Rapid and quantitative detection of the microbial spoilage in milk using Fourier transform infrared spectroscopy and chemometrics

Microbiological safety plays a very significant part in the quality control of milk and dairy products worldwide. Current methods used in the detection and enumeration of spoilage bacteria in pasteurized milk in the dairy industry, although accurate and sensitive, are time-consuming. FT-IR spectroscopy is a metabolic fingerprinting technique that can potentially be used to deliver results with the same accuracy and sensitivity, within minutes after minimal sample preparation. We tested this hypothesis using attenuated total reflectance (ATR), and high throughput (HT) FT-IR techniques. Three main types of pasteurized milk - whole, semi-skimmed and skimmed - were used and milk was allowed to spoil naturally by incubation at 15 degrees C. Samples for FT-IR were obtained at frequent, fixed time intervals and pH and total viable counts were also recorded. Multivariate statistical methods, including principal components-discriminant function analysis and partial least squares regression (PLSR), were then used to investigate the relationship between metabolic fingerprints and the total viable counts. FT-IR ATR data for all milks showed reasonable results for bacterial loads above 10(5) cfu ml(-1). By contrast, FT-IR HT provided more accurate results for lower viable bacterial counts down to 10(3) cfu ml(-1) for whole milk and, 4 x 10(2) cfu ml(-1) for semi-skimmed and skimmed milk. Using FT-IR with PLSR we were able to acquire a metabolic fingerprint rapidly and quantify the microbial load of milk samples accurately, with very little sample preparation. We believe that metabolic fingerprinting using FT-IR has very good potential for future use in the dairy industry as a rapid method of detection and enumeration.     

Cascade Enzymatic Hydrolysis Coupling with Ultra ne Grinding Pretreatment for Sugarcane Bagasse Sacchari cation

The biorefinery process for sugarcane bagasse saccharification generally requires significant accessibility of cellulose. We reported a novel method of cascade cellulase enzymatic hydrolysis coupling with ultrafine grinding pretreatment for sugarcane bagasse saccharification. Three enzymatic hydrolysis modes including single cellulase enzymatic hydrolysis, mixed cellulase enzymatic hydrolysis, and cascade cellulase enzymatic hydrolysis were compared. The changes on the functional group and surface morphology of bagasse during cascade cellulase enzymatic hydrolysis were also examined by FT-IR and SEM respectively. The results showed that cascade enzymatic hydrolysis was the most efficient way to enhance the sugarcane bagasse sacchari cation. More than 65% of reducing sugar yield with 90.1% of glucose selectivity was achieved at 50 oC, pH=4.8 for 72 h (1200 r/min) with cellulase I of 7.5 FPU/g substrate and cellulase II of 5 FPU/g substrate.     

Rapid impedance method for predicting the potential shelf life of packaged pasteurized milk

The potential shelf life of packaged pasteurized milk was studied using a rapid impedance method. The results showed a high correlation between the shelf life of Fengxing and Xiang Man-lou pasteurized milks and between the detection time and the logarithm of colony-forming units per milliliter. The impedance detection time was measured after preliminary incubation at 30 degrees C for 6 h for 100 and 200 microL milk samples, or at 37 degrees C for 6 h for 100-400 microL milk samples for Fengxing pasteurized milk, and after 6 h preliminary incubation at 30 degrees C for 300 and 400 microL milk samples or 6 h at 37 degrees C for 100-400 microL milk samples for Xiang Man-lou pasteurized milk. Regressive equations were then constructed to predict the potential shelf life. Compared with the traditional method, the impedance method plus pre-incubation of milk at elevated temperatures (30 and 37 degrees C) was rapid, accurate, and convenient. The entire estimation process was completed within 11-14 and 14-20 h for Fengxing and Xiang Man-lou pasteurized milks, respectively.     

