Pasteurization Step Research Articles

Germination-induced bioluminescence, a route to determine the inhibitory effect of a combination preservation treatment on bacterial spores.

In this work, we have used spores of Bacillus subtilis that specifically induce bioluminescence upon initiation of germination as a rapid, real-time monitor of the effects of preservative treatments on germination. Using this tool, we have demonstrated that the combination of mild acidity (pH 5.5 to 5.0), lactic acid (0. 5%), and a pasteurization step (90 degrees C for 5 min) results in enhanced inhibition of spore germination compared with the effects of the individual treatments alone. Inhibition by the combination treatment occurred as a result of both direct but reversible inhibition, entirely dependent on the physical presence of the preservative factors, and permanent, nonreversible damage to the L-alanine germination apparatus of the spore. However, we were able to restore germination of the preservative-damaged spores unable to germinate on L-alanine by supplementing the medium with the nonnutrient germinant calcium dipicolinic acid. The demonstration that simple combinations of preservative factors inhibit spore germination indicates that food preservation systems providing ambient stability could be designed which do not adhere to the strict limits set by commonly accepted processes and which are based on precise understanding of their inhibitory action.

Aspects of manufacture, composition and safety of orubisi: a traditional alcoholic beverage in the north-western region of Tanzania

The processing technology and characteristics of orubisi/amarwa, an opaque beer commonly consumed in Kagera region in the north-western part of Tanzania is described in detail. The protein content of orubisi increased from 2.0 to 2.7% after 120 hours of fermentation. The maximum alcohol content of orubisi as determined by specific gravity method was 2.5%. The alcohol profile of orubisi analysed by gas liquid chromatography (GLC) was found to contain ethanol and iso-butanol. The test for methanol was negative. Orubisi was characterised as product with relatively high acidity ranging from 0.35-0.89 g/100 ml and a final pH of 3.7. The levels of fermentable sugars-sucrose, maltose, glucose and fructose-were 0.5, 0.7, 1.8 and 0.6 g/100 ml after 120 hours of fermentation, respectively. High microbial counts were encountered in orubisi. The viable counts included yeasts: 2.0 × 107 cfu/ml, moulds 7.4 × 106 cfu/ml, coliforms 1.18 × 102 cfu/ml, lactic acid bacteria 6.5 × 107 cfu/ml and total aerobic count 2.95 × 107 cfu/ml. Based on these results, orubisi poses a serious danger to public health due to the presence of high numbers of total count and coliforms. In order to improve the safety of orubisi the pasteurisation step is recommended in the process of preparing orubisi. Hygienic handling of the final product is necessary in order to avoid contamination before consumption. The presence of trace amount of iso-butanol can lead to irritation of mucous membranes and depression of central nervous system.

Purification, characterization, and application of an acid urease from Arthrobacter mobilis

It has been shown that urea in fermented beverages and foods can serve as a precursor of ethylcarbamate, a potential carcinogen, and acid urease is an effective agent for removing urea in such products. We describe herein the purification and characterization of a novel acid urease from Arthrobacter mobilis SAM 0752 and show its unique application for the removal of urea from fermented beverages using the Japanese rice wine, sake, as an example. The purified acid urease showed an optimum pH for activity at pH 4.2. The enzyme exhibited an apparent K m for urea of 3.0 mM and a V max of 2370 μmol of urea per mg and min at 37°C and pH 4.2. Gel permeation chromatographic and sodium dodecyl sulfate gel electrophoretic analyses showed that the enzyme has an apparent native molecular weight ( M r) of 290 000 and consisted of three types of subunit proteins ( M r, 67 000, 16 600, 14 100) denoted by α, β, and γ. The most probable stoichiometry of the subunits was estimated to be α:β:γ=1:1:1, suggesting the enzyme subunit structure of (αβγ) 3. The enzyme also existed as an aggregated form with an M r of 580,000. The purified enzyme contained 2 g-atom of nickel per αβγ unit of the enzyme. Enzyme activity was inhibited by acetohydroxamic acid, HgCl 2, and CuCl 2. The isoelectric point of the native enzyme was estimated by gel electrofocusing to be 6.8. Urea (50 ppm), which was exogenously added to sake (pH 4.4, 17±1% (v/v) ethanol), was completely decomposed by incubation with the enzyme (0.09 U ml −1) at 15°C for 13 days. The enzyme was unstable at temperatures higher than 65°C and pHs lower than 4, and was completely inactivated under the conditions of a pasteurization step involved in the traditional sake-making processes. These results indicate that the enzyme is applicable to the elimination of urea in fermented beverages with minimal modification to the conventional process.

