Monday, September 20, 2010

LABORATORY QUALITY ASSURANCE

INTRODUCTION

The objective of laboratory quality assurance is to verify the accuracy and precision of information obtained from analyses and to ensure that data obtained from analyses are suitable for use in decision-making. The accuracy and precision required may vary, depending on the use to be made of the information, and however strong the effort for precision may be, and some variation in results from biological analyses is to be expected.

Generally, food microbiology laboratories-governmental, industrial, academic, or commercial-are active in one or more of the following areas:

1. Basic research determines the characteristics of microorganisms important in foods and the factors that affect their growth and survival.
2. Applied research uses basic information to develop and test new food products and microbiological methods.
3. Quality control monitors food production to detect deviations from good manufacturing practices, to assure conformance to criteria, and to detect contaminants.
4. Investigative activities seek to determine the causes of food-borne illness and spoilage problems.

Although the principal objective of a laboratory quality assurance program is to ensure the correctness of data, the systems, and procedures required for an effective program of this sort provide additional benefits. For example, using good microbiological techniques not only prevents cross-contamination of the samples being examined, but also protects personnel against infection and their working environment against contamination. Monitoring and maintaining equipment to ensure proper functioning decreases the risk of electrical and fire hazards. Another indirect benefit of a laboratory quality assurance system is the standardization of analytical methods, resulting in decreased intra- and inter-laboratory variation. Finally, the record keeping activities required for laboratory quality assurance provide information that helps management and individual bench-workers evaluate proficiency. The ability of management and workers to monitor the quality of work promotes confidence and pride in the laboratory.

This article introduces the principal concepts in quality assurance for the food microbiology laboratory. It is not possible to present here specific quality assurance programs to meet the needs of all food microbiology laboratories. Rather, this chapter is concerned with the particular laboratory functions that must be controlled and the probable sources of laboratory error. The selection of appropriate methods for quality assurance will be addressed, but specific methods will be referenced rather than discussed in detail. Thus, the reader should be mindful that this chapter is intended to serve as a guide for the design and implementation of laboratory quality assurance programs, not as; detailed statement of the system itself.


MANAGEMENT'S ROLE IN LABORATORY QUALITY ASSURANCE

Direct responsibility for the design and implementation of a laboratory quality assurance program lies within the management of the laboratory. Management must evaluate the risks associated with laboratory error and the costs and benefits of reducing error through systematic quality assurance. Measurement of the cost of these activities requires a system to account for material, labor, and overhead costs. The likelihood and significance of errors arising from each particular activity of the laboratory and the costs associated with controlling and monitoring a particular activity must be considered in development and operation of a quality assurance program. The principles Hazard Analysis Critical Control Points (HACCP) are useful for the development of concepts for a laboratory quality assurance system.

The following steps describe a general approach that should be followed establishing a laboratory quality assurance program:

1. Formalize objectives and policies that specify the accuracy of work required, including the selection of appropriate methods and sample handling procedures.
2. Select appropriate personnel to perform laboratory functions to maintain the desired level of quality.
3. Provide facilities and equipment necessary for the performance of laboratory functions.
4. Establish monitoring methods and record keeping protocols to verify the accuracy and consistency of the laboratory work.
5. Establish procedures for the initiation of corrective measures when unacceptable quality is discovered.


GENERAL LABORATORY OPERATIONS

The first step in implementing a quality assurance program for the laboratory is to make a formal statement of the objectives and scope of activities for the laboratory, after which general operating procedures are developed to meet the objectives. Specific standard operating procedures are installed to ensure that approved sample preparation and analytical methods are chosen and followed. Within any analytical method, procedural steps may be subject to interpretation that can result in minor modifications having a pronounced influence on the outcome of an analysis. Standard operating procedures should be sufficiently described, to prevent minor modifications or deviations that could arise from misinterpretation of a method and to provide an accurate record of each stage of a procedure. For research laboratories, general standard operating procedures may be established for development of a research proposal, approval of the project, and periodic evaluation of the progress made on the project. Specific operating procedures, such as method validation and execution, may need to be modified during a project as new data are obtained and interpreted.

Sample Management
This section discusses the criteria used to determine the acceptability of samples received by the laboratory and the proper handling of samples accepted for analysis.

1. Criteria for acceptance of a sample
a. Adequate documentation and identification must accompany samples, including a description of the place of collection, manufacturer, date and time of collection (especially for perishable samples), reason for collection (such as, compliance with legal standards or routine surveillance), sampling plan followed, analysis requested, and storage conditions.
b. The original condition of samples and the integrity of sample containers must be maintained from collection until receipt at the laboratory. The manner of shipment must be appropriate for the type of sample.
c. Other considerations for acceptance of samples are (1) the samples must be representative of the lot of product and processes sampled, and (2) the laboratory must have the capability to do the requested analyses.

2. Handling of samples in the laboratory
a. After receipt, samples must be stored to maintain their original condition until analyzed. Samples should be tested as soon as possible after receipt.
b. It may be necessary for an individual in the laboratory to have the responsibility as sample custodian to maintain accountability records for samples in the laboratory. This individual may (1) receive samples, (2) record the date and time received, (3) initially verify the identity of samples, (4) store according to instructions accompanying samples, (5) record the date and time when samples are delivered to analyst(s) for examination and the date and time when they are returned to storage following analyses, (6) maintain a long-term sample storage system, and (7) dispose of samples as necessary.

Laboratory Standard Operating Procedures
Each laboratory should have a standard operations manual that describes (l) procedures for the acceptance or rejection of samples, (2) methodology to be followed for specific analyses, (3) appropriate control procedures, (4) all quality assurance procedures, (5) procedures for cleaning and sterilizing equipment, (6) procedures for preparing media and reagents, and (7) procedures for handling and disposing of contaminated materials.

