PapQuin-50 a Quinoa grain-based bacterial growth medium nitrogen source

Abstract

Commercially available Quinoa grain powder is subjected to proteolytic digestion using papain enzyme. Derived hydrolysate is ultrafiltered using two sequential ultrafiltration steps of 50 KDa and 5 KDa membranes to remove high and low molecular impurities respectively. Concentrated filtrate is 0.2 μ filter-sterilized and used as a component growth medium for fastidious microorganisms Neisseria meningitidis and Streptococcus pneumoniae. The product is tested for its ability to be used as meat free nitrogen and carbon source for the fastidious bacterial growth and found to be working well.

Claims

1. Claims of the invention: i) Quinoa grain flour papain enzyme hydrolysate is claimed to be used as nitrogen source in bacterial growth media. ii) Methods of preparation include enzyme hydrolysis and purification using Specific MWCO (molecular weight cut-off) sequential membrane filtration steps are claimed to purify the PapQuin-50 as growth media component.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0009] Process used in the current invention: [0010] Water solubilized quinoa powder is prepared at a concentration of 50 mg/ml by constant mixing at 37° C. for 2 to 3 h. The solution is then filtered through 0.8 μm filter to separate undissolved portion. [0011] Filtered Quinoa solution is then pH adjusted to 7.0 and treated with papain [Thermo Scientific Cat #AC416761000] enzyme at a final concentration of 50 μg/ml at 65° C. for 6 h with constant shaking in a shaker incubator at 150 RPM. [0012] Centrifuged the hydrolysate at 8000 g at 4° C. for 30 minutes to separate any insoluble sediment. [0013] Transferred the supernatant to ultrafiltration step in a Viva-flow [Sartorius item #VF05P3] 50 KDa MWCO PES membrane cassette on a laboratory scale cross flow system. [0014] Collected the flow-though (Permeate) at a twice starting concentration (100 mg/ml) based on collected flow-through volume. [0015] The Permeate is further purified with Viva-flow [Sartorius item #VF05P1] 5 KDa MWCO membrane cassette ultrafiltration system for 5 diafiltration-volumes of purified water to remove small molecular contamination to collect the retentate. [0016] 0.2 micron Filter sterilized product PapQuin-50 was used as a medium component substituting yeast extract in Frantz medium of composition given below table 1. [0017] Tested the medium agar plates and liquid growth for fastidious organism Neisseria meningitidis serogroups A, C, W-135 and Y and Streptococcus pneumoniae serotypes 7F, 9V, 12F and 23F. 15 g/L Bacto agar is used for making agar plates. [0018] Results showed that Papquin-50 can replace yeast extract in Frantz medium to support the agar plate and liquid medium growth of Neisseria meningitidis and Streptococcus pneumoniae strains used.

TABLE-US-00001 TABLE 1 Medium composition: Frantz medium replacing Yeast extract with PapQuin-50 *Frantz media Current-work Component g/L medium g/L L-glutamic acid 1.60 1.60 L-Cysteine .Math. HCl 0.02 0.02 NaCl 6.00 6.00 Na.sub.2HPO.sub.4, 12H.sub.2O 6.24 6.24 KCl 0.09 0.09 NH.sub.4Cl 1.25 1.29 MgSO.sub.4, 7H.sub.2O 1.23 1.23 Glucose 5.00 5.00 PapQuin-50 — 2.00 Yeast Extract 2.00 — [0019] Frantz I. D. Jr Growth Requirements of the meningococcus. J. Bact (1942). 43:757-761, 1

Results:

[0020] Papquin-50 used to replace the yeast extract in Frantz medium worked very well for both Neisseria and Streptococcus strains used. [0021] Papquin-50 is shown to be working for agar plate growth and liquid medium growth during the invention.

CONCLUSIONS

[0022] Plant origin papain enzyme digested quinoa flour nitrogen source named ‘PapQuin-50’ preparation is described in this patent application. Commercially available Quinoa grain powder used in this claim is digested with commercially available plant origin enzyme papain. A MWCO 50 KDa and 5 KDa membrane diafiltration steps are used to purify this nitrogen source to make consistent preparation. Growth promoting activity of the PapQuin-50 medium replacing yeast extract in a standard medium supporting fastidious Gram negative (meningococci) and Gram positive (Pneumococci) bacteria has been presented. This nutritious medium supplement Papquin-50 may support the growth of other bacterial species as well.