Identification of the geographical origin of pumpkin seed oil by the use of rare earth elements and discriminant analysis

The aim of this work was to develop an analytical method to assure the geographical origin of Styrian pumpkin seed oil, a high priced local product which is protected by the European Union. The development of such a method is not only of interest for scientists but also of importance for producer that want to guarantee the purity and quality of their products as well as the consumer wanting to know the origin of the food products.Since the rare earth elements (REE) concentrations are extremely low in pumpkin seed oil a fast and sensitive analytical method with ICP-MS had to be developed and validated. In the current project, pumpkin seeds from different regions in Austria were sampled. The REE patterns in the extracted oil of these seeds were determined and a preliminary classification with discriminant analysis was successfully done on a statistical basis.The study demonstrates that pumpkin seed oils from different geographical origin show variable rare earth element patterns making it possible to trace the origin of these oils.     

The potential of multielement stable isotope analysis for regional origin assignment of butter

 Regional origin assignment of highly valuable milk products such as butter, is of considerable importance for legal, fiscal and trade controls within the European Union (EU). It is also of value for ensuring fair competition and as a means of protecting consumers against fraud due to mislabelling. Conventional chemical methods of analysis are not able to determine the regional provenance of butter unambiguously. Therefore, stable isotope ratio determinations of the light elements (C,N,O,S; bioelements) and the heavy element, Sr (a trace element), have been used for this purpose. Stable isotope ratios of these elements in natural cycles, their variations due to climate and geology, the abiotic and biological fractionation of isotopes and, finally, the reasons for regional differences in multielement stable isotope ratios of butter are discussed. Results are given for butter from several European countries and from outside the EU. The results indicate that stable isotope ratios and subsequent discriminant analysis based on data for samples of certified origin can enable the reliable detection of the regional provenance of butter. Received: 11 May 1999 / Revised version: 17 August 1999     

EXTERNAL VALIDATION AND TRANSFERABILITY OF NIRS MODELS DEVELOPED FOR DETECTING AND QUANTIFYING MBM IN INTACT COMPOUND FEEDING STUFFS

ABSTRACT

The ban on the use of animal‐origin by‐products such as meat and bone meal (MBM) in compound feeds was one of the measures implemented in the European Union to stop the spread of bovine spongiform encephalopathy (BSE) and to prevent its reoccurrence. It is now clear that the blanket ban will only be lifted if reliable analytical methods are available that ensure the detection of animal by‐products. Near‐infrared spectroscopy (NIRS) is likely to be the most rapid method for testing feedingstuffs, enabling a substantial increase in the number of samples tested and providing instant detection of adulteration, especially when samples are analyzed in intact form. This study aimed to demonstrate the feasibility of NIRS predictive models for detecting and quantifying MBM in intact compound feedingstuffs, and to demonstrate the transferability of calibration models between two NIRS instruments.

PRACTICAL APPLICATIONS

This study shows the potential of near infrared spectroscopy (NIRS) as a fast screening method for detecting the adulteration of compound feedingstuffs with animal‐origin meals, useful for the feed industry and the inspection bodies. NIRS could provide the first line of defense of the food chain, allowing a large‐scale analysis and making more costly methods to be used more productively on suspect specimens.     

Tracing the geographical origin of food: The application of multi-element and multi-isotope analysis

The globalisation of food markets and the relative ease with which food commodities are transported through and between countries and continents, means that consumers are increasingly concerned about the origin of the foods they eat. A growing number of research articles have been published in the last 5 years detailing the use of natural abundance isotope variation and elemental concentrations as geographic ‘tracers’ to determine the provenance of food. These investigations exploit the systematic global variations of stable hydrogen and oxygen isotope ratios in combination with elemental concentrations, including heavy isotope variations (e.g. strontium-87) and other biogeochemical indicators. This article reviews the developments in the application of multi-isotopic and multi-element methods in the emerging field of ‘Food Forensics’.     

