In vitro osteocytic microdamage and viability quantification using a microloading platform.

Bone remodeling is a process in which bone is resorbed by osteoclasts and formed by osteoblasts. This is normally a paired process, although it can be disrupted by changes in mechanical load. One theory is that osteocytes play a key role in the cellular regulation of this process. Mechanotransduction studies, which investigate how cells convert mechanical stimuli into biophysical effects and cellular activity, offer one way to investigate this theory. Mechanotransduction work is commonly done by applying an isolated mechanical load to cells grown in vitro, and quantifying the response. While in vitro work does not fully replicate the natural environment, it does allow the study of isolated factors. In this study, a mechanical loading platform was designed, fabricated, and characterized for bone mechanotransduction studies. This platform was designed to tent cell-seeded substrates from below, loading using out of plane distension. This introduced a nonuniform strain profile, enabling the study of cells cultured under identical conditions and variable strains as a function of substrate location. An alphanumerically gridded polydimethylsiloxane well substrate was designed and fabricated for cellular loading experiments. Following initial characterization, a study was run to quantify the cellular activity of osteocyte-like MLO-Y4 cells as a function of strain field. The results indicated that regions with lower strains led to an increase in cellular activity while higher strains led to a reduction in cellular activity. This demonstrated that cells could be exposed to mechanically-induced microdamage using the microloading platform.

Impact of Gap Junctional Intercellular Communication on MLO-Y4 Sclerostin and Soluble Factor Expression.

Bone remodeling is a continual process in which old bone is resorbed by osteoclasts and new bone is formed by osteoblasts, providing a mechanism for bones' ability to adapt to changes in its mechanical environment. While the role of osteoblasts and osteoclasts in bone remodeling is well understood, the cellular regulation of bone remodeling is unclear. One theory is that osteocytes, found within bone, play an important role in controlling the bone remodeling response. Osteocytes possess gap junctions, narrow channels that extend between nearby cells and allow communication between cells via the transfer of small molecules and ions. This work investigated the potential role of gap junctional intercellular communication in bone remodeling by exposing osteocyte-like MLO-Y4 cells to mechanical strains and quantifying the expression of soluble factors, including sclerostin, a protein closely associated with bone remodeling. The soluble factors and sclerostin expression were further examined after inhibiting gap junctional intercellular communication to study the impact of the communication. At supraphysiologic strains, the inhibition of gap junctional intercellular communication led to increases in sclerostin expression relative to cells in which communication was present, indicating that the communication may play a significant role in regulating bone remodeling.

Performance metrics based on signal intensity for ion mobility spectrometry--based explosive trace detectors using inkjet printed materials.

Commercial off-the-shelf (COTS) explosive trace detectors (ETDs) have become an integral part of security practices aimed at protecting the public, transportation, and facilities. Despite their widespread deployment, quality control procedures that can evaluate day-to-day instrument performance or differences among units of the same manufacture are in need for development. In this work, we describe the preparation of test materials (TMs) using inkjet printing that have fixed dosing levels of two explosives; 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and pentaerythritol tetranitrate (PETN). The uncertainty in the mass of dispensed solute is 0.8% (nominal 1 ng RDX and 5 ng or 20 ng PETN depending on ETD). TMs are stable under storage for at least 20 days at temperatures consistent with indoor and outdoor environments, and can be used by field personnel at deployed locations. Inkjet printing is shown to provide the necessary control over the spatial distribution of analyte on the substrate, thus limiting the variability in the signal response due to the sample. Measurements of signal intensities for two COTS ETDs were obtained from TMs over multi-year time spans and for multiple units of each ETD. Reproducibility in the signal response is shown to be between 6% and 15% RSD, or approximately double the within-day variability. The large datasets allow for the first time modeling of signal intensities with respect to normal distributions, which support the use of standard 3-sigma control practices.

Seed train development for the fermentation of bagasse from sweet sorghum and sugarcane using a simplified fermentation process.

A process was developed for seed culture expansion (3.6 million-fold) using 5% of the hemicellulose hydrolysate from dilute acid pretreatment as the sole organic nutrient and source of sugar. Hydrolysate used for seed growth was neutralized with ammonia and combined with 1.0mM sodium metabisulfite immediately before inoculation. This seed protocol was tested with phosphoric acid pretreated sugarcane and sweet sorghum bagasse using a simplified process with co-fermentation of fiber, pentoses, and hexoses in a single vessel (SScF). A 6h liquefaction (L) step improved mixing prior to inoculation. Fermentations (L+SScF process) were completed in 72 h with high yields (>80 gal/US ton). Ethanol titers for this L+SScF process ranged from 24 g/L to 32 g/L, and were limited by the bagasse concentration (10% dry matter).

Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160.

Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L+SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190°C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180°C).

Assessment of breast cancer-related arm lymphedema--comparison of physical measurement methods and self-report.

PURPOSE: To determine the relationship between physical methods of measuring lymphedema and self-reported swelling, their reliability, and standard error of measurement. METHOD: Lymphedema in each arm of women with (n = 33) and without (n = 18) unilateral arm lymphedema, secondary to breast cancer was measured by self-report, bioimpedance spectroscopy (BIS), perometer, and the truncated cone method. RESULTS: The physical measurement tools were highly reliable (ICC((2,1)): 0.94 to 1.00) with high concordance (r(c): 0.89 to 0.99). Self-report correlated moderately with physical measurements (r = 0.65 to 0.71) and was moderately reliable (ICC((2,1)): 0.70). CONCLUSIONS: Lymphedema assessment methods are concordant and reliable but not interchangeable.

Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli.

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K(m) values for NADPH (8 microM and 23 microM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K(m) for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli.

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.

Phase II study of rabbit anti-thymocyte globulin, cyclosporine and granulocyte colony-stimulating factor in patients with aplastic anemia and myelodysplastic syndrome.

We investigated efficacy and safety of rabbit anti-thymocyte globulin (rATG), cyclosporine and granulocyte colony-stimulating factor (G-CSF) as first-line therapy for patients with aplastic anemia (AA) and low or intermediate-1 or hypoplastic myelodysplastic syndrome (MDS). rATG 3.5 mg/kg (or 2.5 mg/kg per day for patients >or=55 years with MDS) was given for 5 days. Cyclosporine (5 mg/kg) and G-CSF (5 microg/kg) were given daily and continued for up to 6 months or longer. Responses were assessed about 3 and 6 months after therapy. Thirty-six patients have been enrolled on study and 32 patients treated; 25 were evaluable for a response (13 with AA, 12 with MDS); the rest are too early. The median age was 62 years (range, 20-83) for patients with AA and 63 (range, 42-80) for patients with MDS. Of 13 patients, 12 (92%) patients with AA responded (5 complete response (CR), 7 partial response (PR)), whereas of 12 patients, 4 (33%) patients with MDS responded (1 CR, 3 PR). For patients with AA, the median time to response (TTR) was 93 days (range, 79-623), whereas in the MDS group the median TTR was 111 days (range, 77-139). Grade III/IV toxicities were mainly cytopenias and neutropenic fever. Combination of rATG, cyclosporine and G-CSF is safe and effective as first-line treatment of AA and has significant activity in low-risk MDS.

Single frequency versus bioimpedance spectroscopy for the assessment of lymphedema.

BACKGROUND: The aims were to determine (i) whether single frequency bioimpedance analysis (SFBIA) is as accurate as bioimpedance spectroscopy (BIS) in measurement of extracellular fluid and (ii) whether change in extracellular fluid was specific to only the limb directly affected by surgery. METHODS: Arms of the control (n = 28) and arm lymphedema group (n = 28) and legs of the leg lymphedema group (n = 16) were assessed with SFBIA. All four limbs in all participants were assessed with BIS. All measurements occurred in a single session. RESULTS: BIS-measured ratios were highly concordant with those obtained with SFBIA (r (c) = 0.99, P < 0.001). Repeated measures ANOVA revealed that the ratio involving the lymphedema limb was different to the ratio of the non-oedematous limbs which was not significantly different to the arm or leg ratios of the control group. CONCLUSIONS: SFBIA is a simple accurate alternative to BIS for the clinical assessment of unilateral lymphedema. BIS discriminates those with clinical diagnosis of unilateral lymhoedema from those without the diagnosis.

Re-engineering Escherichia coli for ethanol production.

A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160(KO11, Deltafrd::celY(Ec) DeltaadhE DeltaldhA, DeltaackA lacA::casAB(Ko) rrlE::(pdc( Zm)-adhA(Zm)-adhB(Zm)-FRT-rrlE)pflB(+)). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.

Low salt medium for lactate and ethanol production by recombinant Escherichia coli B.