The manual should provide instructions to cover most of the normal situations that the laboratory will encounter. It will inform personnel about appropriate methodology, ensure uniformity of sample analysis, and promote quality assurance programs.

The selection of methods to he included in the manual will depend to a certain extent on the type of laboratory, i.e., governmental, commercial, or industrial. The description of each method in the manual should be complete and detailed enough that reference to other publications is unnecessary. Included should be all the necessary controls and checks on materials, media, reagents, positive and negative controls, the desired response from each control, and corrective measures that should be taken it a control is not correct.

Limitations of each test should be included, where known, as well as a list of precautions to be taken. Possible interferences should be described, such as natural inhibitory substances in foods that must be diluted out for growth of any organism to occur.

The manual should fully describe the duality assurance program in use. Included should be each quality assurance procedure, the frequency of use, specific analytical methods that may be required in each procedure, applicable tolerances for each procedure and, if possible, remedial steps to correct out-of-control procedures, and the names or titles of personnel to be notified if out-of-control procedures are found.


PERSONNEL

Quality results require quality personnel. The personnel selected must have the education, experience, and motivation necessary to perform their jobs and to carry out the requirements of the quality assurance program. The number of persons and their technical training may vary considerably from laboratory to laboratory, depending on the types and numbers of analyses to be performed. However, general guidelines for selection of laboratory personnel are available.

Successful management of laboratory personnel through motivation, training, supervision, and workload direction is as important as selection of the personnel. Workers must be properly trained to perform their duties, and the first step in training is to specify as completely as possible the duties of the position and the importance of these duties to the quality of results generated by the whole laboratory. The goals of training are that the workers know the exact duties they are to perform, with sufficient instruction and time to learn how to perform these duties to obtain results of the highest quality. Employees must be made fully aware of their quality assurance vies and especially of the adverse consequences that will arise from failure to carry out their duties carefully.

Motivation of laboratory personnel to do high-quality work is essential for quality assurance program. To achieve this requires a safe, efficiently designed facility, sufficient supplies and equipment, workloads that are not excessive, and suitable compensation.

Evaluation of Personnel
Uniform application of laboratory procedures by all analysts is very important for consistent results. The routine evaluation of the accuracy of each analyst’s performance is necessary for such consistency. The supervisor is the best person to evaluate worker performance on a day-to-day basis. An appropriate agency or individual may occasionally check on-site worker performance. However, experience has shown that on-site evaluations alone are not enough to minimize variation among analysts.

The proficiency of each analyst should be rated by having the laboratory participate in split/check sample programs in which the performance of each analyst can be compared to the performance of other analysts in the same laboratory or other laboratories. Split or check samples to verify analyst performance can be generated internally or supplied by external sources. Comparison of results obtained from laboratories participating in an external split/check program provides a valid evaluation of the proficiency of the laboratory as a whole. Statistically acceptable performances by individual analyst and the laboratory as a whole give the best indication that all elements associated with diagnostic procedures (personnel, media, reagents, and equipment) are satisfactory. Critics have asserted that split/check sample proficiency testing programs in clinical laboratories do not truly measure day-to-day capabilities because the laboratory staff usually is aware of the source of tends to be more careful than usual. A more realistic measure of performance is the introduction of internal "blind" unknown samples.

Split samples can be naturally or artificially contaminated. Naturally contaminated samples have the advantage that they represent the kinds of samples routinely received and examined by analysts. However, their microbiological character may not be known, and it may be difficult to obtain large enough quantities of such samples to distribute to several analysts or laboratories.

Studies of clinical laboratories suggest that continuous participation in proficiency surveys results in improved analyst performance. Participation in the national milk laboratory proficiency-testing program has resulted in marked improvement in analyst performance. Although ongoing proficiency studies have not been conducted in food microbiology laboratories, it is likely that improvement in analyst and laboratory performance can be obtained.


FACILITIES

The safety of workers is of utmost importance in design and construction of laboratory facilities. The food microbiology laboratory should be designed and built to meet this priority and to provide for the convenience of tile workers and operations. It should be adequately equipped to carry out the stated objectives of the laboratory. The following points should be considered when designing a laboratory.


Laboratory Design
1. Ventilation, temperature, and humidity
Laboratories should be well ventilated, preferably by use of central air-conditioning, to reduce the amount of particulates in the air and minimize temperature variation. Temperature and relative humidity should be comfortable for workers and suitable to the requirements of the laboratory equipment. Normally, an ambient temperature of 21° to 23°C and a relative humidity of 45% to 50% are recommended.

2. Lighting
Laboratory lighting should be maintained at an average intensity of at least 50, and preferably 100, foot-candles. Dependence upon natural sunlight during the day should be discouraged because of high variability in its intensity. Because direct sunlight is known to have deleterious effects on media, reagents, and specimens, preparation or storage of these items in direct, sunlight should be avoided.

3. Laboratory space and bench areas
Laboratory space should be organized to maximize usefulness. Where possible, media preparation and glassware cleaning areas should be separated from the analytical areas. Equipment and materials should be positioned to make the maximum amount of bench space available. For most routine work, six linear feet is the recommended minimum work area for each analyst. The ideal bench top height is 36 to 38 in with a depth of 28 to 30 in. The walls and ceiling of the laboratory should be covered with good-grade enamel or epoxy paint, or other material that provides a smooth, impervious surface that is easily disinfected. Floors should be covered with high-quality tile or other impenetrable material. Cracks and crevices should be minimized, as they provide an opportunity for the buildup of debris that may contribute to cross-contamination of samples. Unnecessary traffic through the laboratory should be prohibited. Eating or smoking should never be permitted in a microbiology laboratory.