Chromatographic removal and heat inactivation of hepatitis B virus during the manufacture of human albumin

The purpose of the present study was to examine the efficacy of the chromatographic and pasteurization steps, employed in the manufacture of human albumin, in the removal and/or inactivation of hepatitis B virus (HBV). Most human albumins manufactured today are prepared from donor plasma by fractionation methods that use precipitation with cold ethanol. CSL Limited, an Australian biopharmaceutical company, has recently converted its method of manufacture for albumin from a traditional Cohn fractionation method to a method employing chromatographic techniques. A step‐by‐step validation of virus removal and inactivation was performed on this manufacturing process, which includes a DEAE‐Sepharose® and CM‐Sepharose® Fast Flow ion‐exchange step, a Sephacryl® S200 High‐Resolution gel‐filtration step and a bulk pasteurization step where product is held at 60 °C for 10 h. HBV partitioning experiments were conducted on scaled‐down chromatographic columns with hepatitis B surface antigen (HBsAg) as a marker, whereas the HBV model virus, duck HBV, was used to study the inactivation kinetics during pasteurization. Reductions for HBsAg through the three chromatographic steps resulted in a total log10 decrease of 1.5 log10, whereas more than 6.5 log10 decrease in duck HBV in Albumex®5 was achieved during pasteurization.

Chromatographic removal and heat inactivation of hepatitis B virus during the manufacture of human albumin.

The purpose of the present study was to examine the efficacy of the chromatographic and pasteurization steps, employed in the manufacture of human albumin, in the removal and/or inactivation of hepatitis B virus (HBV). Most human albumins manufactured today are prepared from donor plasma by fractionation methods that use precipitation with cold ethanol. CSL Limited, an Australian biopharmaceutical company, has recently converted its method of manufacture for albumin from a traditional Cohn fractionation method to a method employing chromatographic techniques. A step-by-step validation of virus removal and inactivation was performed on this manufacturing process, which includes a DEAE-Sepharose(R) and CM-Sepharose(R) Fast Flow ion-exchange step, a Sephacryl(R) S200 High-Resolution gel-filtration step and a bulk pasteurization step where product is held at 60 degreesC for 10 h. HBV partitioning experiments were conducted on scaled-down chromatographic columns with hepatitis B surface antigen (HBsAg) as a marker, whereas the HBV model virus, duck HBV, was used to study the inactivation kinetics during pasteurization. Reductions for HBsAg through the three chromatographic steps resulted in a total log10 decrease of 1.5 log10, whereas more than 6.5 log10 decrease in duck HBV in Albumex(R)5 was achieved during pasteurization.

Preclinical Assessment of Immunoreactivity of a New Purified Equine F(ab ′)2 against European Viper Venom

□ The immunological and pharmacokinetic properties of a new, further purified, pasteurized preparation of equine F(ab′)2 (VIPERFAV) against Vipera aspis, Vipera berus, and Vipera am-modytes venom were compared with the current equine F(ab′)2 preparation (IPSER Europe). Affinity constants of the V. aspis-specific F(ab′)2 were determined using biosensor technology and found to be in the range of 108M-1 for the four antigenic fractions of V. aspis toxins and for both F(ab′)2 preparations. The improvement of 51% in the specific activity (LD50 mg-1) of the new F(ab′)2 was in close agreement with the 1.8-fold increase in the immunoreactive fraction of the new preparation. In vivo investigations of venom immunocom-plexation by F(ab′)2 in rabbits confirmed the ability of F(ab′)2 to neutralize and redistribute toxin venom. Infusion of a stoichiometric molar ratio (i.e., 1mgkg-1) of the new antivenom induced a 2.3-fold elevation of the plasma venom concentration with a Tmax observed 8h after F(ab′)2 administration and a decline in the terminal half-life from 31.92±4.49h to 16.73±4.34h, in contrast, for the venom alone. The area under the curve was 1.4-fold greater in the VIPERFAV group than in the IPSER Europe group during the post-F(ab′)2 infusion period. Increasing the F(ab′)2 dose to 3mgkg-1 increased by 27% the percent of venom bound to F(ab′)2. Finally, the greater the venom distribution, the smaller and less pronounced the plasma redistribution. These results demonstrate that the purification and pasteurization steps involved in the preparation of the new F(ab′)2 have no deleterious influence on F(ab′)2 affinity but, on the contrary, improve the protective efficacy. Alteration of viper venom kinetics by specific F(ab′)2 antivenom was also shown to be dependent on the interval between of F(ab′)2 administration and venom bite and on the specific F(ab′)2 dose administered.

