Lactulose Concentrations Research Articles

Determination of lactose and lactulose isomers in UHT milk by CZE-UV

A capillary zone electrophoresis method with indirect detection in ultraviolet region (CZE-UV) for determination of lactose and lactulose in seventeen UHT milk brands using single-point standard addition was proposed. An average lactulose content of 455.52 mg L−1 and a relative standard deviation (RSD) of 45% were found, while ca. 23% of brands showed lactulose concentrations above 600 mg L−1. High RSD values were found in intra-brands analysis (17–49%) indicating the existence of many variations in UHT thermal processing. The mean lactose content was 4.45 g 100 mL−1 with a RSD of 9.2%. Validation results appointed good precision (RSD of 3.19% and 3.82%), and low limits of quantification (62.53 ± 4.32 mg L−1 and 0.083 ± 0.003 g 100 mL−1) for lactulose and lactose, respectively. Recoveries rates for lactose were found considering three concentration levels (96.5–102.2%) and for standard added in all samples during quantification step (100.02 ± 1.39)%. Lactulose recovery average of standard added was (99.24 ± 1.46)%. The method can be useful for thermal processing quality control as well lactose hydrolysis monitoring.

Green method using water for lactose and lactulose extraction and determination in milk by high-performance liquid chromatography with refractive index detection

Lactose is the main carbohydrate found in milk. However, the ultra-high temperature (UHT) process might convert lactose to lactulose by isomerization. Lactulose can be prebiotic; nevertheless, at high concentrations, it can be fermented in the gastrointestinal tract because it is not hydrolyzed by enzymes. In view of the increase in lactose intolerant consumers, it becomes necessary to determine the presence and amounts these compounds in UHT milk. Hence, this study aims to propose and validate a method with simple sample preparation to measure lactose and lactulose contents in milk. Following green chemistry principles, the proposed method is distinguished by the use of only water in the extraction step, with an effective sample clean-up step performed via ultracentrifugation; likewise, water was also employed as the mobile phase for high-performance liquid chromatography with refractive index detection analysis. Moreover, the proposed method allows for the verification of the efficiency of the lactose-free milk process by monitoring lactose residual levels in addition to measuring the degradation contents of glucose and galactose. Further, the developed method even represents an alternative for detecting counterfeit milk made with added sucrose, ensuring the quality of milk for consumers.

Sensory profile and technological characterization of boneless dry-cured ham with lactulose added as a prebiotic.

ObjectiveThis study investigates the technological and sensory profile of boneless dry-cured ham with different contents of lactulose added as a prebiotic ingredient.MethodsIn addition to the control samples (without the addition of lactulose), three treatments were formulated to contain 2%, 4%, or 6% lactulose. Technological (lactulose content, instrumental color and texture profile analysis) and sensory (acceptance and check-all-that-applies tests) analyses were performed on the final product.ResultsThe lactulose content in the finished product (1.86%±0.23%, 3.16%±0.18%, and 2.51%±1.35%) was lower than the lactulose originally added (2%, 4%, and 6%, respectively). The addition of 4% and 6% lactulose made (p<0.05) the products darker (lower L*) and redder (lower h) with higher hardness and chewiness values, when compared to control samples. The additions of 2% and 4% lactulose reduce the appearance acceptability of the products, but overall the treatments were well accepted.ConclusionThe use of up to 4% lactulose as a prebiotic in the production of boneless dry-cured hams provides an alternative to improving its nutritional value with little alteration in the technological characteristics and still meeting the sensory characteristics desired by consumers.

Immobilization of β-galactosidase on chitosan-coated magnetic nanoparticles and its application for synthesis of lactulose-based galactooligosaccharides

