Detection of added beet or cane sugar in maple syrup by the site-specific deuterium nuclear magnetic resonance (SNIF-NMR) method: collaborative study

Results of a collaborative study are reported for the detection of added beet or cane sugar in maple syrup by the site-specific natural isotope fractionation-nuclear magnetic resonance (SNIF-NMR) method. The method is based on the fact that the deuterium content at specific positions of the sugar molecules is different in maple syrup from that in beet or cane sugar. The syrup is diluted with pure water and fermented; the alcohol is distilled with a quantitative yield and analyzed with a high-field NMR spectrometer fitted with a deuterium probe and fluorine lock. The proportion of ethanol molecules monodeuterated at the methyl site is recorded. This parameter (D/H)I is decreased when beet sugar is added and increased when cane sugar is added to the maple syrup. The precision of the method for measuring (D/H)I was found to be in good agreement with the values already published for the application of this method to fruit juice concentrates (AOAC Official Method 995.17). An excellent correlation was found between the percentage of added beet sugar and the (D/H)I isotopic ratio measured in this collaborative study. Consequently, all samples in which exogenous sugars were added were found to have a (D/H)I isotopic ratio significantly different from the normal value for an authentic maple syrup. By extension of what is known about plants having the C4 cycle, the method can be applied to corn sweeteners as well as to cane sugar. One limitation of the method is its reduced sensitivity when applied to specific blends of beet and cane sugars or corn sweeteners. In such case, the C13 ratio measurement (see AOAC Official Method 984.23, Corn Syrup and Cane Sugar in Maple Syrup) may be used in conjunction.     

Eliminating false positive C4 sugar tests on New Zealand Manuka honey

Carbon isotope analyses (δ¹³C) of some New Zealand Manuka honeys show that they often fail the internationally recognised Association of Official Analytical Chemists sugar test (AOAC method 998.12) which detects added C₄ sugar, although these honeys are from unadulterated sources. Failure of these high value products is detrimental to the New Zealand honey industry, not only in lost export revenue, but also in brand and market reputation damage. The standard AOAC test compares the carbon isotope value of the whole honey and corresponding protein isolated from the same honey. Differences between whole honey and protein δ¹³C values should not be greater than +1.0‰, as it indicates the possibility of adulteration with syrups or sugars from C₄ plants such as high fructose corn syrup or cane sugar. We have determined that during the standard AOAC method, pollen and other insoluble components are isolated with the flocculated protein. These non‐protein components have isotope values which are considerably different from those of the pure protein, and can shift the apparent δ¹³C value of protein further away from the δ¹³C value of the whole honey, giving a false positive result for added C₄ sugar. To eliminate a false positive C₄ sugar test for Manuka honey, prior removal of pollen and other insoluble material from the honey is necessary to ensure that only the pure protein is isolated. This will enable a true comparison between whole honey and protein δ¹³C isotopes. Furthermore, we strongly suggest this modification to the AOAC method be universally adopted for all honey C₄ sugar tests. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of the carbon-13 content of sugars and pulp from fruit juices by isotope-ratio mass spectrometry (internal reference method). A European interlaboratory comparison

An interlaboratory comparison by ¹³C-isotope ratio mass spectrometry was organised by working group No. 1 of the European Commission of Standardisation (CEN/TC 174) in order to define the repeatability (r) and the reproducibility (R) of the ¹³C determination in sugars and pulp, isolated from the same fruit juice. Six unlabelled juices were analysed in 19 laboratories in the European Union, Australia and the USA. Three samples were authentic juices (orange, grapefruit, pineapple) and the remaining samples were the same juices with an addition of sugar at 15 g l⁻¹ (orange, pineapple) or 11.8 g l⁻¹ (grapefruit). The sugar was prepared by mixing equal amounts of beet and cane sucroses. The different laboratories used the same experimental protocol under different conditions (operator, conversion system for CO₂ preparation, mass spectrometer). The results for the sugars (r = 0.27%., R = 0.82%.) were comparable to those from an earlier ring test (r = 0.30%., R = 0.70%.), while the results for the pulp presented higher interlaboratory variability (r = 0.38%., R = 1.89%.) possibly due to experimental difficulties. Nevertheless, the RSD_R for free sugars ranged 0.9–3.2% and for pulp, 0.9–9.3%. On the basis of the Horwitz equation and taking account of the concentration of the isotopes measured, an RSD_R of 7–8% might have been expected. The experimentally determined RSD_R values were less than twice the theoretically expected values, which is a criterion of an acceptable method. The results were therefore considered to be acceptable by specialists in the field of isotopic analysis, and the method applied was found to improve the sensitivity of the ¹³C determination for the detection of sugar addition in fruit juices.     