Individual nutrient salts were experimentally varied to determine the minimum requirements for efficient L (+)-lactate production by recombinant strains of Escherichia coli B. Based on these results, AM1 medium was formulated with low levels of alkali metals (4.5 mM and total salts (4.2 g l(-1)). This medium was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18-19 mmol l(-1) h(-1) for both (initial 48 h of fermentation).

Development of industrial-medium-required elimination of the 2,3-butanediol fermentation pathway to maintain ethanol yield in an ethanologenic strain of Klebsiella oxytoca.

Fermentation efficiency and nutrient costs are both significant factors in process economics for the microbial conversion of cellulosic biomass to commodity chemicals such as ethanol. In this study, we have developed a more industrial medium (OUM1) composed of 0.5% corn steep liquor (dry weight basis) supplemented with mineral salts (0.2%), urea (0.06%), and glucose (9%). Although the growth of strain P2 was vigorous in this medium, approximately 14% of substrate carbon was diverted into 2,3-butanediol and acetoin under the low pH conditions needed for optimal cellulase activity during simultaneous saccharification. Deleting the central region of the budAB genes encoding alpha-acetolactate synthase and alpha-acetolactate decarboxylase eliminated the butanediol and acetoin coproducts and increased ethanol yields by 12%. In OUM1 medium at pH 5.2, strain BW21 produced over 4% ethanol in 48 h (0.47 g ethanol per g glucose). Average productivity (48 h), ethanol titer, and ethanol yield for BW21 in OUM1 medium (pH 5.2) exceeded that of the parent (strain P2) in rich laboratory medium (Luria broth).

Transferring AAC intervention to the home.

PURPOSE: To evaluate the acquisition of AAC skills during an initial clinical trial and assess subsequent transfer of the training to the home setting. METHOD: A 12-year-old male with autism was first seen in a clinical setting to establish the use of a voice-output communication device. After learning to use the device to request access to preferred objects in the clinical trial, the intervention was transferred to the home. Follow-up with the parent was conducted via e-mail and telephone. Videotapes were made of initial home-based sessions to enable evaluation of the participant's progress. RESULTS: The programme was successful in teaching the participant to use a portable AAC device to make requests during the clinical trial and then in two home-based activities. CONCLUSION: An initial clinical trial with follow-up support for parents may be an efficient method for beginning AAC intervention and transferring the training procedures to the home setting.

Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant).

Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p < 0.10) between KO11 and LY01 under each of six different growth conditions (p < 0.000001). Statistical evaluation of differentially expressed genes according to various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from choline and choline uptake, respectively). Addition of glycine (10 mM) increased ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11. Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of the cell envelope components were also noted, which may contribute to increased ethanol tolerance.

Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase.

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).

Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose.

The simplicity of the fermentation process (anaerobic with pH, temperature, and agitation control) in ethanologenic Escherichia coli KO11 and LY01 makes this an attractive system to investigate the utility of gene arrays for biotechnology applications. By using this system, gene expression, glycolytic flux, and growth rate have been compared in glucose-grown and xylose-grown cells. Although the initial metabolic steps differ, ethanol yields from both sugars were essentially identical on a weight basis, and little carbon was diverted to biosynthesis. Expression of only 27 genes changed by more than 2-fold in both strains. These included induction of xylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR and the cyclic AMP-CRP system and repression of Mlc-regulated genes encoding glucose uptake (ptsHIcrr, ptsG) and mannose uptake (manXYZ) during growth on xylose. However, expression of genes encoding central carbon metabolism and biosynthesis differed by less than 2-fold. Simple statistical methods were used to investigate these more subtle changes. The reproducibility (coefficient of variation of 12%) of expression measurements (mRNA as cDNA) was found to be similar to that typically observed for in vitro measurements of enzyme activities. Using Student's t test, many smaller but significant sugar-dependent changes were identified (p < 0.05 in both strains). A total of 276 genes were more highly expressed during growth on xylose; 307 genes were more highly expressed with glucose. Slower growth (lower ATP yield) on xylose was accompanied by decreased expression of 62 genes concerned with the biosynthesis of small molecules (amino acids, nucleotides, cofactors, and lipids), transcription, and translation; 5 such genes were expressed at a higher level. In xylose-grown cells, 90 genes associated with the transport, catabolism, and regulation of pathways for alternative carbon sources were expressed at higher levels than in glucose-grown cells, consistent with a relaxation of control by the cyclic AMP-CRP regulatory system. Changes in expression of genes encoding the Embden-Meyerhof-Parnas (EMP) pathway were in excellent agreement with calculated changes in flux for individual metabolites. Flux through all but one step, pyruvate kinase, was predicted to be higher during glucose fermentation. Expression levels (glucose/xylose) were higher in glucose-grown cells for all EMP genes except the isoenzymes encoding pyruvate kinase (pykA and pykF). Expression of both isoenzymes was generally higher during xylose fermentation but statistically higher in both strains only for pykF encoding the isoenzyme activated by fructose-6-phosphate, a key metabolite connecting pentose metabolism to the EMP pathway. The coordinated changes in expression of genes encoding the EMP pathway suggest the presence of a common regulatory system and that flux control within the EMP pathway may be broadly distributed. In contrast, expression levels for genes encoding the Pentose-Phosphate pathway did not differ significantly between glucose-grown and xylose-grown cells.