Application of controlled mesocosms for understanding mercury air-soil-plant exchange

Whole system elemental mercury (Hg0) flux was measured for approximately 1.5 years using two large gas exchange mesocosms containing approximately 100 two-year old aspen trees (Populus tremuloides) planted in soil with elevated mercury concentrations (12.3 microg/g). We hypothesized that during leafout, whole mesocosm Hg0 flux would increase due to movement of Hg0 in the transpiration stream from the soil to the air. This hypothesis was not supported; plants were found to assimilate Hg0 from the contaminated air, and whole system Hg0 emissions were reduced as plants leafed-out due to shading of the soil. Surface disturbance, watering, and increases in soil moisture, light, and temperature were all found to increase whole system Hg0 flux, with light being a more significant factor. Although surface soils were maintained at 15-20% moisture, daily watering caused pulses of Hg0 to be released from the soil throughout the experiment. Data developed in this experiment suggested that those processes acting on the soil surface are the primary influence on Hg emissions and that the presence of vegetation, which shields soil surfaces from incident light, reduces Hg emissions from enriched soils.     

Identification of cherry tomatoes growth origin by means of magnetic resonance imaging

Magnetic resonance imaging (MRI) spectroscopy is a promising non‐invasive and non‐destructive analytical technique in food science, since it offers the opportunity of studying vegetables and fruits in their wholeness without any manipulation. The aim of this preliminary study was to demonstrate the possibility of assessing unambiguously the place of origin of fresh cherry tomatoes by means of MRI experiments. The innovative approach has been successfully used to distinguish PGI (Protected Geographical Indication) cherry tomatoes from non‐PGI ones, as well as cv. Naomi from cv. Shiren samples. The method determines informative physical and morphological parameters, transverse relaxation times and thicknesses, respectively, which can be combined into four empirical equations; two of them are used to determine the cultivated variety and the other two to assess the place of origin. This approach has successfully recognize the cultivated variety of c. 90% of the analyzed samples and the geographical origin of c. 80% of the investigated cherry tomatoes. Copyright © 2006 Society of Chemical Industry     

EVALUATION OF THE NUTRITIONAL QUALITY OF NONTOXIC KERNEL FLOUR FROM JATROPHA CURCAS L. IN RATS

ABSTRACT

The origin of Jatropha curcas L. is in Central America, probably Mexico, although it is also distributed in South America, Africa and Asia. In Mexico, it grows as nontoxic and toxic J. curcas genotypes. In this work, the protein quality including protein efficiency ratio (PER), net protein ratio (NPR) and true digestibility (TD) of nontoxic genotype defatted flour was assessed using Wistar rats. The probed diets contained flour (3), flour‐lysine, 1% (4), flour‐phytase, 500 FTU (5) and two control diets: nitrogen‐free (1) and casein (2). The rats were fed for 28 days. The PER (1.37, 1.77 and 1.61) and NPR (1.80, 2.29 and 2.12) obtained values for diets (3.4 and 5) were lower than those obtained for casein (2.07 and 2.46), respectively. No statistical differences were found in TD.

PRACTICAL APPLICATIONS

Jatropha curcas defatted flour may be used in the food industry for the development of diets for human and animal consumption. Besides achieving fortification of foods that are made from wheat, which has a low protein content, with the addition of Jatropha meal, these foods will improve the protein quality of many food products.     

BAKING AND SENSORY CHARACTERISTICS OF MUFFINS INCORPORATED WITH APPLE SKIN POWDER

ABSTRACT

Apple fruit skin, a rich source of dietary fiber and phenolics, is a by‐product of apple processing. The feasibility of incorporating dried apple skin powder (ASP) as a value‐added food ingredient in bakery food products using a model system of muffins was investigated. The blanched, dehydrated and ground ASP was incorporated into muffins at 0, 4, 8, 16, 24 or 32% (w/w) levels with replacement of equivalent amount of wheat flour of a standard muffin mixture. The highest level of replacement (32% w/w) had a significant adverse effect on the baking characteristics. A taste panel of 66 panelists showed that the replacement of wheat flour with 8, 16 or 24% ASP in the muffin mixture did not affect the overall acceptability.

PRACTICAL APPLICATIONS

Identification of ways to incorporate apple skins, one of the by‐products of apple pie and sauce manufacturing, as a health food ingredient in human diet could provide many health benefits. Furthermore, better use of the by‐product will also provide benefits to the apple industry as well as solutions for environment concerns associated with disposal. The results of the present study indicate that blanched and dehydrated apple skin powder (ASP) could be considered as an alternative dietary fiber source or specialty food ingredient for muffins. Therefore, the potential for the industrial exploitation of ASP as a health food ingredient for the bakery industry and selected functional foods is promising.     