13C-IRIS: an improved method to detect the addition of low levels of C4-derived sugars to juices.

A method to improve the detection of cane or corn sugar addition to fruit juices has been developed. This involves the isolation of the individual sugars and measurement of their carbon isotope ratios. Using the Isotopic Relations of Individual Sugars approach, the additions of cane sucrose or cane invert as low as 4% of the total sugars can be detected in orange juice. This method offers a significant improvement over the conventional carbon isotopic method that has a detection limit of about 10%.     

Detection of royal jelly adulteration using carbon and nitrogen stable isotope ratio analysis

Stable isotope ratios ((13)C/(12)C and (15)N/(14)N) were measured in royal jelly (RJ) samples by isotope ratio mass spectrometry (IRMS) to evaluate authenticity and adulteration. Carbon and nitrogen isotope contents (given as delta values relative to a standard, delta(13)C, delta(15)N) of RJ samples from various European origins and samples from commercial sources were analyzed. Uniform delta(13)C values from -26.7 to -24.9 per thousand were observed for authentic RJ from European origins. Values of delta(15)N ranged from -1.1 to 5.8 per thousand depending on the plant sources of nectars and pollen. High delta(13)C values of several commercial RJ samples from -20.8 to -13.3 per thousand indicated adulteration with high fructose corn syrup (HFCS) as a sugar source. Use of biotechnologically produced yeast powder as protein source for the adulterated samples was assumed as delta(15)N values were lower, as described for C(4) or CAM plant sources. RJ samples from authentic and from adulterated production were distinguished. The rapid and reliable method is suitable for urgent actual requirements in food monitoring.     

A site-specific and multi-element isotopic approach to origin inference of sugars in foods and beverages

A strategy is presented for the characterization of sugars according to their botanical origin. The samples fermented in standardized conditions can be described in the multi-dimensional space of the overall carbon isotope ratio of ethanol measured by isotope ratio mass spectrometry (IRMS) and of the specific hydrogen isotope parameters of the methyl and methylene sites derived from nuclear magnetic resonance investigation of site-specific natural isotope fractionation (SNIF-NMR method). In the comparison of natural juices, the deuterium and oxygen-18 parameters of water extracted from the juice and from the end fermentation medium also contain information on the origin of the product. The isotopic effects of the concentration processes leading to concentrated juices, musts and syrups can be estimated and taken into account in interpreting the data.The classification power of this multi-element and multi-site approach is illustrated by discriminant analyses involving selected isotopic variables associated with pineapple, apple and barley sugars, compared to beet and cane sugars which are common sources of enrichment. The ability of the method to detect adulteration by exogeneous sugars is improved when environmental conditions can be taken into account.     