Pharmacokinetic properties of two factor VIII concentrates with single and double viral inactivation.

In order to assess whether addition of a modified pasteurization step to the manufacture of a solvent/detergent-treated factor VIII concentrate of high purity has affected its properties, three sets of pharmacokinetic studies were performed. In an open, controlled study with seven patients and a double-blind cross-over study with nine patients, the pharmacokinetic parameters of the double virus-inactivated preparation were compared with those of a mono-inactivated product, using a one-stage clotting assay. In a longitudinal study comparison was made between baseline and 8 months after treatment with the double virus-inactivated product in six patients, using three different factor VIII assays. There were no differences in recovery, clearance or half-life between the single- and double-inactivated products. After 8 months of treatment, the half-life and mean residence time were longer with the one-stage clotting assay, whereas the chromogenic assay showed a lower recovery, a faster cleareance and smaller area under the curve with the double virus-inactivated product. All differences were small and none was consistent in all three methods. In conclusion, the double virus inactivation does not appear to have affected the pharmacokinetic characteristics of this factor VIII preparation.

Chemical and Immunochemical Characterization of Polymers or Aggregates in Preparations of Human Serum Albumin

The polymer/aggregate fractions of three human serum albumin preparations (HSA) from different manufacturers were isolated by size-exclusion high-performance liquid chromatography (HPLC) and analyzed by different electrophoretic and immunochemical methods. All of these polymer/aggregate fractions contained only 30-50% albumin. The rest seemed to be mainly heat-denatured haptoglobin which did not react with an anti-haptoglobin serum, whereas the albumin part reacted with an anti-albumin serum. It could be shown that these polymers or aggregates are formed by disulfide bonds between albumin and the small amounts of denatured impurity globulins (haptoglobin, transferrin) during the pasteurization step. The higher the amount of these heat labile globulins in the final preparation before the pasteurization step, the higher was the polymer/aggregate content of the pasteurized albumin preparation.

Assay of possible formation of antigenic components in heat-treated plasma protein preparations.

To eliminate the risk of hepatitis infections, human plasma protein preparations can be heated in solution to 60 degrees C for 10 h thus inactivating several viruses. Preclinical safety experiments were performed in order to exclude the possibility of the formation of antigenic components, not present in normal human plasma, through this pasteurization step. Rabbits were immunized with either the heated or the unheated product. Sera were investigated in the Ouchterlony test against the homologous antigen to demonstrate precipitating antibodies. Antisera of rabbits immunized with the heated product were adsorbed with the preparation without the heating step. After centrifugation, the incubates were retested in the Ouchterlony assay. In passive cutaneous anaphylaxis assays guinea-pigs received the adsorbed antisera i.v., and both preparations were injected intracutaneously. The diameters of skin reactions were measured and did not show differences between those treated with the heated and non-heated product. No neoantigens could be detected with the following heated plasma protein fractions: prothrombin, coagulation factors VIII, IX, X, XIII, antithrombin III, fibrinogen, and Cl-inactivator. These results did not apply to heat-denaturated rabbit serum in a validation experiment.

Influence of Drying Plant Environment on Salmonellae Contamination of Dry Milk Products

This study was done to correlate incidence of salmonellae found in the dry milk processing plant environment with finished product contamination. Three plants with various histories of environmental salmonellae incidence were chosen for the study. The daily plant samples representing one lot of production were placed in a 1500-g composite in a sterile sample container and submitted to the central analytical laboratory for analysis. Two samples of nonfat dry milk were found to contain salmonellae in 8 continuous months of sampling and testing. In each instance of finished product positive, the environment had at least four positive samples recorded in the routine environmental program during the week or on the day in which the positive product was noted. Repeat tests of the positive product were negative on one lot and confirmed the positive in two of three retests of the other lot. It may be concluded from this study that controlling salmonellae in the dry milk plant environment will effectively preclude finished product contamination since dry milks are produced in essentially closed systems in a process that includes a pasteurization step.