EFFECTS OF 1‐METHYLCYCLOPROPENE AND MODIFIED ATMOSPHERE PACKING ON POSTHARVEST LIFE AND QUALITY IN TOMATOES

ABSTRACT

The effects of 1‐methylcyclopropene (1‐MCP), modified atmosphere packaging (Xtend, StePac L.A. Ltd., Tefen, Israel) and their combination were investigated on storage and quality maintenance of tomatoes cv. “Perla” harvested at two maturity stages. 1‐MCP treatment was performed in 1,000 nL/L doses for 24 h while untreated fruits were considered as control. Fruits were stored at 12C with 90% relative humidity for 21 days. Weight loss, skin color, elasticity, lycopene, ascorbic acid, soluble solid content (SSC), titratable acidity (TA) and SSC/TA were evaluated with the intervals of 7 days. MAP with and without 1‐MCP reduced weight loss and maintained to elasticity compared with control and 1‐MCP alone. Nevertheless, 1‐MCP or its combination with MAP significantly delayed changes in parameters related to fruit ripening, such as skin color, lycopene, TA and SSC/TA increase in both ripening stages. Overall results indicated that the combination of 1,000 nL/L 1‐MCP and modified atmosphere package was the most effective treatment in both ripening stages.

PRACTICAL APPLICATIONS

Tomato is a climacteric fruit and its ripening depends on ethylene production physiology. 1‐Methylcyclopropene (1‐MCP) prevents ethylene action by blocking ethylene receptors and extends the storage life of tomatoes. Modified atmosphere packaging (MAP) of fresh fruits and vegetables refers to the still evolving technique of matching the respiration of the product with the O₂ and CO₂ permeability of packages in order to modify the oxygen and carbon dioxide concentrations of the atmosphere to desired levels within the package. Recent studies revealed that combined applications of 1‐MCP and MAP have significant effects on maintaining storage quality of banana, litchi and plums. This study was conducted on the investigation of the effects of 1‐MCP and MAP combination on storage duration and quality of tomatoes harvested at different maturity stages.     

INFLUENCE OF COMBINED MICROWAVE‐VACUUM DRYING ON DRYING KINETICS AND QUALITY OF DRIED TOMATO SLICES

ABSTRACT

The study investigated the influence of four microwave powers on the drying kinetics and quality of tomato slices in a microwave‐vacuum dryer at vacuum pressures of 0.04, 0.05 and 0.06 MPa. The results showed that increase in microwave‐vacuum drying conditions increased drying rates and resulted in decreased drying time from 84 to 14 min. The nonenzymatic browning index increased with microwave power whereas the overall flavor degradation was between 18.99 and 20.80%. The brightness and the yellowness of the dried tomatoes generally increased but there was a slight reduction in redness when compared with the fresh. The effective moisture diffusivity increased with microwave power and was 7.22 × 10^?9, 9.10 × 10^?9, 14.99 × 10^?9 and 25.19 × 10^?9 m²/s at respective microwave powers of 200, 300, 500 and 700 W.

PRACTICAL APPLICATIONS

Microwave‐vacuum drying is one of the advanced methods with numerous advantages including high thermal efficiency, shorter drying time and improved product quality. Tomato is the second most important vegetable with enormous health benefits, which include reduction of cholesterol, improvement of vision, maintenance of gut, lowering of hypertension, alleviation of diabetes, and prevention of urinary tract infections and gallstones. The quality of dried products obtained using the microwave‐vacuum dryer provides industries with method that can be used to enhance the quality of dried tomatoes. The dried product is a valuable ingredient in the preparation of soups and sauces, as well as in cosmetics and pharmaceutical formulations.