4. Storage areas
Storage space for equipment, materials, and samples should be sufficient for needed media, reagents, glassware, and plastic ware. The use of cabinets with doors and drawers will minimize dust buildup and allow easier cleaning and disinfection of laboratory surfaces. Test samples must be stored under conditions outlined in the particular analytical procedure being followed. Samples stored at room temperature should be placed in sealed containers to prevent the proliferation of pests. A written standard policy should outline the conditions and length of storage time for samples. Storage areas should be routinely inventoried, and superfluous samples, and outdated media and reagents should be disposed of according to an established, documented policy.

5. Other utilities
Every laboratory should be equipped with enough electrical outlets of the appropriate voltage and amperage, enough natural gas jets for Bunsen burners, a waste disposal system, and laboratory-grade water. The laboratory also should have an adequate number of sinks with hot and cold tap water and, preferably, lines for de-ionized or distilled water. Sinks with foot-operated hand-washing taps are recommended.

6. Laboratory-grade water
Laboratory-grade water, which should be available in the food microbiology laboratory, is defined as water that has been treated to free it from nutritive and toxic materials. Laboratory-grade water may be produced by distillation, reverse osmosis, ion exchange, filtration, or a combination of these. Viable microorganisms have been shown to accumulate in laboratory water systems such as ion exchange systems, and they should be monitored routinely for microbial growth according to a written standard operating procedure. In addition to this microbiological monitoring, the following physical-chemical elements should be monthly and documented to have met the indicated parameters:
a. Trace metals, a single metal not greater than 0.05 mg/L
b. Total metals, equal to or less than 1.0 mg/L
c. pH 5.5 to 7.5
d. Residual chlorine less than 0.1 mg/L

7. Personnel safety
To ensure the safety of personnel, all facilities should be designed according to established federal, state, and local building and safety codes. All laboratories should be equipped with fire extinguishers and alarms, sprinkler systems, eyewash stations, and safety showers. Approved safety glasses should he available to laboratory workers and visitors. A comprehensive safety program, including worker training, should be a vital part of laboratory procedures.

8. Animal facilities
Some food microbiology procedures require the use of laboratory animals. Laboratory animals should be maintained in separate areas other than those where routine analytical tests are performed. Animal rooms should have all air discharged to the outside without recirculation. A minimum of 15 air changes per hour is recommended. A specific written operating procedure should be designed outlining the details of animal maintenance.

Housekeeping
A routine cleaning and disinfection schedule for the entire laboratory should he established, documented, and monitored for effectiveness. Disinfectants such as iodophor, quaternary ammonium compounds, or phenolic disinfectants should be employed. All laboratory benches and equipment should be disinfected before and after each use.

Laboratory materials should be stored after use in order to maintain a clutter-free work area. Unneeded and outdated materials should be discharged according to a written procedure that includes a description of the methods of disposal, safety precautions, and frequency of inventory measurement.

Dust and soil should not be allowed to build up in a microbiology laboratory. Close attention should be paid to corners and hard-to-clean areas. Floors should be wet-mopped, preferably with a suitable disinfectant-detergent solution not dry-mopped, or swept with a broom because these practices will contribute to airborne contamination.

Environmental Monitoring
To assess the efficacy of the established laboratory disinfection schedule and to determine the microbial profile of the laboratory, a written operating procedure on environmental monitoring should be followed. This operating procedure should contain a description of the environmental sampling procedure and statements of locations to be sampled, tolerance limits, and frequency of monitoring.


EQUIPMENT AND INSTRUMENTATION

The reliability of an analytical procedure is only as good as the reliability the equipment and instruments used for the procedure. A protocol should be established to verify the reliability of the equipment and instruments. Equipment should be used only by properly trained personnel. All equipment: (balances, pH meters, etc.) should be cleaned before and after use.

1. Thermometers and temperature recorders
Thermometers should meet the minimum specifications outlined in "Standard Methods for the Examination of Dairy Products." Their accuracy should be checked at least annually with a thermometer certified by the National Bureau of Standards (NBS). Mechanical windup temperature-recording devices are preferred for incubators and water baths that are used continuously. Such mechanically driven temperature recorders are recommended over electrical plug-in recorders in order to measure temperature fluctuations during power failures. This is especially important during nonworking hours. The recorders should be validated annually against a certified NBS thermometer and after any repair work. Written specifications should be established for these recorders, and at least daily, if not continuously, temperatures should be monitored in all active incubators, water baths, refrigerators, freezers, and ambient laboratory environments. Results of such monitoring should be placed in the permanent quality assurance records.

2. Balances
Laboratory balances should be sensitive to 0.1 g with a 200 g load. An analytical balance having a sensitivity of 1 mg with a 10 g load should be used for weighing small quantities. Single pan balances are preferred. The accuracy of laboratory balances should be checked routinely, preferably daily, by standard reference weights that are calibrated annually against a certified NBS set of weights. Generally, the balance should be checked with several different weights. Written documentation should be maintained on the calibration of the standard reference set of weights as well as on routine accuracy checks of balances.

3. pH meters
pH meters should be standardized with a minimum of two standard buffers (pH 4.0, pH 7.0, or pH 10.0) before use. Aliquots of buffer solution should be used once and discarded. Standard buffer solutions should be dated upon receipt and an expiration date established after opening the container. The pH meter should be accurate within 0.1 pH unit. The life of pH electrodes will vary by type, brand, and frequency of use. Manufacturers directions should be followed for servicing pH electrodes.

4. Autoclaves
Autoclaves should be equipped with accurate pressure and temperature gauges. They should be equipped with a calibrated thermometer located properly on the exhaust line to register the minimum temperature within the sterilizing chamber. It is preferable that the autoclave be equipped with a temperature recorder in order to provide a permanent record for each sterilization cycle. A permanent record keeping system should be established to document each sterilization cycle. This consists of a daily chart listing for each cycle such items as temperature and time settings, materials in the chambers, pressure and temperature readings once the autoclave has reached the sterilizing region of the cycle, and date and time that the sterilizing cycle is started and finished, followed by the signature or initials of the operator.