Abstract β-Galactosidase was covalently immobilized on the chitosan-coated magnetic nanoparticles activated with glutaraldehyde. The effect of three factors including protein content, pH and time on the yield of immobilization was investigated, and the optimum values were 0.45 mg/ml, 4.9 and 4.9 h, respectively. The maximum yield of immobilization reached 98.8% in term of enzyme activity. The stability of β-galactosidase significantly was improved after immobilization from 2.5 days to over 6 days of half-life at 60 °C and was active in wider ranges of temperature (45–60 °C), and pH (4.0–5.5) compared to soluble form. The activity of immobilized enzyme remained 85% of origin after eight recycles (at 60 °C, pH 4.5 for 4 h). Different initial lactulose concentrations (0.58–2.34 M) were applied for synthesis of galactooligosaccharides. The maximum yield of galactooligosaccharides was 17% mol/mol at the initial lactulose concentration of 2.34 M at 36 h of reaction. It showed that both hydrolytic and trans-galactosylation activities of β-galactosidase remained well after immobilization. The kinetic model of immobilized enzyme was determined. These results revealed that immobilized enzyme on chitosan-coated magnetic nanoparticles has application potentials in the production of prebiotic lactulose-based galactooligosaccharides.

Effect of selected prebiotics on the growth of lactic acid bacteria and physicochemical properties of yoghurts

The feasibility of manufacturing yoghurts with three prebiotics, i.e., fructooligosaccharides (FOS), galactooligosaccharides (GOS) or lactulose added at two concentrations (2 and 4%, w/v) was investigated. Physicochemical and microbial characterisation was conducted on the effect of prebiotics on acidification, viability of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, lactose and prebiotic consumption and production of organic acids during fermentation (up to 6 h) and cold storage (for 28 d). GOS and FOS remained unaltered throughout fermentation and cold storage, while the viability of starter culture was similar to that observed in the control yoghurt. Yoghurts manufactured with 4% lactulose showed a significant decrease of lactulose content associated with a lower decrease of lactose relative to the control. This effect was associated with a significant increase (2.2 log10 cfu mL−1) in L. delbrueckii ssp. bulgaricus and a moderate decrease (1.0 log10 cfu mL−1) in S. thermophilus at the end of cold storage.

High-Temperature Short-Time Pasteurization System for Donor Milk in a Human Milk Bank Setting.

Donor milk is the best alternative for the feeding of preterm newborns when mother's own milk is unavailable. For safety reasons, it is usually pasteurized by the Holder method (62.5°C for 30 min). Holder pasteurization results in a microbiological safe product but impairs the activity of many biologically active compounds such as immunoglobulins, enzymes, cytokines, growth factors, hormones or oxidative stress markers. High-temperature short-time (HTST) pasteurization has been proposed as an alternative for a better preservation of some of the biological components of human milk although, at present, there is no equipment available to perform this treatment under the current conditions of a human milk bank. In this work, the specific needs of a human milk bank setting were considered to design an HTST equipment for the continuous and adaptable (time-temperature combination) processing of donor milk. Microbiological quality, activity of indicator enzymes and indices for thermal damage of milk were evaluated before and after HTST treatment of 14 batches of donor milk using different temperature and time combinations and compared to the results obtained after Holder pasteurization. The HTST system has accurate and simple operation, allows the pasteurization of variable amounts of donor milk and reduces processing time and labor force. HTST processing at 72°C for, at least, 10 s efficiently destroyed all vegetative forms of microorganisms present initially in raw donor milk although sporulated Bacillus sp. survived this treatment. Alkaline phosphatase was completely destroyed after HTST processing at 72 and 75°C, but γ-glutamil transpeptidase showed higher thermoresistance. Furosine concentrations in HTST-treated donor milk were lower than after Holder pasteurization and lactulose content for HTST-treated donor milk was below the detection limit of analytical method (10 mg/L). In conclusion, processing of donor milk at 72°C for at least 10 s in this HTST system allows to achieve the microbiological safety objectives established in the milk bank while having a lower impact regarding the heat damage of the milk.

Lactulose determination in UHT milk by CZE-UV with indirect detection

Lactulose is not found naturally in milk, but it has been detected in small quantities in heated dairy products. It can therefore be used as an indicator of the severity of heat treatment for dairy products. This study proposes an alternative method for lactulose quantification in milk using capillary zone electrophoresis with ultraviolet detection (CZE-UV). The investigation used several Brazilian UHT milk samples, and various sample pretreatment methods and injection conditions were considered. Optimal separation results were achieved using Carrez clarification solutions. Low detection and quantification limits were obtained, and good precision and high recovery rates indicated the applicability of the method for analysis of milk. Due to matrix effects, single-point standard addition was used for lactulose quantification. High lactulose concentrations (>1000 mg L−1) suggested that recirculation may have occurred during milk processing. The findings demonstrate that the CZE-UV method is a viable alternative for quality process control.