Effects of hemicellulose and lignin on enzymatic hydrolysis of cellulose from dairy manure

This study focused on the effect of hemicellulose and lignin on enzymatic hydrolysis of dairy manure and hydrolysis process optimization to improve sugar yield. It was found that hemicellulose and lignin in dairy manure, similar to their role in other lignocellulosic material, were major resistive factors to enzymatic hydrolysis and that the removal of either of them, or for best performance, both of them, improved the enzymatic hydrolysis of manure cellulose. This result combined with scanning electron microscope (SEM) pictures further proved that the accessibility of cellulose to cellulase was the most important feature to the hydrolysis. Quantitatively, fed-batch enzymatic hydrolysis of fiber without lignin and hemicellulose had a high glucose yield of 52% with respect to the glucose concentration of 17 g/L at a total enzyme loading of 1300 FPU/L and reaction time of 160 h, which was better than corresponding batch enzymatic hydrolysis.     

Fluorimetric determination of alkaline phosphatase in solid and fluid dairy products

A new assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products. The method proposed is simple, rapid and directly applicable to solid and liquid dairy samples. ALP in the test sample hydrolyzes a non fluorescent substrate, trifluoromethyl-β-umbelliferone phosphate, to its highly fluorescent phenolate product. The assay is performed in a reverse micellar medium composed of mixed buffer (2-amino-2-methyl-1-propanol buffer pH 9.0 and borate buffer pH 9.0) in AOT/isooctane, at a temperature of 38 °C. Total test time is 450 s. Reaction rates are linear (except for butter) up to 8.5 and 11% (v/v) raw milk, for whole milk and chocolate milk, respectively. The detection limits are 0.04, 0.4 and 0.22% (v/v) raw milk, for whole milk, chocolate milk and butter, respectively. The precision of the fluorimetric method was assessed by repeated analysis of a pasteurized milk sample spiked with mixed herd raw milk. The accuracy of the method was evaluated by comparison with an official colorimetric assay using p-nitrophenylphosphate as ALP substrate.     

Interlaboratory validation of the Vidas SET2 kit for detection of staphylococcal enterotoxins in milk products

An earlier intralaboratory validation study based on the EN ISO 16140 Standard conducted by the Community Reference Laboratory for coagulase-positive staphylococci including Staphylocococcus aureus showed that, after an extraction step using dialysis concentration, the Vidas SET2 detection kit could be used to screen staphylococcal enterotoxins in milk and milk products. In order to fully validate Vidas SET2, an interlaboratory study was organized. Six freeze-dried samples and 3 ready-to-use concentrated extracts were analyzed by 21 laboratories according to the method, including a detection with Vidas SET2. Results did not show false-positive or -negative results. Accordance and concordance parameters were equal to 100%, corresponding to a concordance odds ratio of 1. This interlaboratory study confirmed the satisfactory outcome of the preliminary tests and of the intralaboratory study performed previously. The Vidas SET2 detection kit can be used as a method for the detection of staphylococcal enterotoxins in milk and milk products as well as the Transia Plate SET detection kit in the European screening method for official control purposes, after an extraction step followed by dialysis concentration.     

N-methyl-2-pyrrolidonium-based Brönsted-Lewis acidic ionic liquids as catalysts for the hydrolysis of cellulose

We experimentally studied the catalytic performances of a series of Br?nsted-Lewis acidic N-methyl-2-pyrrolidonium metal chlorides([Hnmp]Cl/MCl_x, where M=Fe, Zn, Al, or Cu) for the hydrolysis of microcrystalline cellulose(MCC) and cotton to produce reducing sugar. A variety of factors, such as temperature, time, ionic liquid(IL) species, IL dosage, and the concentration of the metal chloride were investigated. [Hnmp]Cl/FeCl_3 presented the best hydrolysis performance, affording a 98.8% yield of total reducing sugar from MCC(1 h, 100 °C, 0.1 g MCC, 0.2 g acidic IL, 2.0 g [Bmim]Cl as solvent), which is better than or comparable to results previously obtained with other –SO_3H functionalized acidic ILs. The hydrolysis performances of [Hnmp]Cl/MClx were rationalized using density functional theory calculations, which indicated that interactions between the metal chlorides and the cellulose, including charge-transfer interactions are important in the hydrolysis of cellulose and degradation of glucose. This work shows that Br?nsted-Lewis acidic ILs are potential catalysts for the hydrolysis of cellulose to produce sugar.     

