Toxicological Evaluation of Lactase Derived from Recombinant Pichia pastoris

Abstract

A recombinant lactase was expressed in Pichia pastoris, resulting in enzymatic activity of 3600 U/mL in a 5 L fermenter. The lactase product was subjected to a series of toxicological tests to determine its safety for use as an enzyme preparation in the dairy industry. This recombinant lactase had the highest activity of all recombinant strains reported thus far. Acute oral toxicity, mutagenicity, genotoxic, and subchronic toxicity tests performed in rats and mice showed no death in any groups. The lethal dose 50% (LD50) based on the acute oral toxicity study is greater than 30 mL/kg body weight, which is in accordance with the 1500 L milk consumption of a 50 kg human daily. The lactase showed no mutagenic activity in the Ames test or a mouse sperm abnormality test at levels of up to 5 mg/plate and 1250 mg/kg body weight, respectively. It also showed no genetic toxicology in a bone marrow cell micronucleus test at levels of up to 1250 mg/kg body weight. A 90-day subchronic repeated toxicity study via the diet with lactase levels up to 1646 mg/kg (1000-fold greater than the mean human exposure) did not show any treatment-related significant toxicological effects on body weight, food consumption, organ weights, hematological and clinical chemistry, or histopathology compared to the control groups. This toxicological evaluation system is comprehensive and can be used in the safety evaluation of other enzyme preparations. The lactase showed no acute, mutagenic, genetic, or subchronic toxicity under our evaluation system.

Effective treatment of hypertension by recombinant Lactobacillus plantarum expressing angiotensin converting enzyme inhibitory peptide

“…To evaluate the safety of RLP, ATT and BMCMT assessments were performed as described previously with some modifications [ 51 , 52 ]. Specifically, 6–8 week old BALB/c mice were divided randomly into three groups with 10 mice per group; the mice were subjected to either 4.5 × 10 9 CFU or 4.5 × 10 7 CFU RLP resuspended in 0.1 mL PBS.…”

“…The RLP group was orally treated with a dose of 4.5 × 10 8 CFU in 0.1 mL PBS for 5 continuous days, whereas the negative control group was treated with 0.1 mL PBS for 5 days and the positive control group was intraperitoneally injected with CP 50 mg/kg (BW) on the 5th day. Twenty-four hours after the last dose, the mice were sacrificed by cervical dislocation, and bone marrow cells were collected immediately as described before [ 52 ]. One thousand polychromatic erythrocytes (PCE) were recorded per mouse, and the frequency of micronucleated cells was calculated by counting them and dividing by the total number of polychromatic erythrocytes.…”