Proof of Concept
Overview
Monogastric animals can not digest the xylan contained in grain feed, which may
lead to a series of digestion problems for monogastric animals. Poultry are typical monogastric animals.
Poultry breeding industry impact our daily life, thereafter, how to provide poultry breeders with
high-quality feed is a critical issue. To address this issue, we started our project, aiming at developing a
probiotic (Lactobacillus) containing xylanase to produce feed additives and poultry beverages.
The application of our additives in grain feeds will then enhance poultry’s
digestion. We believe that our additives will be a good choice for feed producers, poultry breeders and even
the general public, who consumes poultry everyday.
Supporting Experiment Results
A. Secretory expression of pSIP403-PUS-xyn AM in Lactobacillus reuteri
1. After the PCR identification sequence of pSIP 403-PUS-xynA AM bacteria liquid is
correct, two parallel test groups are inoculated into 10ml of MRS liquid medium containing 5 ug/mL
erythromycin, and cultured in a shaker at 37°C for 15 hours until the OD600 is 1.8722. And 2.2173;
2. Then transfer 1ml to 200ml MRS liquid containing 5 ug/mL erythromycin, and continue to cultivate until
the OD600 is 0.5304 and 0.6718;
3. At this time, start induction. The recombinant strain is added with 25 ng/mL
SppIP to induce expression. After shaking at 37°C and 220r/ml for 3 hours, samples are collected at
different time points to establish a function model of time and secreted expression;
4. Take 20ml of the recombinant strain after induction culture, of which 1ml is
used to detect the OD600 of the sample with an ultraviolet spectrophotometer, the remaining 19ml is
centrifuged, washed twice with 1 mL of 0.1 mol/L PBS (pH 6.0), and the pellet is resuspended in 2ml of PBS
and ultrasonic broken.
5. Take the broken supernatant for DNS enzyme activity detection.
Induction Time
From the experiment results above, the growth of pSIP403-PUS-xyn AM reaches its
optimal status at around 24h.
DNS enzyme activity detection
1. Detection principle: 3,5-dinitrosalicylic acid solution and reducing sugar
solution are reduced to brown-red amino compound after being heated together. Within a certain range, the
amount of reducing sugar and the degree of brown-red substance color are reduced. In a certain proportional
relationship, the absorbance of the resulting compound can be measured, and the concentration of reducing
sugar can be deduced from the standard curve.
2. Configuration of reagents
(1) Commercial DNS reagents
(2) Glucose standard solution (1mg/ml): accurately weigh 0.1mg of anhydrous
glucose, and dilute to a 100ml volumetric flask after dissolving; glucose standard solution (0.1mg/ml): take
10ml of the above 10-2mol/l The solution is diluted to a 100ml volumetric flask; according to the above
dilution rule, the solution is diluted to different concentrations of glucose (shown in the table below).
3. Xylanase extraction: Centrifugal supernatants of broken samples at different
induction time points;
4. Glucose standard curve drawing: take 1ml glucose standard solution into a 15ml
centrifuge tube, add 2ml DNS reagent, boil water bath for 5 minutes, cool tap water to room temperature,
take 1ml into a cuvette, and measure the OD value at 540nm. Take the absorbance value as the ordinate and
each standard concentration (mg/ml) as the abscissa to obtain the glucose standard curve.
5.Sample detection DNS: Take 1ml of the broken bacteria induced at different time
points and add it to a 15ml centrifuge tube, add 2ml DNS reagent, boil water bath for 5min, cool tap water
to room temperature, take 1ml into a cuvette, and measure the OD value at 540nm .
Calculation of Unit Enzyme Activity, Enzyme Activity
n=Dilution Times
K: Curve Slope
T: Reaction Time, min
1000: mg is converted to ug
K: Curve Slope
T: Reaction Time, min
1000: mg is converted to ug
From the experiment results above, the DNS color method was used to detect the unit
enzyme activity of two parallel group samples at different induction time points, and the average unit
enzyme activity was calculated. The experimental data showed that the enzyme activity was maximum when 25
ng/mL SppIP was induced for 8-12 hours. Is the best induction time.
In summary, our experiment results fully indicate that we have successfully
obtained a probiotic, namely pSIP403-PUS-xyn AM in Lactobacillus reuteri, which is functional in secreting
xylanase.
Relying on this experiment results, we may further test the feasibility of our plan
to make feed additives and poultry beverages.