATAATACAAAGGGCAGCGAATCCGCGAGGCCAAGCAAATCCCA
TAAAATTATTCTCA

Table 2
Summary of the BLAST result

Samples Sequence length blasted (bp) % identity (Accession no.) Identified name of samples
TKMFT 8 817 99 (AB 233331) Staphylococcus sciuri
TKMFT 22 816 98 (KU 323661) Staphylococcus sciuri
TKMFT 25 919 98 (KX 023250) Staphylococcus sciuri
TKMFT 39 905 98 (KX 023250) Staphylococcus sciuri

Qualitative analysis of amino acids in gluten fermented with Staphylococcus sciuri by TLC
According to the results obtained in TLC chromatograms for amino acids in gluten fermented with the isolate TKMFT
8, the maximum production of amino acids was observed on 48 hours (Day-2) of fermentation and after that the
production of amino acids started decreasing. According to the results shown in Table 4.5 and Plate 4.4, it was clear that
as the fermentation progresses amino acid production increased and reached maximum production of amino acids on 48
hours of fermentation. Moreover the highest protease activity by the isolate was obseved on 48 hours of incubation.
Hence sample taken from the second day of fermentation was selected for RP-HPLC analysis to quantify the amino
acids. TLC analysis of gluten fermented with TKMFT 8 showed the presence of Glutamic acid (E), Isoleucine (I),
Tryptophan (W), Cysteine (C), Valine (V), Threonine (T), Lysine (K), Phenylalanine (F), Leucine (L), Methionine (M),
Isoleucine (I), Alanine (A), Serine (S) and presence of unidentified amino acids were also observed and labelled as X1,
X2, X3, X4, X5, X6, X7 and X8. The identification and quantification of these amino acids were carried out by RP-
HPLC analysis. TLC results showed that highest production of amino acids was observed on 48 hours of gluten
fermentation with Staphylococcus sciuri. Moreover, the highest protease activity by the isolate was observed on 48
hours of incubation. Hence sample taken from the second day of fermentation was selected for RP-HPLC analysis.
 9

TLC chromatogram of unfermented Sample Gluten (Control)

Unfermented sample gluten and commercially purchased vital wheat gluten used as control-1 and control-2
respectively. In order to detect the presence of amino acids in control samples, qualitative analysis of amino acids using
TLC was performed. As shown in Table 4.11 and Plate 4.10, standard amino acid spots were obtained and showed
negative result for sample which revealed that protein present in the samples were not dgraded into amino acids.
According to these results, it can be concluded that bacterial isolates played the major role in the degradation of gluten
protein into aminoacids. Gluten samples may contain trace amount of free amino acids and the presence of these amino
acids were analyzed using RP-HPLC and results were recorded and compared with the samples treated with the
bacterial isolates.

RP-HPLC analysis of amino acids

The chromatographic separation, identification, and quantification of amino acids was carried out using RP-HPLC
system from Waters, USA.10 A similar study has been reported the determination of amino acid concentrations by RP-
HPLC analysis.7 The % area calculation protocol reports the area of each peak in the chromatogram as a % of the total
area of all peaks. The area % provides a suitable approximation of the relative amounts of components.In the present
study, the RP-HPLC results showed the presence of 17 amino acids in varying concentrations in sample gluten
fermented with Staphylococcus sciuri as shown in table 3. This is in good agreement with the previous results, which
reported that the contents of total and free amino acids of enzymatic wheat gluten hydrolysates (g/100 g hydrolysate)
and observed varying concentrations of 17 amino acids (Histidine, Arginine, Threonine, Valine, Methionine, Isoleucine,
Leucine, Phenylalanine, Lysine, Aspartic acid, Glutamic acid, Serine, Glycine, Alanine, Proline, Tyrosine and
Cystine).10 The study conducted by Sari et al. (2014)16 also reported the presence of above mentioned 17 amino acids in
wheat gluten which have been obtained after acid hydrolysis of wheat gluten as well as combined enzymatic and dilute
acid hydrolysis of wheat gluten.
Moreover, similar findings have been observed in the study conducted by Di cango et al. (2002) 5 where amino acid
composition in lactic acid bacterial enzymes fermented doughs composed of 16 amino acids. The RP-HPLC
chromatogram of sample gluten fermented with Staphylococcus sciuri is depicted in fig.2. It is presumed that
 10

