Gluten-degrading bacteria are microbes capable of breaking down gluten proteins, which can be beneficial for individuals with gluten intolerance or sensitivity. These bacteria help digest gluten, reducing the risk of adverse reactions in susceptible individuals. However, it's important to note that these bacteria may not completely eliminate the effects of gluten for those with celiac disease, and a strict gluten-free diet is typically still necessary. Always consult with a healthcare professional for personalized dietary advice.

1. Gluten Degrading
Bacteria
(GDB)

2. INTRODUCTION
Background
• Celiac disease (CeD) is a small-intestinal T-cell-mediated
autoimmune disorder affecting approximately 1% of the world’s
population.
• Adhering to a gluten-free diet (GFD) is the only known treatment
for CeD.
• Only 2–3% develop CeD during their lifetimes.
• The aim of this research study is to isolate GDB from the small
intestine of non-celiac FDR subjects.
Research objective
• Isolate gluten degrading bacteria from the small intestine.
• Characterize the gluten degrading capabilities of the isolated
bacteria.

3. METHODOLOGY
Sample collection
- Duodenal biopsies were taken from four different categories of
patients and were transported for microbial analysis.
Enrichment culture
- Serial dilutions of the FDR biopsy samples were prepared in
sterile phosphate buffer saline (PBS), and aliquots of 100
microliter and were spread on sterile nutrient agar (NA) and
Reasoner’s 2A agar (R2A) plates.
- The plates were incubated aerobically at 37C for 72 h.
Isolation
- After the incubation period, morphologically distinct
colonies were sub cultured on respective media. Finally,
the bacterial isolates were preserved in liquid nitrogen for
further work.

4. Determination of glutenase activity
- The bacterial cultures were streaked on an in-house-prepared
synthetic gluten agar (GA) medium and incubated for 24–72 h.
- A zone of clearing of gluten around the bacterial colonies was
considered evidence of glutenase activity.
- GA medium was prepared by replacing 23 g peptone in the
nutrient agar media with 23 g gluten so that gluten was the only
protein source to support bacterial growth.
Identification
- Bacterial genomic DNA extraction was performed using the
phenol–chloroform DNA extraction method.
- PCR was accomplished using the following conditions: initial
denaturation at 95C for 5 min, followed by 30 cycles of 95C for 1
min, 55C for 30 s, and 72C for 30 s with a final extension at 72C
for 7 min. Finally, the amplified products were purified and
sequenced.

5. Hydrolysis And Genome Sequencing
- Enzyme activity was determined from the proteolytic removal of
the paranitroanilide group which was monitored
spectrophotometrically at 405 nm
- The NAB46 and R2AA77 bacterial strains were cultured on NA and
R2A culture media at 37C under aerobic conditions for 24–72 h of
incubation. Genomic DNA was extracted, and high-quality DNA was
processed for whole-genome sequencing.
Extraction of crude enzyme from bacterial strains
- In R2AA77 bacterial strain, clear zone of gliadin utilization observed
around the bacterial colony indicated bacterial glutenase activity.
- In NAB46 bacterial strain, glutenase activity was detected by
spotting the culture supernatant on a gliadin agar plate.

6. Protease assay and protein estimation
- Enzyme protease activity was assayed by measuring the release
of tyrosine from casein as the substrate using the Folin-Ciocalteu
reagent, the standard was developed using pure tyrosine.
Western blot analysis
- The gliadin-degrading potential of partially purified PEP and GEP
enzymes was recognized by detecting hydrolysed gliadin peptides
in western blotting.