液相色谱/元素分析-同位素比值质谱联用法鉴定蜂蜜掺假

采用液相色谱/元素分析-同位素比值质谱联用法(LC/EA-IRMS)对国内蜂蜜掺假情况进行了研究。基于测定得到的38个纯正蜂蜜样品的碳同位素δ13C值数据,提出了纯正蜂蜜样品的δ13C值要求: 蛋白质和蜂蜜的δ13C差值(Δδ13CP-H)≥~0.95‰,果糖和葡萄糖的δ13C差值(Δδ13CF-G)在~0.64‰至0.53‰范围内,各个组分间的δ13C最大差值(Δδ13Cmax)＜2.09‰。对150个日常检测样品、蜂农和蜂蜜供应商的蜂蜜样品分别采用本文建立的LC/EA-IRMS和国家标准方法(EA-IRMS)进行鉴定,LC/EA-IRMS方法检出58个掺有C3或C4植物糖浆的阳性样品,而EA-IRMS方法仅检出7个掺有C4植物糖浆的阳性样品,可见新方法大大提高了对蜂蜜掺假的鉴别能力。     

Combination of sugar analysis and stable isotope ratio mass spectrometry to detect the use of artificial sugars in royal jelly production

The effects of feeding bees artificial sugars and/or proteins on the sugar compositions and ¹³C isotopic measurements of royal jellies (RJs) were evaluated. The sugars fed to the bees were two C4 sugars (cane sugar and maize hydrolysate), two C3 sugars (sugar beet, cereal starch hydrolysate), and honey. The proteins fed to them were pollen, soybean, and yeast powder proteins. To evaluate the influence of the sugar and/or protein feeding over time, samples were collected during six consecutive harvests. ¹³C isotopic ratio measurements of natural RJs gave values of around −25 ‰, which were also seen for RJs obtained when the bees were fed honey or C3 sugars. However, the RJs obtained when the bees were fed cane sugar or corn hydrolysate (regardless of whether they were also fed proteins) gave values of up to −17 ‰. Sugar content analysis revealed that the composition of maltose, maltotriose, sucrose, and erlose varied significantly over time in accordance with the composition of the syrup fed to the bees. When corn and cereal starch hydrolysates were fed to the bees, the maltose and maltotriose contents of the RJs increased up to 5.0 and 1.3 %, respectively, compared to the levels seen in authentic samples (i.e., samples obtained when the bees were fed natural food: honey and pollen) that were inferior to 0.2% and not detected, respectively. The sucrose and erlose contents of natural RJs were around 0.2 %, whereas those in RJs obtained when the bees were fed cane or beet sugar were as much as 4.0 and 1.3 %, respectively. The combination of sugar analysis and ¹³C isotopic ratio measurements represents a very efficient analytical methodology for detecting (from early harvests onward) the use of C4 and C3 artificial sugars in the production of RJ.     

GasBench/isotope ratio mass spectrometry: a carbon isotope approach to detect exogenous CO(2) in sparkling drinks

A new procedure for the determination of carbon dioxide (CO(2)) (13)C/(12)C isotope ratios, using direct injection into a GasBench/isotope ratio mass spectrometry (GasBench/IRMS) system, has been developed to improve isotopic methods devoted to the study of the authenticity of sparkling drinks. Thirty-nine commercial sparkling drink samples from various origins were analyzed. Values of delta(13)C(cava) ranged from -20.30 per thousand to -23.63 per thousand, when C3 sugar addition was performed for a second alcoholic fermentation. Values of delta(13)C(water) ranged from -5.59 per thousand to -6.87 per thousand in the case of naturally carbonated water or water fortified with gas from the spring, and delta(13)C(water) ranged from -29.36 per thousand to -42.09 per thousand when industrial CO(2) was added. It has been demonstrated that the addition of C4 sugar to semi-sparkling wine (aguja) and industrial CO(2) addition to sparkling wine (cava) or water can be detected. The new procedure has advantages over existing methods in terms of analysis time and sample treatment. In addition, it is the first isotopic method developed that allows (13)C/(12)C determination directly from a liquid sample without previous CO(2) extraction. No significant isotopic fractionation was observed nor any influence by secondary compounds present in the liquid phase.     