     

A research note: the potential for transfer of Salmonella from irrigation water to tomatoes

The transmission of Salmonella Enteritidis from soil to fruit by contaminated irrigated water was studied using 20 patio tomato plants. In order to track the presence of Salmonella in the soil and plants a luminescent strain transformed with the full luxCDABE gene cassette from Photorhabdus luminescens was used. The tomato plants were irrigated every other day by direct application of water containing Salmonella Enteritidis (10⁵ CFU ml^?1) to the soil. Samples of soil, stem, leaf and fruit were taken weekly and assayed for Salmonella by plating onto Luria Bertani agar containing 50 µg ml^?1 ampicillin. There was a significant difference (P < 0.05) in Salmonella counts from soils sampled during the course of the study. No Salmonella were recovered from the leaf, stem, and fruit samples taken from the tomato plants. This indicates that, under these test conditions, watering with contaminated water directly into the soil does not result in the transmission of Salmonella, and possibly other pathogens, to tomatoes. Copyright © 2004 Society of Chemical Industry     

Presence of benzodiazepine-like molecules in food and their implication in the nutrition of cirrhotic patients

Benzodiazepine (BDZ)-like compounds, present in trace amounts in normal subjects increase in the blood of liver cirrhotic patients. The origin of these compounds is still unknown but they are present in medicinal plants and foods. Herein we report the detection of BDZ-like molecules in fruits, vegetables, cereals, meat, milk and cheeses and in different cultivars of potatoes, tomatoes and carrots. The extracted food was separated by HPLC purification and the collected fractions were tested by radioreceptor binding assay in order to evaluate their ability to selectively bind the central benzodiazepine receptors. The mean value was 14.80 ng of diazepam equivalent (DE)/g in fruits, 4.34 ng DE/g in vegetables, 6.35 in cereals and 4.09 in meat. BDZ-like compounds are poorly present in cheeses and completely absent in olive and seeds oil. From these findings it is possible to select food with low amount of BDZ-like molecules useful for cirrhotic diet in order to prevent hepatic encephalopathy.     

CHANGES IN Pb, Cd, Fe, Cu AND Zn LEVELS DURING THE PRODUCTION OF KAŞAR CHEESE

ABSTRACT

Levels of Pb, Cd, Fe, Cu and Zn in milk, curd, pressed curd, fresh cheese, whey, rennet and scalding water taken from two different Ka?ar cheese plants (A and B) in Ankara, Turkey were investigated. The milk used in plant A contained higher amount of Pb, Fe and Zn than the milk used in plant B. Pb level during processing in both dairy plants showed a significant increase from milk to curd (626.2–912.3 µg/kg for plant A and 265.2–371.8 µg/kg, dry weight, for plant B) (P < 0.01). Similarly, Fe, Cu and Zn contents of the curds in plant A and B showed an important increase with respect to the milk (P < 0.01). During transition of the curd to pressed curd and of pressed curd to fresh cheese, almost all metals tested showed a decrease because of the loss of these metals into whey and scalding water. The results showed that curdling the milk was the most important contamination step.

PRACTICAL APPLICATIONS

Heavy metals may enter the human body through food, water, air or absorption through the skin, and can cause metabolic anormalies. Heavy metals may reach our foods from a number of sources. The more important of these are: soil; the chemicals applied to agricultural land; the water used in food processing or cooking; and the equipment, containers and utensils used for food processing, storage or cooking. Milk and milk products are the basic components of the human diet, and among milk products cheese holds an important place. Ka?ar cheese ranks second with respect to consumption, significantly contributing to the Turkish diet. In order to prevent the health risk of consuming contaminated cheeses, it is very important to determine the effect of equipment and process variation on the heavy‐metal content of Ka?ar cheese. The results of this study would help the regulatory authorities to establish a Hazard Analysis and Critical Control Points (HACCP) plan and to identify important contamination sources for Ka?ar and similar kind of cheeses.

     

Consumer attitude and behaviour towards tomatoes after 10 years of Flandria quality labelling

In recent years, trust in food safety and food quality has decreased as a result of consecutive food crises. Consequently, numerous quality labels signalling process-related credence characteristics have been established. One of these labels is the Belgian Flandria quality label for fresh fruit and vegetables. Based on cross-sectional data collected through a self-administered consumer survey ( n  = 373), this paper addresses consumer attitudes, behaviour and perception towards tomatoes in general, and the Flandria tomato label in particular. Buyers, who constitute 26.8% of the sample, perceive Flandria tomatoes as superior to other tomatoes because of their guarantee of origin, better taste and stricter production control. However, they also report the strongest perception of Flandria as an ordinary tomato as compared to non-buyer segments. Overall, findings indicate that the Flandria label – after being intensively used for 10 years for a wide range of other fruits and vegetables besides tomatoes – has become fairly standard for tomatoes with little perceived differentiation apart from its certified production and origin.     