Immobilization and some properties of commercial enzyme preparation for production of lactulose-based oligosaccharides

The commercial enzymatic preparation, Pectinex Ultra SP-L, was successfully immobilized on activated chitosan particles for production of lactulose-based galacto-oligosaccharide (LuOS). The main degrees of polymerization (DP) of LuOS produced in this bioreactor were DP3 and DP4. The immobilization yield in term of expressed activity and recovery activity were 71% and 36.05%, respectively. The optimum temperature (60°C) was not affected by immobilization process, while the optimum pH was shifted from pH 5.5 to pH 4.5. In addition, a thermal stability of the immobilized enzyme was better than that of free enzyme. The half-life time of hydrolytic activity increased from 2.5days (free enzyme) to 14days (immobilized enzyme). Hydrolytic KM values were 651.3mM and 187.9mM lactulose for free enzyme and immobilized enzyme, respectively. Furthermore, the immobilized enzyme showed high reusability. The yield of LuOS formation was more than 60% after 33 cycles of reuses. In the case of trans-galactosylation, a model with 8 reaction rates was build using five-step reaction pathways with different initial lactulose concentrations. This catalytic model was validated well for a long-term period (27 days). These results strongly support to develop a continuous bioconversion to synthesize functional food ingredients, prebiotic LuOS.

Effect of electro-activated sweet whey on growth of Bifidobacterium, Lactobacillus, and Streptococcus strains under model growth conditions

Recently, we demonstrated the efficacy of electro-activation to improve the functionalities of whey that can be used as a prebiotic and antioxidant agent through lactulose and Maillard reaction products formation. The aim of the present study was to evaluate the effect of electro-activated sweet whey (EA-whey) on growth of probiotics of Bifidobacterium, Lactobacillus, and Streptococcus strains in pure cultures and to compare EA-whey with non-electro-activated whey, lactulose, lactose, sucrose, glucose and galactose at different concentrations (1.25, 2.5 and 5%). The bacterial growth was monitored through maximum optical density (ODmax) and maximum growth rate (μmax) measurements. Moreover, the effects of EA-whey on the growth of L. johnsonii La-1 in the presence of oxygen was assessed. FTIR spectroscopy analyses of the bacterial membrane structure were monitored as a function of EA-whey concentration. The results showed that EA-whey enhanced the growth of all the test bacteria. They clearly demonstrated a promoting bifidogenic effect of EA-whey compared to lactulose. The growth of L. johnsonii La-1 was greatly enhanced under aerobic conditions by the supplementation of the growth medium with EA-whey. This growth promoting effect could be related to the ability of EA-whey to prevent the accumulation of hydrogen peroxide, its high antioxidant capacity and lactulose content. Moreover, FTIR spectra showed that EA-whey acts as an antioxidant in regards to cell membrane lipids oxidation by oxygen species and limited their adverse effect on probiotic bacteria during their growth. Thus, EA-whey, a potential prebiotic and antioxidant, could be used as active ingredient in manufacturing functional fermented dairy products.

Lactulose production by a thermostable glycoside hydrolase from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167.

Lactulose has various uses in the food and pharmaceutical fields. Thermostable enzymes have many advantages for industrial exploitation, including high substrate solubilities as well as reduced risk of process contamination. Enzymatic synthesis of lactulose employing a transgalactosylation reaction by a recombinant thermostable glycoside hydrolase (GH1) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was investigated. The optimal pH for lactulose production was found to be 4.5, while the optimal temperature was 85 °C, before it dropped moderately to 83% at 90 °C. However, the relative activity for lactulose synthesis dropped sharply to 35% at 95 °C. At optimal reaction conditions of 70% (w/w) initial sugar substrates with molar ratio of lactose to fructose of 1:4, 15 U mL-1 enzyme concentration and 85 °C, the time course reaction produced a maximum lactulose concentration of 108 g L-1 at 4 h, corresponding to a lactulose yield of 14% and 27 g L-1 h-1 productivity with 84% lactose conversion. The transgalactosylation reaction for lactulose synthesis was greatly influenced by the ratio of galactose donor to acceptor. This novel GH1 may be useful for process applications owing to its high activity in very concentrated substrate reaction media and promising thermostability. © 2017 Society of Chemical Industry.