Staphylococcus sciuri was found to be the most effective organism for gluten degradation and subsequent production of
amino acids and great liberation of amino acids during fermentation further supported these findings. Furthermore, the
amino acid concentration determined by RP- HPLC agreed well with the above results. The above findings encouraged
the use of Staphylococcus sciuri for the degradation of gluten waste from the halwa manufacturing units

Table 3
RP-HPLC analysis data of amino acids in gluten sample fermented with
Staphylococcus sciuri (TKMFT 8)

Peak Name RT (min) Area (V*sec) Height ( V) %Height

% Area
Asp 1.802 1163988 2.29 306336 3.83
Glu 1.935 16971664 21.07 3652149 25.15
Ser 3.150 1906081 4.33 343607 4.29
Gly 3.352 5271489 11.97 900393 11.25
His 3.565 819191 1.76 116207 1.45
Arg 3.974 1484793 3.37 234811 2.93
Thr 4.084 1309683 2.97 231674 2.90
Ala 4.254 2377840 5.40 417051 5.21
Pro 4.434 7068265 18.54 1258583 15.73
NH3 4.949 1222046 2.78 163514 2.04
Tyr 6.026 1101939 2.88 168660 2.11
Val 6.631 2235767 4.84 370998 4.64
Met 7.000 958694 2.18 135559 1.69
Cys 7.639 379848 0.86 50314 0.63
Ile 8.050 1957973 5.03 333405 4.17
Leu 8.202 2862003 7.52 539252 6.74
Phe 8.971 1899982 4.31 333555 4.17
Lys 9.835 2238041 5.08 328828 4.11
 11

Fig. 2: RP-HPLC chromatogram of sample gluten fermented with Staphylococcus sciuri (TKMFT 8)

Conclusion

In the present study, the potent protease producing microorganism is identified as Staphylococcus sciuri. The selected
bacterial isolate may be considered as a potential commercial source of protease and also as a very promising candidate
for gluten degradation. Microbial fermentation of gluten has yielded most of the essential amino acids required by
plants. The crude extract of post fermented gluten solution contains nutrients as amino acids which can be directly
applied as foliar application with suitable concentration according to the requirement of the crop. The use and
requirement of amino acids in agriculture is quite large. This is the first report on gluten degradation into amino acids
by protease of Staphylococcus sciuri. The environmental pollution as well as waste disposal problem is solved.

Acknowledgement
Authors acknowledge the CEPCI, Kollam and TKM Institute of Technology for providing all the necessary facilities for
carrying out this research work.

References
1. Altschul S.F., Gish W., Miller W., Myers E.W. and Lipman D.J., Basic local alignment search tool, Journal of
Molecular Biology. 215(3), 403–410 (1990)

2. Ammar A.S.M., Food Processing Wastes: Characteristics, Treatments and Utilization Review, Journal of
Agricultural and Veterinary Sciences Qassim University.7(2),71-84 (2014)

3. Arvanitoyannis I.S. and Tserkezou P., Corn and rice waste: A comparative and critical presentation of
methods and current and potential uses of treated waste, International Journal of Food Science and Technology.
43(6),958-988 (2008)

4. Day L., Augustin M.A., Batey I.L.and Wrigley,C.W., Wheat-gluten uses and industry needs, Trends Food Sci.
Technol. 17(2),82-90 (2006)

5. Di Cagno R., De Angelis M., Lavermicocca P., De Vincenzi M., Giovannini C., Faccia M.and Gobbetti M.,
Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides
involved in human cereal intolerance, Appl. Environ. Microbiol. 68(2),623–633 (2002)
6. Funke G., Monnet D., deBernardis C., von Graevenitz A.and Freney, J., Evaluation of the VITEK 2 system
for rapid identification of medically relevant gram-negative rods, J. Clin. Microbiol.36(7),1948–1952 (1998)