Origin of nitrogen in reforested lignite-rich mine soils revealed by stable isotope analysis

Restoration of the nitrogen cycle is an important step in the recovery of an ecosystem after mining. Carbon and nitrogen in rehabilitated lignite containing mine soils can be derived from plant material as well as from lignite inherent to the parent substrate. We assessed the use elemental and stable carbon and nitrogen isotope measurements to trace the orgin of soil nitrogen and applied these techniques to elucidate the origin of mineral N in the soil and the soil solution. The conceptual approach of this study included physical fractionation in addition to sampling of vegetation and soil from a lignite-containing mine site rehabilitated in 1985 with Pinus Nigra. We studied the elemental and isotopic composition of bulk samples as well as isolated fractions and soil solution. Our data indicate that the stable carbon and nitrogen isotopic composition of the soil samples are the result of mixing between plant material and substrate inherent lignite. delta15N isotopes may be used as indicators of nitrogen contribution from plants to solid samples as well as soil solution. N-isotope composition of ammonia shows low spatial and interannual variability, despite strong concentration changes. Plant-derived nitrogen contributes in higher amounts to the soil solution compared to the bulk mineral soil.     

A SPECIFIC QUALITATIVE DETECTION METHOD FOR PEANUT (ARACHIS HYPOGAEA) IN FOODS USING POLYMERASE CHAIN REACTION

A qualitative method for detection of peanuts in foods using polymerase chain reaction was developed. A universal primer pair CP 03‐5′/CP 03‐3′ was designed to confirm the validity of the DNAs for PCR. The plant‐specific amplified fragments were detected from 13 kinds of plants using the universal primer pair. In addition, for the specific detection of peanuts with high sensitivity, the primer pair agg 04‐5′/agg 05‐3′ was designed to detect the gene encoding the peanut agglutinin precursor. The primer pair specifically generates a 95‐bp amplified fragment from peanut genomic DNA. Five hundred femto grams of peanut genomic DNA can be detected using the established method. The same qualitative results were obtained from both model processed and nonprocessed food samples containing 0.001, 0.01 and 0.1% of peanut. Moreover, it was shown that the trace amount of peanut in the commercial food products could be qualitatively detected using this method. The reproducibility and applicability of the proposed methods were verified in a six‐laboratory collaborative study.     

PRODUCT DEVELOPMENT AND NUTRACEUTICAL ANALYSIS TO ENHANCE THE VALUE OF DRIED FRUIT

ABSTRACT

Value‐added fruit products suitable for production by small‐scale farmers and processors were developed and evaluated. Apples, blueberries, peaches and strawberries from local growers were processed into a dried fruit product. The total phenolics, total anthocyanins and oxygen radical absorbing capacity (ORAC) were determined. Drying the fruit concentrated total phenolics, total anthocyanins and ORAC levels as compared to fresh fruit. The nutraceutical components of the blueberries, peaches and strawberries of the local dried fruit were higher than those of commercial dried fruit purchased at a natural food store. The dried fruit from both the local and commercial fruits was used to create a fruit pie filling for consumer evaluation. Consumer acceptance of fruit pie filling from dried fruit grown locally and dried commercial fruits was evaluated using a 9‐point verbal hedonic scale and a 5‐point Just About Right scale. In terms of consumer evaluation, the pie fillings made from local dried fruits were either better or at parity with those from commercial products. In addition to the nutraceutical benefits, the pie fillings were consumer acceptable products that could represent an avenue for small‐scale farmers to add value to surplus fruit.

PRACTICAL APPLICATIONS

Complete utilization of fruit crops is a key component to the success of small farm operations. Excess fruit crops can be dried (or processed in other manners) and used to create many value‐added products. Increased consumer awareness of healthy food products provides a niche market for these fruit products. In this research, the nutraceutical benefits of fresh and dried apples, blueberries, peaches and strawberries were investigated and the consumer overall acceptance of a pie filling produced from these dried fruit was shown.