Ensuring the proper functioning of autoclaves is essential. This can be done with biological indicators and through physical measurements. Biological indicators are available from several commercial sources. Physical measurements can be made with thermocouples and maximum registering thermometers. A combination of both biological indicators and physical measurements should be used to validate sterilization processes. Physical validations employing thermocouples located in various areas of the autoclave chamber should be performed annually. Biological indicators and maximum registry thermometers should be employed with each use.

5. Hot air sterilizing ovens
Each sterilizing oven should be equipped with a thermometer and, preferably, a temperature recorder, both calibrated against an NBS thermometer. A time and temperature record should be maintained for each sterilization cycle. In addition, periodic physical and biological validations are suggested.

6. Other equipment
Equipment such as water baths, incubators, and refrigerators should be equipped with thermometers or temperature recorders, or both, calibrated against an NBS thermometer. Operating procedures should be established so that proper written records are maintained for each piece of equipment.
Laminar flow hoods should be checked with a particle counter on a routine basis. Specifications should be established on the filtering efficiency of the hoods. They should be routinely disinfected and monitored. Airflow rates should be monitored periodically with a certified flow meter.

Indicator strips, which are commercially available, should be changed daily. Written records should be maintained to daily document the existence of anaerobic conditions in the chamber.

Preventive Maintenance of Equipment
It is important that the food microbiology laboratory have a formal, written preventive maintenance program. If the laboratory is small and does not have a maintenance department, preventive maintenance agreements with the manufacturers or dealers from whom the equipment was purchased should be arranged. Reputable independent maintenance firms also can provide preventive maintenance services. Regardless of laboratory size, the following points, as outlined in "Quality Assurance Practices for Health Laboratories," should be considered when establishing a preventive maintenance program:

1. Inventory
Each piece of equipment, with its location, complete name, age, and description, and the appropriate supervisor or person responsible for the item, should be listed in the inventory. The pertinent information should be outlined on a separate page or card for each item of equipment. This record constitutes a portion of the inventory control.

2. Definition of service tasks.
For each item of equipment, the tasks needed to keep the equipment calibrated, operating, and clean must be defined. Supportive information may be obtained from manufacturers' brochures, product guides, and journal reprints. These specific tasks should be included in the laboratory's written statement of standard operating procedures.

3. Interval establishment
The frequency with which the defined tasks should be performed must be determined. This will vary with the item of equipment, the type of installation, and the workload of the particular item of equipment. However, even equipment used infrequently must have minimum standards of preventive maintenance.

4. Personnel
Those individuals should be listed who are immediately available, or may be ultimately available, for function verification tasks, cleaning, preventive maintenance, troubleshooting, and repair. This assignment should be customized to the laboratory's own situation. In general, it is preferable to depend upon in-house personnel who can perform maintenance activities economically and efficiently. However, for some instruments, one must depend on a manufacturer's services or on an independent service company.

5. Job assignments
If the program is to succeed, responsibilities for the tasks outlined above must be assigned so that each person will know the responsibilities. Job assignments should be matched with training, experience, and aptitude.

6. Training
Laboratories having a large number of well-trained personnel who are familiar with laboratory equipment are indeed fortunate. Most laboratories must carry out some in-service training to teach personnel the use of special monitoring devices and the performance of some of the more difficult service tasks.

7. Special instruments
The monitoring devices, techniques, materials, and types of special equipment used to check each type of instrument in the laboratory should be listed. If the laboratory does not have the necessary monitoring equipment, it must be acquired.

8. Setting up the system
Once the effort has been made to inventory the equipment, define the tasks, and train the personnel on special instruments, the program should continue year after year. A record for each item of equipment should be established in which all entries are made. In addition, it may be necessary to develop a reminder system so that appropriate personnel are notified when certain tasks are to be performed.

9. Records and documentation
Documentation is needed to record that the appropriate service tasks have been accomplished. This may be the appropriate place to incorporate a system of reminders to ensure that the tasks are performed on time. Index card systems are good for this purpose and are inexpensive. Some laboratories use a computer to remind technologists of these tasks. The documentation scheme must be tailored to the laboratory's specific needs.

10. Surveillance
After setting up a program, periodic surveillance should be carried out to ensure that the records are legible and complete.


LABORATORY GLASSWARE AND PLASTIC WARE

Specifications of laboratory glassware and plastic ware should be established and followed. For example, the calibration of newly purchased glass or plastic pipettes should be checked upon receipt in the laboratory. The calibration marks on dilution bottles should be checked with NBS-certified volumetric glassware.

Glassware should be made of high-quality, low-alkali borosilicate glass. Glassware composed of soft glass presents problems because of leaching of components and the presence of surface alkali, which may interfere with some analytical procedures. Etched or chipped glassware should be discarded. Plastic ware should be free of defects and toxic residues.

Procedures must be established to sterilize and wash microbiologically contaminated reusable glass- or plastic ware. Microbiologically contaminated reusable lab ware must be sterilized by autoclaving or other suitable means before being washed.

Reusable glassware and plastic ware should be washed manually or mechanically with hot water containing a suitable detergent. Stubborn residues may be removed by soaking in a potassium dichromate cleaning solution on glassware before washing. Screw caps, test tube caps, and other reusable closures also should be washed in a detergent solution and rinsed thoroughly. Many detergents have a high affinity for glassware and plastic ware and some are highly bacteriostatic; it is imperative to ensure their removal after washing. Glassware and plastic ware should be checked routinely for alkaline, or acidic residues by applying a few drops of bromthymol blue pH indicator. This indicator is useful because it displays color changes from yellow to blue-green to blue in a pH range of 6.5 to 7.3. Since most cleaning solutions are either acidic or alkaline, this simple test assures proper rinsing.