7. Gerez C.L., Rollan G.C. and de Valdez1 G.F., Gluten breakdown by lactobacilli and pediococci strains
isolated from sourdough, The Society for Applied Microbiology. 42(5),459-464 (2006)

8. Heinrikson R.L.and Meredith S.C., Amino acid analysis by Reversed-phase high-performance liquid

chromatography: precolumn derivatization with phenylisothiocyanate, Anal Biochem. 136, 65-74 (1984)

9. Itagi H.B., Singh V., Indiramma A.R.and Prakash, M., Shelf stable multigrain halwa mixes: preparation of
halwa, their textural and sensory studies, J Food. Sci Technol. 50, 879-889 (2013)

10. Koo S.H., Bae I.Y., Lee S., Lee D., Hur B. and Lee H.G., Evaluation of wheat gluten hydrolysates as taste-
active compounds with antioxidant activity, J Food Sci Technol.51(3),535–542 (2014)

11. Lamacchia C., Camarca A., Picascia S., Di Luccia A. and Gianfrani, C., Cereal-based gluten-free food: how
to reconcile nutritional and technological properties of wheat proteins with safety for celiac disease
patients, Nutrients (Review). 6(2),575–590 (2014)

12. Lowry O.H., Rosebrough N.J., Farr A.L. and Randall,R.J., Protein measurement with Folin phenol reagent, J.
Biol. Chem.193(1),265-275(1951)
 12

13. Murray H.G.and Thompson W.F., Rapid isolation of high molecular weight DNA, Nucleic Acids Res.
8(19),4321-4325(1980)

14. Rizzello C.G., De Angelis M., Di Cagno R., Camarca A., Silano M., Losito I., De Vincenzi M., De Bari
M.D., Palmisano F.and Maurano, F., Highly efficient gluten degradation by lactobacilli and fungal proteases
during food processing: new perspectives for celiac disease, Appl Environ Microbiol.73(14),4499–4507(2007)

15. Russ N. and Pittroff,R.M., Utilizing waste products from the food production and processing industries,
Crit.Rev Food Sci Nutr. 44(1),57-62(2004)

16. Sari Y.W., Alting A.C., Floris.R., Sanders J.P.M. and Bruins M.E., Glutamic acid production from wheat by-
products using enzymatic and acid hydrolysis, Biomass and bioenergy.67,451-459 (2014)
17. Sharma A.K., Sharma V., Saxena J., Yadav B., Alam A.and Prakash A.,Isolation and Screening of
Extracellular Protease Enzyme from Bacterial and Fungal Isolates of Soil, International Journal of Scientific
Research in Environmental Sciences. 3(9),334-340 (2015)
18. Simgamsetty S., Yarlagadda P., Yenigalla B.M.and Myneni, R.B., Ease with VITEK 2 systems, biomerieux in
identification of non-lactose fermenting bacteria including their antibiotic drug susceptibility: our experience,
International Journal of Research in Medical Sciences. 4(3),813-817(2016)

19. Tovoli F., Masi C., Guidetti E., Negrini G., Paterini P. and Bolondi L., Clinical and diagnostic aspects of
gluten related disorders, World J Clin Cases (Review). 3(3),275–284 (2015)

20. Tsuchida O., Yamagota Y., Ishizuka J., Arai J., Yamada J., Takeuchi M.and Ichishima E., An alkaline
protease of an alkalophilic Bacillus sp. Curr. Microbiol. 14(1),7-12 (1986)

21. Wallet F., Lorez C., Renaux E., Lemaitre N.and Courcol R.J., Performances of VITEK 2 Colorimetric Cards
for Identification of Gram-Positive and Gram-Negative Bacteria, Journal of Clinical Microbiology. 43(9),4402–
4406 (2005)