Deuterium nuclear magnetic resonance spectroscopy and stable carbon isotope ratio analysis/mass spectrometry of certain monofloral honeys

Quantitative deuterium nuclear magnetic resonance spectroscopy (NMR) has been used in conjunction with stable carbon isotope ratio analysis/mass spectrometry to refine the detection of sugars that have been added to monofloral honeys. The 13C content of sugars indicates the type of photosynthetic metabolism of the plant that synthesized them; the deuterium content is more characteristic of secondary metabolism and of environmental factors. Consequently, determination of the 13C content of honeys and of proteins extracted from the honeys can be used to detect the addition of C4 plant sugars (cane or corn), but it does not reveal the addition of C3 plant sugars such as beet sugar. Deuterium NMR gives useful information for some monofloral honeys. NMR measurement is performed on ethanol obtained from fermentation of the honey and extracted by distillation. The isotopic composition of the ethanol indicates the nature of the sugars from which it was derived. Various types of monofloral honeys were studied, and the results obtained with commercially available honeys demonstrate the usefulness of isotopic analysis and the need to compile a database of authentic honeys to validate or affirm certain results.     

Measurement of the 13C/12C ratio of soil-plant individual sugars by gas chromatography/combustion/isotope-ratio mass spectrometry of silylated derivatives

Carbohydrate is an important pool in the terrestrial carbon cycle. The potential offered by natural and artificial 13C-labelling techniques should therefore be applied to the investigation of the dynamics of individual sugars in soils. For this reason, we evaluated the method of 13C sugar analysis by gas chromatography/combustion/isotope-ratio mass spectrometry (GC/C/IRMS) after hydrolysis and direct trimethylsilylation. Trimethylsilylation involved the addition of several carbon atoms per sugar. These atoms have to be taken into account in the estimation of the carbon isotope ratio. The analysis of standard and natural pentoses and hexoses of known 13C enrichments revealed that the number of analysed added carbon atoms was less than expected from stoichiometry. This was attributed to incomplete derivatization and/or incomplete oxidation of methylsilyl carbon before IRMS. Using a calibration of the number of analysed added carbon atoms, the isotope excess of enriched samples could be determined with a relative error close to 5%. Concerning the determination of natural abundances by GC/C/IRMS, we could measure the delta 13C of standard C3- and C4-derived sugars with an accuracy of +/-1.5 per thousand using the previous calibration. We were able to apply this technique to plant-soil systems labelled by pulse-chase of 13CO2, revealing the nature and dynamics of sugars in the plant rhizosphere.     

Minimization of carbon addition during derivatization of monosaccharides for compound-specific delta13C analysis in environmental research

Little is known about the delta13C composition of monosaccharides representing the largest carbon reservoir in the biosphere. The main reason for this might be that monosaccharides have to be derivatized prior to gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses and that a large isotopic correction is necessary for the carbon that has to be added to the original molecule during derivatization, resulting in large uncertainty of the calculated delta13C values of individual monosaccharides. The amount of added derivatization carbon is twice (alditol acetates) or even three times (trimethylsilyl (TMS) derivatives) as high as the amount of the original monosaccharide carbon. In addition, isotope fractionation occurs during acetylation. Therefore, the objectives of this study were (i) to minimize carbon addition during derivatization for GC/C/IRMS measurements of monosaccharides in soil and sediment samples and (ii) to quantify improvements in accuracy and precision of the final results. Minimization of carbon addition was accomplished by derivatization with methylboronic acid (MBA) and TMS thereafter (MBA method). Monosaccharides derivatized with the MBA method instead of TMS reduced the number of added carbon atoms from 2.2-2.7 to 0.3-0.8 per sugar carbon atom. Although the precision of GC/C/IRMS measurements with both methods is comparable (about 0.3 per thousand), delta13C values of an internal standard indicated that the newly developed MBA method is about 2 per thousand more accurate than the TMS method. delta13C comparison between soil samples that differed only slightly in their bulk carbon isotope signature showed that the MBA method is better in proving these small differences on a significant level. Total precision of the whole MBA method including all analytical and calculation steps is better by a factor of almost three than the TMS method.     