Toxicity Testing
Disposable glassware and plastic ware may be sterilized by ethylene oxide gas. If these items, pipettes, etc., are not properly rinsed after the sterilization treatment, toxic residues may remain. Therefore, it is important to check these items periodically for toxic residues and to request certification from the supplier that no toxic residues are present. Similarly, glass items washed and sterilized in the laboratory may contain toxic detergent residues not detected by the bromthymol blue pH test. These items should le checked periodically for toxic detergent residues. Procedures for toxicity testing are detailed in several publications. The washing procedures should be checked at least annually by performing toxicity tests and should be modified if necessary.

Sterility Testing as a Quality Assurance Tool
The sterility of sterilized supplies and equipment must be ensured. A sterility test may be performed on a portion of the sterilized items. Sterility Control tests on Petri dishes may be performed by simply pouring a nonselective medium such as Standard Methods Agar into several randomly selected plates from a case. Upon solidification, the plates are then incubated aerobically or an-aerobically and examined for growth. Sterility controls on sampling containers, utensils, and dilution bottles may be performed by the rinse filtration technique. According to this technique, the items are aseptically rinsed with sterile phosphate buffer that is filtered through a membrane filter. The filter is placed on the surface of a nonselective agar and incubated under prescribed conditions.


MEDIA AND REAGENTS

Food microbiology laboratories use many different media and reagents to detect and enumerate microorganisms, and most of them are purchased already prepared or in dehydrated form. Reagents and media should be tested before using to validate their efficacy. Those media and reagents formulated in the laboratory also should be prepared carefully and validated for performance.

Common errors that occur in preparation of media and reagents are listed here:
1. Incorrect weighing of dry material.
2. Use of dry material that has deteriorated because of exposure to heat, moisture, oxidation, or other environmental factors.
3. Incorrect measurement of water volume, or use of tap water or water from a malfunctioning still or de-ionizing resin column. Water must meet the requirements for laboratory pure water and be proven microbiologically suitable.
4. Use of containers and glassware that are contaminated with detergent or other chemicals.
5. Incomplete mixing of ingredients during preparation of media or solutions. This may result in excessive or insufficient gel strength of the medium and uneven concentrations of constituents among aliquots.
6. Overheating during preparation at in the molten state before dispensing into plates, tubes, or bottles. Overheating can result in loss o hydrolysis of the agar, caramelization of carbohydrates, lowering of pH, increase or decrease in inhibitory content in selective or differential precipitates.
7. Improper determination of pH, resulting in the addition of too much acid or alkali.
8. Improper addition or incorporation of unsatisfactory supplements or enrichments, or addition of supplements at the wrong temperature, possibly causing chemical changes in the supplements if the temperature is too high, or solidification of media before proper mixing if too cold.
9. Failure of the laboratory to subject samples of finished media to quality control procedures before the media are used.
10. Failure of the laboratory to test samples of dehydrated media purchased from suppliers to ensure that the media are productive.

Receipt of Media, Reagents, and Ingredients
Containers of media and reagents should be dated upon receipt. The laboratory should maintain a media/reagent control file where the following information is recorded for each shipment of media or reagents received:
1. Manufacturer and manufacturer’s code.
2. Quantity received, i.e., size, and number of containers.
3. Date received.
4. Date opened.
5. Location where medium/reagent is to be stored.
6. Initials of person receiving and placing the item into stock.
7. Results of productivity, and selectivity testing, if performed

Each lot of medium/reagent should be inspected before use for volume, tightness of closure, clarity, color, consistency, and completeness of label.

Storage of Dehydrated Media, Reagents, and Ingredients
Directions for the storage of most media and ingredients are generally listed by the supplier on the label of each container. In addition, the supplier will often indicate an expiration date after which the item should not be used. When available, the supplier’s directions should be followed. Some general guidelines are listed below:

1. Store dehydrated media in tightly capped bottles or tightly closed plastic liners in a cool, dry place protected from light. If specified, keep under refrigeration and in the dark.
2. Keep no more than 6 months’ to a year’s supply on hand, being sure to use older stocks first. Do not exceed supplier’s expiration date.
3. Dehydrated media and reagents should be free-flowing powders or crystals. If a change is noted in this property or in the color, the item in question should be discarded.
4. Media containing dyes should be protected from light by storage in a dark room or a dark bottle or by wrapping the container with foil or brown paper.


RECORD KEEPING

It is essential for a food microbiology laboratory to maintain accurate and permanent records of sample analyses and quality assurance programs. The nature of the records can vary from individual worksheets and data books to entire electronic data processing systems. The length of time and the manner in which records are retained will depend on the laboratory's objectives, the nature of the records, the scope of the work performed, and the space available for storage.

Sample Analytical Data
1. Records should be kept documenting the care and disposition of samples during their time in the laboratory. These should show the storage conditions, the personnel with custody of samples, and the final disposition of samples when no longer required.
2. Records of all aspects of sample analyses, including sample descriptions, storage conditions and reverse sample retention, descriptions of analytical methods, all raw data, mid observations, calculations, and conclusions, are required. The analyst(s) responsible for each segment of the procedure should be identified in the record. These records may be in the form of worksheets that become a part of the entire record for each sample or a notebook that can be referred to in the sample records and correspondence.
3. Analytical records should be reviewed for completeness and accuracy before the results are reported. This review should be at least a two-step operation; with the first review done by should analyst in the laboratory and a second by the supervisor.