     

Jaboticaba byproduct encapsulation by lyophilization: pH and food application stability

The study aimed to microencapsulate jaboticaba byproduct extract by lyophilization, followed by the characterization, pH stability and food application of the prepared microcapsules. The products were visualized by scanning electron microscopy and the yield, encapsulation efficiency (EE%), and bioactive compounds (high‐performance liquid chromatography) were determined. The major compounds found (in all samples) were cyanidin‐3‐glucoside, acids ellagic and gallic, and quercetin. The EE% was 80.0% and yield 67.2%. The capsules presented smooth surfaces and varied morphologies, such as broken glass of irregular sizes. The extracts revealed brighter and lighter‐colored solutions in low pH buffers, while microencapsulation protected the monomeric anthocyanins and phenolic compounds, particularly at pH 6.5. At 4 °C, maria‐mole desserts produced with the extract, showed a higher color variation (ΔE) than the product prepared with the microcapsules. Thus, microencapsulation may be an alternative to maintain the stability of the jaboticaba extract, as a way to reuse the agroindustrial byproduct.

Practical applications

The nutritional and functional properties of a vast biodiversity of plants (and their byproducts) have not yet been fully studied. Jaboticaba byproduct has great potential for economic exploitation and could be a valuable source of natural antioxidants, however, more studies of this byproduct, are required. Moreover, protecting the antioxidants during the fruit extraction process and storage, can improve the utilization and application of bioactives in food products. In this study, the extraction and microencapsulation of jaboticaba byproduct were realized. The results showed microencapsulation may be a feasible alternative to maintain the stability of the jaboticaba extract and provide a way to reuse the agroindustrial byproduct, as an ingredient in the food and, potentially, cosmetics and pharmaceutical industries.     

Evaluation of elemental profile coupled to chemometrics to assess the geographical origin of Argentinean wines

Traceability of wines requires knowledge of their characteristics, which are associated with the geographical origin of grape, soil, water, climate as well as the winery techniques. The aim of this work was to classify wines and soil from three production areas of Argentina according to multielement data. The influence of the provenance soil on the wine element composition was also investigated.Eleven elements were determined in 31 wine samples and 137 soil samples from regions under study. Stepwise discriminant analysis allows us to correctly classify 100% of the wines analysed from the three regions using only seven parameters (K, Fe, Ca, Cr, Mg, Zn and Mn) and 92% correct classification for soils using seven variables (Ca, Cr, K, Fe, Cu, Zn and, Mg). Canonical analysis between soils and wines datasets affords a correlation coefficient of 0.85 (P-value < 0.001). Thus, almost 85% of variability observed amongst wines could be attributed to the soil in which the vines were cultivated.The analysis of elemental concentrations in the wines and soils, in combination with chemometrics, provides a powerful tool to verify the geographical origin of wines.     

CHANGES IN TOTAL PHENOLIC AND FLAVONOID CONTENTS OF ROWANBERRY FRUIT DURING POSTHARVEST STORAGE

ABSTRACT

Postharvest storage conditions of rowanberry (Sorbus aucuparia) fruits were investigated. The influences of temperature and storage time on total phenolic content were observed. Both variables affected phenolic content of rowanberries, and their total phenolic contents significantly reduced during the 20‐day storage at 4 and 22C. The loss of total phenolic was found to be almost 50% of its initial amount (3,619.8 mg/kg fresh weight [fw]) at 22C, which was higher than occurred loss (30%) at 4C. An investigation of rowanberry flavonoids showed the presence of rutin, quercetin‐3‐glucoside and quercetin‐3‐D‐galactoside (Q3DG). Quantitative analysis of berry flavonoids showed Q3DG as a major compound, being 24.55 mg/kg fw. The highest reduction (86% of its initial amount) was also seen in that compound level compared with other ones. Degradation kinetics of flavonoids was studied at 22C over 16 days of storage. Checking of model adequacies indicated that their reductions followed a first‐order kinetic.

PRACTICAL APPLICATIONS

Postharvest storage has been taken, increasing the interest of scientists because of various reasons, one of which is explained next. The presented results indicated the direct and/or indirect influence of storage conditions on the quality of berries, implying that whoever is planning to store plant and/or plant origin food products in a refrigerator or at room temperature should consider the conditions and their possible effects on the quality parameters from the aspects of economics and nutritional properties of products. In the present study, the stability of an important bioactive group, phenolics, in rowanberries and the influences of environmental conditions were examined, and additionally, degradation kinetics of major phenolic group, flavonoids, was analyzed. The results are valuable and helpful for the evaluation of the postharvest storage and its effect on rowanberries. Introduced model may be helpful for the prediction of storage time required for the degradation of phenolics up to the acceptable limit.