The use of stable isotopes ratios for authentication of fruit juices

The determination of the content of stable isotopes, ¹⁸O and ²H, respectively, in juice water facilitates the distinction between authentic juices and juices made from concentrates by redilution with tap water. At the same time, the detection of C₄ cane or corn-derived sugar syrups in fruit juices which are produced from C₃ fruit types is thus facilitated by the characteristic differences in ¹³C/¹²C, expressed as δ ¹³C (‰) values due to photosynthetic CO₂ assimilation via the C₃₋, C₄₋, and crassulacean acid metabolism pathways. In this study, the quantitative determination of water added to an authentic juice, on the basis of δ ¹⁸O, and δ ²H values, respectively, was successfully performed. Also, the δ ¹⁸O, and δ ²H of juice water and δ ¹³C of the whole juice in 18 samples were also determined. The results obtained provided us with the possibility of distinguishing between authentic fruit juices and those obtained by redilution of concentrated fruit juices and the detection of C⁴ type added sugar.     

Sensitive Determination of the Diquat Herbicide in Fresh Food Samples on a Highly Boron‐Doped Diamond Electrode

The highly boron‐doped diamond electrode (HBDD) combined with square wave voltammetry (SWV) was used in the development of an analytical procedure for diquat determination in potato and sugar cane samples and lemon, orange, tangerine and pineapple juices. Preliminary experiments realised in a medium of 0.05 mol L^?1 Na₂B₄O₇ showed the presence of two voltammetric peaks around ?0.6 V and around ?1.0 V vs. Ag/AgCl/Cl^? 3.0 mol L^?1, where the first peak could be successfully used for analytical proposes due the facility in the electrode surface renovation. After the experimental and voltammetric optimisation, the calculated detection and quantification limits were 1.6×10^?10 mol L^?1 and 5.3×10^?10 mol L^?1 (0.057 µg L^?1 and 0.192 µg L^?1, respectively), which are lower than the maximum residue limit established for fresh food samples by the Brazilian Sanitary Vigilance Agency. The proposed methodology was used to determine diquat residues in potato and sugar cane samples and lemon, orange, tangerine and pineapple juices and the calculated recovery efficiencies indicated that the proposed procedure presents higher robustness, stability and sensitivity, good reproducibility, and is very adequate for diquat determination in complex samples.     

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.     

A selective unsaturated hydrocarbon subtraction technique for stable carbon isotopic analysis of atmospheric methyl chloride, methyl bromide, and C2-C5 saturated hydrocarbons using continuous-flow isotope ratio mass spectrometry

Using continuous-flow isotope ratio mass spectrometry, we have developed a new analytical system which enables us to determine the stable carbon isotopic composition of CH3Cl, CH3Br, and C2-C5 saturated hydrocarbons in gas samples even if they contain substantial amounts of unsaturated hydrocarbons, using an I2O5 reagent for their selective subtraction. The analytical precision of the delta13C determinations is better than 0.5 per thousand for >300 pmolC injections and better than 5 per thousand for 20 pmolC injections. Using the system, delta13C values for CH3Cl and CH3Br were found in burning exhaust that contain a substantial quantity of unsaturated hydrocarbons. CH3Cl and CH3Br measured in exhaust from burning rice plants exhibit highly 13C-depleted values of -56.6 +/- 1.3 per thousand and -48.6 +/- 3.9 per thousand, respectively, while saturated hydrocarbons exhibit delta13C values (-26.4 to -28.9 per thousand) that are comparable with the total delta13C value of the parent material (rice plant; -28.0 per thousand). Using the system, we can determine the delta13C values of methyl halides and hydrocarbons in many kinds of gas samples.     