Research Data
Analytical laboratories are occasionally faced with the need to develop a new method of analysis or to modify an existing method. The details of these search activities must be properly documented. One should also consider adopting applicable elements of the following recommendations to other types record keeping in the laboratory.

The standard, bound laboratory notebook is the preferred medium for the recording of research data. At times, however, other media may replace or be used in conjunction with the laboratory notebook. Record maintenance procedures are as follows:

1. Each research project should have its own set of notebooks.
2. The first pages should be reserved for a brief table of contents that should include dates, type of information, and page numbers.
3. Notebook entries are to be made only in black or blue-black ink, with a fountain pen or ballpoint pen. Pencil or pen with water-soluble ink is not to be used. All entries must be dark and clear enough to be photocopied.
4. Illustrations such as charts, graphs, photographs, etc., may be pasted securely in the notebooks if the they approximate the size of the page. Voluminous printouts, photographs, charts, etc., may be maintained in supplemental files and referenced in the notebook.
5. All entries made by someone other than the notebook owner should be initialed.
6. Entries are to be neat and legible. No erasures are to be made; errors will be marked through with a single line, initialed, and dated.
7. Experimental results will be summarized.
8. All unused notebook pages will be cancelled with diagonal lines.
9. Once a study has been completed, the researcher will maintain the related notebook(s) and other research records. The first-line supervisor is responsible for assuring access and will maintain a log of all such materials under his or her responsibility.
10.A notebook assigned to an employee who is separating from an organization will be returned to the first-line supervisor, who will be responsible for maintaining the notebook and all related information.

Quality Assurance Data
Records should be kept of all quality assurance and control testing. These records can be kept on analytical worksheets as a part of the analytical routine or on separate log sheets. The analysts responsible for each check should be indicated, and the steps taken to bring back into control any procedures or functions out of tolerance should be recorded. Quality assurance records should be maintained for the following:
1. Analytical split or check sample results.
2. Purity and authenticity data on biological standards such as bacterial or fungal stock strains.
3. Calibration records.
4. Calibration/standardization records on analytical instruments such as gas chromatographs, spectrophotometers, pH meters, etc.
5. Annual calibration data and daily use check weighings on analytical balances.
6. Temperature records for freezers, refrigerators, incubators, waterbaths, etc.
7. Moisture level test results in incubators.
8. Time-temperature-pressure records for autoclaves.
9. Thermometer calibration results.

Storage and Retrieval of Data
A system that provides storage and ready retrieval of all the data generated in the laboratory is necessary. The type of system will depend on the type of laboratory and the analyses performed. The length of the time that these records should be kept can vary greatly. In the case of regulatory agency laboratories, they may need to be available for several years.

Enzyme Technology

Approximately 1% of the enzymes so far identified are used commercially as technical enzymes. The largest volume (35%) is proteases for use in detergent manufacture. In food processing, technical enzymes are used to reduce processing cost, to increase yields of extracts from raw materials or improve handling of materials, and to improve the self-life and sensory characteristics of foods.

Enzymes are active at low concentrations and the rates of reaction are easily controlled by adjustment of incubation conditions. However, the cost of enzymes is high, and in some products, enzymes must be inactivated or removed after processing, which adds to the cost of the product. Like other proteins, enzymes may cause allergic responses in some people, and they are usually coated or immobilized on carrier materials to reduce the risk of inhalation of enzyme dust by operations.

Theory
Microbial enzymes have optimum activity under similar conditions to the optimum growth conditions for the microorganism concerned. Enzymes from closely related microbial species have optimum activity under similar conditions, whereas those from unrelated species have widely differing properties. Microbial enzymes are either extra-cellular (secreted by the cells into the surrounding medium) or intracellular (retain within the cell). Extra-cellular enzyme production occurs in either the logarithmic phase or the stationary phase of growth, whereas intracellular enzymes are produced during logarithmic growth but are only released into the medium when cells undergo lysis in the stationary or decline phase.

Enzyme production from microorganisms
The requirements of commercial enzyme production from microorganisms are as follows:
1. Microorganisms must grow well on an inexpensive substrate,
2. They should produce a constant high yield of enzyme in a short time,
3. Methods for enzyme recovery should be simple and inexpensive and
4. The enzyme preparation should be stable
These requirements are met by constitutive mutant strains of microorganism, which permanently retain the required characteristics.

Enzymes are produced by either surface culture on solid substrate (for example rice hulls, fruit peels, soybean meal, wheat flour and peanut meal) or by submerged culture using liquid substrates. Submerged cultures have lower handling costs and a lower risk of contamination and are more suited to automation than are solid substrates. The substrate should contain carbon and energy source and a source of nitrogen for cell growth. In addition, specific nutrients may be required for cell growth and specific minerals may be necessary for enzyme production. In submerged culture, a seed inoculum is produced using similar incubation conditions to those used for production. The substrate (for example molasses starch hydrolysate or corn steep liquor) is low cost and readily available in adequate quantities, with a uniform quality. In batch methods, the inoculum is added to sterile substrate at 3-10% of the substrate volume. Fermenter capacities range from 1000 to 100.000 l. Cells are grown under controlled conditions for 30-150 h. Microprocessors are used to control pH, dissolved oxygen, carbon dioxide and temperature automatically.

Enzyme recovery
Extra-cellular enzymes are recovered from the fermentation medium by centrifugation, filtration, fractional precipitation, chromatographic separation, electrophoresis, membrane separation, freeze drying or a combination of these methods (Skinner, 1975). Intracellular enzymes are extracted by disruption of cells in a homogenizer or mill. Recovery is more difficult and the yield is lower than for extra-cellular enzymes, because some enzymes are retain within the cell mass. If required, the specific activity of the enzyme is increased by precipitation using acetone, alcohols, or ammonium sulfate or by ultra-filtration. The success of commercial enzyme production depends on maximizing the activity of the microorganism and minimizing the costs of the costs and incubation and recovery procedures.