Detoxification of dilute acid hydrolysates of lignocellulose with lime.

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis is accompanied by the production of inhibitors that retard microbial fermentation. Treatment of hot hydrolysate with Ca(OH)(2) (overliming) is an effective method for detoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our results indicate that the optimal lime addition for detoxification varies and depends on the concentration of mineral acids and organic acids in each hydrolysate. This optimum was shown to be readily predicted on the basis of the titration of hydrolysate with 2 N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average composition of 15 hydrolysates prior to treatment was as follows (per L): 95.24 +/- 7.29 g sugar, 5.3 +/- 2.99 g acetic acid, 1.305 +/- 0.288 g total furans (furfural and hydroxymethylfurfural), and 2.86 +/- 0.34 g phenolic compounds. Optimal overliming resulted in a 51 +/- 9% reduction of total furans, a 41 +/- 6% reduction in phenolic compounds, and a 8.7 +/- 4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarity of microorganisms, it is possible that the titration method described here may also prove useful for detoxification and fermentation processes using other microbial biocatalysts.

Use of UV absorbance To monitor furans in dilute acid hydrolysates of biomass.

A simple method based on UV spectra was developed for the estimation of total furans (furfural and hydroxymethylfurfural) in hemicellulose hydrolysates. UV spectra of hemicellulose hydrolysate contained a single dominant peak at around 278 nm. Approximately two-thirds of this peak can be attributed to furan absorbance (furfural and hydroxymethylfurfural). At 284 nm, both furfural and hydroxymethylfurfural have equal absorbance on a weight basis. A comparison of HPLC determinations for different samples of hydrolysate was used to develop a simple equation that allows the accurate prediction of total furans based on the difference in absorbance at 284 and 320 nm. This method may prove useful for quality control applications during the production of biomass syrups using a dilute acid hydrolysis process and during treatments for the amelioration of toxins. Although furans represent only a portion of the toxins present in hemicellulose hydrolysates, the abundance of furans appears to serve as a useful marker to predict relative toxicity.

Effects of Ca(OH)(2) treatments ("overliming") on the composition and toxicity of bagasse hemicellulose hydrolysates.

Hemicellulose syrups from dilute sulfuric acid hydrolysates of hemicellulose contain inhibitors that prevent efficient fermentation by yeast or bacteria. It is well known that the toxicity of these hydrolysate syrups can be ameliorated by optimized "overliming" with Ca(OH)(2). We have investigated the optimization of overliming treatments for sugar cane bagasse hydrolysates (primarily pentose sugars) using recombinant Escherichia coli LY01 as the biocatalyst. A comparison of composition before and after optimal overliming revealed a substantial reduction in furfural, hydroxymethylfurfural, and three unidentified high-performance liquid chromatography (HPLC) peaks. Organic acids (acetic, formic, levulinic) were not affected. Similar changes have been reported after overliming of spruce hemicellulose hydrolysates (Larsson et al., 1999). Our studies further demonstrated that the extent of furan reduction correlated with increasing fermentability. However, furan reduction was not the sole cause for reduced toxicity. After optimal overliming, bagasse hydrolysate was rapidly and efficiently fermented (>90% yield) by LY01. During these studies, titration, and conductivity were found to be in excellent agreement as methods to estimate sulfuric acid content. Titration was also found to provide an estimate of total organic acids in hydrolysate, which agreed well with the sum of acetic, levulinic, and formic acids obtained by HPLC. Titration of acids, measurement of pH before and after treatment, and furan analyses are proposed as relatively simple methods to monitor the reproducibility of hydrolysate preparations and the effectiveness of overliming treatments.