Restructured low-fat cooked ham containing liquid whey fortified with lactulose.

Current health concerns have driven consumers to request products with nutritional and physiological advantages, which can be achieved by using prebiotic ingredients. Lactulose is a prebiotic with excellent functional properties and can be easily incorporated into meat products through the addition of liquid whey. This study investigated the technological and sensorial quality of restructured cooked ham elaborated without liquid whey added (control) and with liquid whey containing different contents (0, 30, 60 and 100 g kg-1 ) of lactulose. Liquid whey did not change any technological or sensorial characteristics of the product, but the general acceptability decreased due to addition of lactulose. Samples with higher lactulose concentrations had lower moisture content, pH and refreezing loss and increased carbohydrate content. Control and whey added samples had higher lightness and lower intense color than samples with lactulose. Liquid whey additions with higher lactulose content increased hardness and chewiness of the samples. Restructured cooked hams formulated with liquid whey and 30 g kg-1 of lactulose had minimal effects on the technological properties and sensory characteristics and, due to the possible benefits conferred by the prebiotic, is a potential alternative to provide meat products with prebiotic activity. © 2017 Society of Chemical Industry.

Increased Intestinal Permeability in Relatives of Patients With Crohn's Disease Is Not Associated With Small Bowel Ulcerations.

Many first-degree relatives of patients with Crohn's disease (CD) have increased intestinal permeability. Video capsule endoscopy (VCE) is the most sensitive imaging test to identify small bowel mucosal lesions that could indicate subclinical CD. We aimed to estimate the association of increased intestinal permeability with small bowel ulcerations detectable by VCE in healthy first-degree relatives of patients with CD. We conducted a cross-sectional study of 223 healthy, asymptomatic first-degree relatives of patients with CD (parents, siblings, and children; 9-45 years old) enrolled at the University of Alberta between 2009 and 2012. Patients were given the lactulose and mannitol test to measure small bowel permeability; we used high-performance liquid chromatography to measure concentrations of lactulose and mannitol in urine samples (increased permeability defined as a ratio of lactulose/mannitol 0.025 or greater). Patients with increased permeability (n= 39) and randomly selected subjects with normal permeability (n= 59) were then examined by VCE for signs of small bowel inflammation and subclinical CD. The prevalence of small bowel lesions was compared among groups. We performed logistic regression analyses to estimate odds ratios for the association of small bowel ulcerations with intestinal permeability. Among 223 first-degree relatives of patients with CD, 30% were found to have increased intestinal permeability; VCE examination found 24% of subjects to have 3 or more small bowel ulcers. Three or more small bowel ulcers were detected in 28% of patients with increased intestinal permeability and 20% of patients with normal intestinal permeability (P= .37). The adjusted odds ratio for the association of 3 or more small bowel ulcers with increased intestinal permeability was 1.5 (95% confidence interval, 0.6-3.8; P= .46). Thirty percent of healthy, asymptomatic first-degree relatives of patients with CD have increased intestinal permeability. However, a strong association of small bowel ulceration seen on VCE with increased intestinal permeability was not observed.

Fed-batch operation for the synthesis of lactulose with β-galactosidase of Aspergillus oryzae

Fed-batch synthesis of lactulose from lactose and fructose with Aspergillus oryzae β-galactosidase was evaluated, obtaining a concentration of 40.4g·L−1, which is 20% higher than obtained in batch, while the concentration of transgalactosylated oligosaccharides (TOS) was reduced by 98%. Therefore, selectivity of lactulose synthesis can be significantly higher by operating in fed-batch mode. The enzyme-limiting substrate mass ratio (E/S) is a critical variable in fed-batch operation. Higher values favor lactose hydrolysis over transgalactosylation, being 400IU/g the limit for proper lactulose synthesis in fed-batch operation. Selectivity of lactulose synthesis increased with E/S being quite high at 800IUH·g−1 or higher. However, this increase was obtained at the expense of lactulose yield. Lactulose synthesis in fed-batch operation was a better option than conventional batch synthesis, since higher product concentration and selectivity of lactulose over TOS synthesis were obtained.