Application of enzymes to foods
In batch operation the enzyme is mixed with food and, after completion of activity, is either retained within the food or inactivated by heat. This method is widely used when the cost of the enzyme is low.

In continuous operation, enzymes are immobilized on support materials by:
1. Micro-encapsulation in polymeric membranes, which retain the enzyme but permit passage of substrates and products,
2. Attachment by electrostatic attraction to ion exchange resins,
3. Cross-linking with for example glutaraldehyde,
4. Adsorption onto colloidal silica and cross-linking with glutaraldehyde,
5. Covalent bonding of non-essential residues on the enzyme to organic polymers, (the most permanent form of attachment),
6. Entrapment in polymer fibers (for example cellulose triacetate and starches)
7. Copolymerization with maleic anhydride and
8. Adsorption onto charcoal, polyacrylamide, or glass.

In adsorption onto charcoal, polyacrylamide, or glass, porous carriers have a high surface area and hence permit higher enzymic activities than non-porous carriers do. They also give protection to the enzyme against variations in the pH or temperature of the substrate but are more difficult to regenerate.

The main advantages of enzyme immobilization are:
1. Enzymes are re-used without the cost of recovery from a food,
2. Continuous processing, and
3. Closer control of pH and temperature to achieve optimum activity.

Immobilization is at present used when an enzyme is difficult to isolate or expensive to prepare. However, because of these advantages, the technique is expanding into areas of processing. The main limitations are:
1. The higher cost of carriers, equipment and process control,
2. Changes to the pH profiles and reaction kinetics of enzymes,
3. Loss of activity (25-60% loss) and
4. Risk of microbial contamination

In operation, either immobilized enzymes are mixed with a liquid substrate and then removed by centrifugation on filtration and reused, or the feed liquor is passed over an immobilized bed of enzyme fixed into a reactor. Immobilized enzymes should have the following characteristics:
1. Short residence times for a reaction,
2. Stability to variations in temperature and other operating conditions over a period of time (for example glucose isomerase is used for 1000 h at 60-650C).
3. Suitability for regeneration and
4. High mass transfer rates between the carrier material and the substrate.

Thursday, September 2, 2010

What are Enzymes and What are They Used for?

Enzymes are protein with enormous catalytic activity. They are synthesized by biological cells and, in all organisms, they are involved in chemical reactions related to metabolism. Therefore, enzyme-catalyzed reactions also proceed in many foods and thus enhance or deteriorate food quality. Relevant to this phenomenon are the ripening of fruits, vegetables, meat and dairy products, and the processing steps involved in the making of dough from wheat or rye flours and the production of alcoholic beverages by fermentation technology.

Enzyme inactivation or changes in the distribution patterns of enzymes in sub-cellular particles of a tissue can occur during storage or thermal treatment of food. Since such changes are readily detected by analytical means, enzymes often serve as suitable indicators for revealing such treatment of food. Examples are the detection of pasteurization of milk, beer or honey, and differentiation between fresh and deep frozen meat or fish.

Enzyme properties are of interest to food chemist since enzymes are available in increasing numbers for enzymatic food analysis or for utilization in industrial food processing.

During industrial-scale production, α-amylase is isolated from the pancreatic gland of swine and cattle from microbial culture e.g. the bacterium Bacillus subtilis or the fungi: Aspergillus oryzae. The high temperature-resistant bacterial amylases, particularly that of Bacillus licheniformis are of interest for the hydrolysis of corn starch, (gelatinization at 105-110 degrees C).

α-amylase is utilized in the baking industry and in the production of starch derivatives. In addition, studies are being conducted in several countries in which malt will be partially replaced by α-amylase and other enzymes of microbial origin, in the production of beer and spirits.

During the malting of cereals, such as barley, β-amylase is bio-synthesized to a greater extent than its counterpart, α-amylase. Because of the presence of these enzymes, malt is used extensively in processes where saccharification of starch is required.

There are three types of pectic enzymes: pectinesterase, polygalacturonase and pectin lyase although the last is not used commercially. Commercially the endo polygalacturonase is more useful as it produces more rapid depolymerization. Fungal enzyme preparations, consisting of polygalacturonase, pectin methylesterase, cellulase, hemicellulaseand protease, are used: (1) to accelerate rates of filtration of fruit juices, (2) to remove pectin from fruit base prior to gel standardization in jam manufacture, (3) to prevent undesirable gel formation in fruit and vegetable extracts and purees, (4) to standardize the characteristics of pectin for the varied uses as a thickener, (5) to recover citrus oils, and (6) to stabilize cloud in fruit juices

Element Facts: Iron

Iron is one of 14 essential trace elements present in hormones, vitamins, enzymes and other proteins which have distinct biological role. In addition, numerous other elements occur in the human body and their physiological roles have not yet been determined.

A deficiency in the essential trace elements results in metabolic disorders that are primarily associated with the absence or decreased activity of metabolic enzymes.

The iron content of the body is 4-5 g. Most is present in the hemoglobin (blood) and myoglobin (muscle tissue) pigments. The metal is also present in a number of enzymes, hence it is an essential ingredient of the daily diet.

The iron requirement depends on the age and sex of the individual, but it is about 1-2.8 mg/day. Iron supplied in the diet must be in the range of 5-28 mg/day in order to meet this daily requirement.

The large variation in intake can be explained by different extents of absorption of the various forms of iron present in food (organic iron compound vs simple salts). The most utilizable source is iron in meat, for which the extent of absorption is 20-30%. The absorption is much less from liver (6.3%) and fish (5.9%), or from cereals, vegetables and milk, from which iron absorption is the lowest (1.0-1.5%). Eggs decrease and ascorbic acid increases the extent of absorption. Bran interferes with iron absorption due to the high content of phytate.