7. RESULT
 Fifteen morphologically different bacterial colonies were isolated.
Only two bacterial isolates showed extracellular proteolytic activity
against gluten on synthetic gluten agar plates.
 The isolates were further identified as B. casei NAB46 and
S.arlettae R2AA77.
 Whole genome sequencing was
done.
 Enzymatic activity of bacterial
strains NAB46 and R2AA77 isolated
on GA plates. . Brevibacterium
casei NAB46 and S. arlettae
R2AA77 efficiently hydrolysed Z-
PPF-pNA and were monitored
spectrophotometrically. No
cleavage was observed when
substrates were incubated in Tris-
HCl buffer only (control)

8.  Gluten-degrading proteases PEP and GEP were partially purified
with considerable gliadin-degrading activity.
 Glutenase activity was evaluated by spreading the crude enzymes
on a gliadin agar plate, followed by observation of the hydrolysis
zone.
 PEP with a specific activity 1.15 U/mg and GEP with specific
activity 0.84 U/mg had successfully degraded the immunogenic
gliadin after 3 and 6 h incubation with respect to control.
 It was observed by western blotting that 3–6 h of incubation of
gliadin with glutenase enzymes provided significant gliadin
hydrolysis.

12. DISCUSSION
• In this study, we screened two bacterial strains, B.
casei NAB46 and S. arlettae R2AA77, from an FDR
subject for their extracellular glutenase activity on a
synthetic gluten agar plate.
• Our examined strains, NAB46 and R2AA77, efficiently
hydrolysed the substrate.
• Genomes annotation suggested that PEP and GEP
were most likely responsible for the B. casei NAB46
and S. arlettaeR2AA77 gluten-degrading activity.
Gluten-detoxifying proteases PEP and GEP were
partially purified, and molecular weight bands 70 kDa
and 51 kDa of concentrated enzymes were observed
in SDS-PAGE that may belong to PEP and GEP,
respectively.

13. • Brevibacterium casei is a human gut colonizer;
an earlier study validated the use of B. casei
AP09 as a human probiotic, exhibiting
cholesterol reduction, bile tolerance, and
antimicrobial properties, and a better survival
rate in gastric and intestinal juice.
• Western blot analysis results suggested that
prolonged incubation may provide complete
hydrolysis of gliadin by these enzymes.

14. POSITIVE VIEW
• In the small intestine, a site of disease development and the
first place of gluten hydrolysis, the partial degradation of
gluten releases proline glutamine-rich peptides, and the
availability of these peptides, might support the growth and
colonization of GDB.
• Being human gut colonizers, B. casei NAB46 and S. arlettae
R2AA77 can be used as oral therapy for patients with CeD.
Both strains are symbiotic human gut colonizers and meet the
survival criteria to remain in the human small intestine.
• Second, PEP and GEP enzymes may respond to a two-enzyme
formulation for maximal hydrolysis of gliadin in the small
intestine. This raises the possibility of deploying these enzymes
in the duodenum to perform local proteolytic activities
individually.
• From a therapeutic point of view, the most intriguing property
of these enzymes is that the source of the enzyme is the
human small intestine.

15. LIMITATIONS
• The most prominent problem with PEPs is the
access of the long peptides to the active site,
which is hidden in a large cavity at the interface
of the catalytic domain.
• Western blot analysis is a sensitive detection of
grain fractions that are toxic to CeD patients.
• The limitation of our study is that these partially
purified enzymes are required for further
investigations using a broader range of
immunotoxic peptides in order to improve the
quality of life of CeD patients.

16. SUMMARY
• Celiac disease (CeD) is an immune-mediated chronic
disorder triggered by the ingestion of wheat gluten
in genetically predisposed individuals.
• Using the gluten agar plate technique, bacterial
strains Brevibacterium casei NAB46 and
Staphylococcus arlettae R2AA77 displaying
glutenase activity were screened, identified, and
characterized.
• These bacteria and their associated glutenase
enzymes efficiently neutralize gliadin immunogenic
epitopes, opening possibilities for their application
as a dietary supplement in treating CeD patients.

17. BIBLIOGRAPHY
Chrome-
extension://efaidnbmnnnibpcajpcglclefindmkaj/https:
//www.ias.ac.in/article/fulltext/jbsc/048/0018#:~:text
=potential%20implication%20in%20celiac%20disease,
SAHABRAM%20DEWALA&text=Gluten%20is%20a%20
major%20food,CeD%2C%20albeit%20with%20many%
20complications.