Predicting lactulose concentration in heat-treated reconstituted skim milk powder using front-face fluorescence

Industrial processing of milk and dairy foods requires heat treatment, which induces undesirable effects in milk such as protein denaturation, organoleptic and nutritional properties alteration, browning, etc. The hypothesis of the present study is that it is possible to use front-face measurement of fluorescent markers such as tryptophan, dityrosine, Maillard intermediate compounds and riboflavin as an inline, cheap, easy and fast way to evaluate lactulose formation in milk. Thus, the specific objective of this study was modeling the kinetics of lactulose formation during heat treatment of milk as well as evaluating the correlation of lactulose concentration with fluorescent markers in order to obtain prediction models of lactulose concentration in heat-treated milk based on front-face fluorescence.Three replicates of the study were performed at three different temperatures (80, 90 and 100 °C) and six different times (0, 10, 15, 30, 45 and 60 min). Standard, low-heat, spray-dried skim milk powder, supplied by Chr. Hansen SL (Barcelona, Spain) was reconstituted at 12% and used in all trials to ensure consistency among the various experimental trials.Lactulose concentration was significantly correlated (P < 0.001) with fluorescence intensity of different markers such as maximum intensity and maximum emission wavelength of tryptophan as well as the fluorescence intensity of riboflavin. Fluorescence changes associated to thermal treatment of milk were characterized and a series of fluorescent markers were identified as predictors allowing prediction of lactulose concentration with an R2 of 0.91 at a temperature of 90 °C.

Development, optimization and validation of an HPLC-ELSD method for the analysis of enzymatically generated lactulose and saccharide by-products

The aim of this study was to develop an HPLC-ELSD method for the quantification of lactulose in complex sugar solutions. Lactulose is a well-known prebiotic and supports the alleviation of digestive disorders. The enzymatic generation of lactulose requires fructose as nucleophilic acceptor. By-products such as glucose and galactose are generated. Four amino-modified silica-columns were tested and compared. The most suitable column based on peak resolution was used to optimize the method. Furthermore, sample preparation was optimized for the recovery of analytes. During the validation step, the following parameters were determined (e.g. for lactulose): recovery (106±7%), precision (98%), correctness (99%), limit of detection (3.9mg/L), limit of quantification (13.4mg/L) and linearity (0.993). The validated method was applied to samples from an enzymatic process for the production of lactulose at the laboratory scale. A final lactulose concentration of 6.7±0.4g/L was determined.

Improving the detection of environmental enteric dysfunction: a lactulose, rhamnose assay of intestinal permeability in children aged under 5 years exposed to poor sanitation and hygiene

BackgroundEnvironmental enteric dysfunction (EED) is an asymptomatic intestinal disorder affecting populations living in conditions of poor sanitation and hygiene. The study tested intestinal barrier function in infants with EED.MethodsWe prospectively...

Analytical assessment of the intense heat load of whipping cream, coffee cream, and condensed milk at retail in Austria and Germany.

Time temperature integrators (TTIs) are useful tools in estimating the heat load applied on differently processed dairy products. The objective of this study was to analyze and assess three TTIs – lactulose, furosine, and acid-soluble β-lactoglobulin (β-Lg) – in 70 high heated dairy products at retail in Austria and Germany comprising whipping cream, coffee cream/milk, and condensed milk products. While β-Lg was not appropriate to evaluate the heat load of these products, furosine and especially lactulose increased with rising intensity of heat treatment, and are appropriate to distinguish between several heating categories analyzed. Pasteurized (n = 8) and “heat treated” (n = 5) whipping cream samples showed lowest furosine (48 ± 14/ 45 ± 19 mg.100 g−1 protein) and low lactulose (29 ± 10/57 ± 28 mg.L−1) concentrations, followed by ESL whipping cream (n = 10), ESL coffee cream (n = 1), and UHT whipping cream (n = 10) (furosine = 72 ± 37/71/161 ± 30 mg.100 g−1 protein; lactulose = 56 ± 41/161/195 ± 39 mg.L−1), respectively. Sterilized condensed milk samples (n = 14) showed the highest concentrations of both TTIs and could be clearly separated from UHT treated samples (n = 5) (furosine = 491 ± 196/216 ± 46 mg.100 g−1 protein; lactulose = 1997 ± 658/409 ± 161 mg.L−1), whereas the so-called heat-treated samples (n = 9) had a heat load in between showing an extreme range of variation for both TTIs.