Apparently, the absorption of iron present in food is, in a healthy organism, regulated by the requirement of the organism. Nevertheless, in order to provide a sufficient supply of iron to persons who require higher amounts (children, women before menopause and pregnant or nursing women), cereals, (bread, flour, rice, pasta products) fortified with iron to the extent of 55-130 mg/kg are recommended.

Extensive feeding tests with chickens and rats have shown that FeeSO4 is the most suitable form of iron, but ferrous gluconate and ferrous glycerol phosphate are also absorbed.

Two food processing problems arising from mineral fortification are the increased probability that oxidation will occur and, in the case of wheat flour, decreased baking quality.

Generally, iron is an undesirable element in food processing; for example, iron catalyzes the the oxidation of fat or oil, increases turbidity of wine and, as a constituent of drinking water, it supports the growth of iron-requiring bacteria.

Enzymes Isolation and Purification

Most of the enzyme properties are clearly and reliably revealed only with purified enzymes. Prerequisites for the isolation of a pure enzyme are selected protein chemical separation methods carried out at 0-4 degrees C since enzymes are often not stable at higher temperatures.

Tissue Disintegration and Extraction
Disintegration and homogenization of biological tissue requires special precautions: procedures should be designed to rupture the majority of the cells in order to release their contents so that they become accessible for extraction. The tissue is usually homogenized in the presence of an extraction buffer which often contains an ingredient to protect the enzymes from oxidation and traces of heavy metal ions. Particular difficulty is encountered during the isolation of enzymes which are bound tenaciously to membranes which are not readily solubilized. Extraction in the presence of tensides may help to isolate such enzymes. As a rule, large amounts of tissue have to be homogenized because the enzyme content in proportion to the total protein isolated is low and is usually further diminished by the additional purification of the crude enzyme isolate.

Enzyme Purification
Removal of protein impurities, usually by a stepwise process, is essentially the main approach in enzyme purification. Often, as a first step, fractional precipitation, e.g. by ammonium sulfate saturation, is used or the extracted proteins are fractionated by molecular weight e.g. column gel chromatography. The fractions containing the desired enzyme activity are collected and, for example, are purified further by ion-exchange chromatography. Other supplemental options are also available, such as various forms of preparative electrophoresis, e.g. disc gel electrophoresis or isoelectric focusing.

The purification procedure can be substansially, shortened by using affinity column chromatography. In this case, the column is packed with a stationary phase to which is attached the substrate or a specific inhibitor of the enzyme. The enzyme is then selectively and reversibly bound and,thus, in contrast to the other inert proteins, its elution is delayed.

Control of Purity
Previously, the complete removal of protein impurities was confirmed by crystallization of the enzyme. This "proof" of purity can be circumstantial and is open to criticism. Today, electrophoretic methods of high separation efficiency are primarily used.

The behavior of the enzyme during chromatographic separation is an additional proof of purity. A purified enzyme is characterized by a symmetrical elution peak in which the positions of the protein absorbance and enzyme activity coincide and the specific activity (expressed as units per amount of protein) remains unchanged during repeated elutions.

During a purification procedure, the enzyme activities are recorded. Data show the extent of purification achieved after each separation step and show the enzyme yield. Such a compilation of data readily reveals the undesired separation step associated with loss of activity and suggests modifications or adoption of other steps.

How Milk is Processed

The processing of raw milk for drinking or into other diary products requires stringent control to obtain a product that is hygienic and of high organoleptic quality.

Milk is produced on the dairy farm under good hygienic conditions, primarily as a result of using milking macines. The warm milk (30°C) is strained in the milk house, cooled and stored in cans or storage tanks to avoid sunlight. Milk is usually transported to the processing plant in cans or in the tank of a milk truck. Transportation of milk from mountains to valleys by pipelines made of polyethylene or PVC was first introduced in Austria, France and Switzerland.

The milk is first purified with a continuously-operated clarifier (centrifuge). Contaminants are removed in the sediment. Cream separation is often achieved simultaneously. The process is conducted at 40°C at 5,500-6,500 rpm. Such centrifuges have a flow production capacity of up to 20,000 kg/h. Back-mixing allows the milk fat content to be adjusted as desired.

The fluid milk is heated after clarification to improve its durability and to kill disease-causing microorganisms. Heat treatments used are:

Pasteurization
The milk is treated: at high temperature (85°C for 2 sec); in a short-time, flash process (71-74°C for 15-40 sec) in plate heaters; or by the low temperature or holder process, in which it is heated at 62-65°C for at least 30 min, with stirring, and it is then cooled.

Ultrahigh temperature (UHT) treatment
The process involves indirect heating by coils or plates at 135-140°C for 6-10 sec, or direct heating by live steam injection at 140-150°C for 2-4 sec, followed by aseptic packaging.

Sterilization
Milk in retail packages is heated in autoclaves at 110-120°C for 10-20 min.

Heat treatment affects several milk constituents. Casein, strictly speaking, is not a heat-coagulable protein; it coagulates only at very high temperatures. Heating at 120°C for 5 h dephosphorylates sodium or calcium caseinate solutions (100% and 85%, respectively) and releases 15% of the nitrogen in the form of low molecular weight fragments.

However, temperature and pH strongly affect casein association and cause changes in micellular structure. An example of such a change is the pH-dependent heat coagulation of skim milk.

All pasteurization processes supposedly kill the pathogenic microorganisms in milk. The inactivation of the alkaline phosphatase is used in determining the effectiveness of pasteurization. At higher temperatures or with longer heating time, the whey proteins start to denature-this coincides with the complete inactivation of acid phosphatase.