Characterization of yeast strains for wine production

Sixteen yeast strains isolated from grapefruit (Citrus paradis), orange (Citrus sinensis) and pineapple (Ananas comosus) were characterized using standard microbiological procedures. The species were identified as Saccharomyces uvarum, S. cerevisiae, S. carlbergensis, and S. ellipsoideus. Their abilities for wine production were tested by using sugar and ethanol tolerance tests. The best biochemically active strain, S. ellipsoideus, was used along with commercially available baker's yeast (S. cerevisiae) to produce wine from grapefruit, orange, and pineapple juices. After fermentation for 14 d with S. cerevisiae and 21 d with S. ellipsoideus, wines produced were compared with Baron de Valls (standard). The highest (10.47% [v/v]) and lowest (7.68% [v/v]) alcohol concentrations with corresponding residual sugar concentrations of 1.88% (w/v) and 7.7% (w/v) were produced from orange after fermentation with S. cerevisiae and S. ellipsoideus, respectively. S. ellipsoideus was found to be the best yeast strain producing wine with the highest acceptable score of 7.41 from orange. The study revealed the possibility of producing wine from our locally available fruits using simple, cheap, and adaptable technology with biochemically characterized yeast strains.     

Determination of hydrogen, carbon and oxygen isotope ratios of ethanol in aqueous solution at millimole levels

Three stable isotope ratios, D/H, (13)C/(12)C and (18)O/(16)O, are measurable in ethanol, an important organic compound that is used as a material for food and beverages, fuel and chemical feedstock, and as a substance related to metabolism. We developed a simple and rapid method of measurement of three isotope ratios of ethanol in aqueous solution at millimole levels using gas chromatography-high-temperature conversion or combustion-isotope ratio mass spectrometry (GC-TC/C-IRMS) combined with solid-phase microextraction (SPME). Using this method, the delta value for ethanol was determined in 30 min for deltaD and delta(13)C, and in 75 min for delta(18)O with precisions of +/-9 per thousand, +/-0.3 per thousand and +/-0.7 per thousand, respectively, for deltaD, delta(13)C, and delta(18)O. An advantage of this process is that it requires no distillation for ethanol purification. The method is useful for small quantities of analyte with low ethanol concentrations, which is expected for environmental and metabolic studies.     

Application of multi-element clustering techniques of five Egyptian industrial sugar products

The concentration of 18 elements in different cane sugar products, i.e., cane sugar plants, crude and syrup juices, molasses, and the end products of the consumer sugar, were analyzed and processed. The samples were collected from five citics, i.e., Kom Ombo, Edfu, Armant, Deshna and Naga Hammady in Upper Egypt where the main Egyptian sugar industry factories are located. INAA was applied for the determination of Al, Ca, Cl, Co, Cr, Fe, Mg, Mn, Na and Sc, while Cu, Li, P, Sn, V and Zn were determined by ICP-AES and Pb and As were determined by AAS. These three analytical methods were applied to optimize the sensitivity and the accuracy of the measurements in order to provide a sound basis for the obtention of reliable clustering results.     

Polygalacturonase and ethanol production in Kluyveromyces marxianus

The coproduction of ethanol and polygalacturonase (PG) in a pilot-scale batch fermentor using yeast extract—glucose (YD)—and sugar beet molasses (SBM)-based media was implemented utilizing a new high-PG-producing strain of Kluveromyces marxianus. A certain growth inhibition was observed in SBM medium, causing ethanol and PG production to be lower. Ethanol productivity and accumulation values of 1.94 g/(L·h) and 40 g/L, respectively, were attained in YD, whereas the best fermentation efficiency (95.1%) was achieved with SBM medium. Maximal PG synthesis occurred at the end of cell growth, with values of 1.08 and 0.46 U/(mg·h) for the YD and SBM media, respectively. When the cultures reached stationary phase, PG production stopped. The highest accumulation level (17 U/mL) occurred in YD medium, in agreement with previous laboratory-scale studies carried out for this strain. The potential applications of the crude enzyme preparations were evaluated with different fruit juices and vegetable slices. The enzyme was able to increase the filtration rate of orange, pear, and apple juices by twofold. Additionally, complete clarification of apple juice was readily accomplished, whereas cucumber, carrot, and banana tissues were macerated to a lesser extent.