Effects of lactulose on growth, carcass characteristics, faecal microbiota, and blood constituents in broilers

This study was conducted to determine the effect of supplementing diets with lactulose on growth performance, carcass characteristics, faecal microbiota, and blood constituents. A total of 324 one-day-old Ross 308 mixed-sex broiler chicks with an average initial body weight of 38 g were used in a 35-day growth assay. There were 18 birds/pen and six pens/treatment with food and water available ad libitum. Treatments consisted of a corn-soybean-meal-based diet with 0, 0.25 and 0.5% of lactulose. The results indicated that body weight gain (BWG) was improved (linear effect, P < 0.05) by increasing the concentration of lactulose in the diet from zero to 0.5% while the feed conversion ratio (FCR) decreased (linear effect, P < 0.05) for Days 8 to 21, 21 to 35, and overall (Day 0 to 35). Chickens fed the diet supplemented with 0.5% lactulose showed a higher relative weight of breast meat compared to other groups. Inclusion of lactulose decreased the count of Salmonella and E. coli in excreta of chickens fed diets containing 0.25 or 0.5% lactulose (P < 0.05), but the count of Lactobacillus was not affected. Drip loss percentage was decreased (P < 0.05) on Day 1 by addition of 0.5% lactulose, but there was no effect on meat colour. Blood characteristics were not influenced. Thus, it was concluded that inclusion of lactulose improves growth performance and alters excreta microbial populations with no adverse effect on broilers.

Quantification of Lactulose and Epilactose in the Presence of Lactose in Milk using a dual HPLC analysis

The valuable lactose derivatives lactulose and epilactose can be derived from lactose either by the Lobry de Bruyn-Alberda van Ekenstein transformation during heat treatments or by enzymatic conversion using cellobiose 2-epimerases (EC 5.1.3.11). The chromatographic determination of lactose, lactulose, and epilactose in milk is challenging, due to the variable ratio of the three saccharides and their similar retention properties. In this work, a dual high-performance liquid chromatography (HPLC) analysis for the quantification of lactose, lactulose, and epilactose in milk samples was developed and validated. The samples originated from an enzymatic lactose conversion using the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Application of this enzyme led to the formation of high lactulose concentrations (28.0 g/L) in milk. The dual HPLC analysis utilized a combination of two chromatographic separation techniques, configured in two parallel systems. After precolumn derivatization, the samples were analyzed as follows: Method 1 determined the concentration of lactose and epilactose using a C18 column with an ion-pair reagent as eluent, coupled with a UV detector. Method 2 determined the concentration of lactulose using a trimodal stationary phase (hydrophilic interaction, anion- and cation-exchange properties) with acetonitrile/ammonium formiate buffer as eluent, coupled with an evaporative light scattering detector. Both methods were validated in terms of linearity, precision and recovery. The revealing detection limits in the milk samples were 3.32 mg/L for lactose, 4.73 mg/L for epilactose and 139 mg/L for lactulose. The dual HPLC analysis presented allows accurate lactose, lactulose, and epilactose separation in complex food matrices such as milk.

Microwave-assisted isomerisation of lactose to lactulose and Maillard conjugation of lactulose and lactose with whey proteins and peptides

Lactose was isomerised to lactulose by microwave heating and purified by a methanolic procedure to a product with approximately 72% lactulose content. Afterwards, lactose and the lactulose-rich product (PLu) were conjugated with either whey protein isolate (WPI) or its antioxidant hydrolysate (WPH) through microwaving. Lactose had a higher Maillard reactivity than PLu, and WPH was more reactive than WPI. The browning intensity of WPI-sugar systems was however higher than that of WPH-sugar pairs. Atomic force microscopy showed larger (up to ≈103nm) particles for WPI-PLu conjugates compared to WPH-PLu counterparts (up to ≈39nm). The Maillard conjugation progressively increased the radical-scavenging activity of WPI/WPH-sugar pairs with increasing conjugation time and improved the foaming properties of WPI and WPH. The WPI/WPH-sugar conjugates showed higher solubility and emulsification index than unreacted counterpart pairs. For native WPI, β-lactoglobulin was not degraded by in vitro gastric digestion, whereas for WPH-PLu conjugates degraded completely.