Studies into using manure in a biorefinery concept

Animal manure is an underutilized biomass resource containing a large amount of organic carbon that is often wasted with the existing manure disposal practices. A research project funded by the US Department of Energy explored the feasibility of using manure via the sugar platform in a biorefinery, converting the carbon from fiber to biochemicals. The results showed that (1) fiber was the major component of manure dry material making up approx 50%, 40%, and 36% of the dry dairy, swine, and poultry manure material, respectively; within dairy manure, more than 56% of the dry matter was in particles larger than 1.680 mm; (2) in addition to being a carbon source, manure could provide a variety of nutrient for fungi T. reesei and A. phoenicis to produce cellulase; (3) the hemicellulose component in the manure fiber could be readily converted to sugar through acid hydrolysis; while concentrated acid decrystallization treatment was most effective in manure cellulose hydrolysis; (4) purification and separation was necessary for further chemical conversion of the manure hydrolysate to polyols through hydrogenation; and (5) the manure utilization strategy studied in this work is currently not profitable.     

Study on the Decreased Sugar Yield in Enzymatic Hydrolysis of Cellulosic Substrate at High Solid Loading

Current technology for conversion of biomass to ethanol is an enzyme-based biochemical process. In bioethanol production, achieving high sugar yield at high solid loading in enzymatic hydrolysis step is important from both technical and economic viewpoints. Enzymatic hydrolysis of cellulosic substrates is affected by many parameters, including an unexplained behavior that the glucan digestibility of substrates by cellulase decreased under high solid loadings. A comprehensive study was conducted to investigate this phenomenon by using Spezyme CP and Avicel as model cellulase and cellulose substrate, respectively. The hydrolytic properties of the cellulase under different substrate concentrations at a fixed enzyme-to-substrate ratio were characterized. The results indicate that decreased sugar yield is neither due to the loss of enzyme activity at a high substrate concentration nor due to the higher end-product inhibition. The cellulase adsorption kinetics and isotherm studies indicated that a decline in the binding capacity of cellulase may explain the long-observed but little-understood phenomenon of a lower substrate digestibility with increased substrate concentration. The mechanism how the enzyme adsorption properties changed at high substrate concentration was also discussed in the context of exploring the improvement of the cellulase-binding capacity at high substrate loading.     

A chemometric approach to the detection of milk adulteration based on protein profiles determined by capillary electrophoresis

The general objective of this study was to utilize chemometrics in the interpretation of capillary electrophoresis milk protein profiles, for the detection of pasteurized milk adulteration with rehydrated milk powder or a rehydrated dairy-based milk substitute. The specific objectives were 1) to collect quantitative data on major casein and whey proteins in authentic and adulterated milks in a single CE analysis; and 2) to apply a pattern recognition procedure, Soft Independent Modeling of Class Analogies (SIMCA), on collected CE protein data, for the development of a statistical model useful in the detection of pasteurized milk adulteration. Authentic samples were fresh milk collected from various farms over a period of six months. Adulterated samples were authentic fresh milk partially or totally substituted with rehydrated milk powder or a rehydrated commercial milk substitute at different levels. Quantitative protein data obtained by capillary free zone electrophoresis for beta-lactoglobulin, alpha-lactalbumin, beta-casein, and alpha-casein of 86 samples, authentic and adulterated samples, were used as a training set to build a SIMCA multivariate statistical model. The detection of sample outliers was useful for the elimination of unusual samples and optimization of the multivariate model. From the 35 commercial pasteurized milks tested, which were treated as unknowns, a total of 14 samples (40%) were not assigned to the authentic or fresh milk group, meaning that these samples had some type of adulteration at the levels included in the training set (> 15%). Decision-making on detecting adulteration of unknown commercial pasteurized milk samples was eased since predictions were based on statistical probabilities.     

提高纤维素酶水解效率和降低水解成本

在我国可大量转化乙醇的是纤维质材料。纤维质材料转化乙醇的关键问题是纤维质转化为糖的过程,提高纤维素酶转化效率的方法有:(1)对纤维质材料进行预处理;(2)研究纤维素酶的最适作用条件;(3)纤维素酶的重复利用;(4)合理的发酵工艺等。本文分析了纤维素的结构以及纤维素酶的作用方式,总结了目前研究较多的几种纤维质材料预处理方法,及其对纤维素酶水解率的影响,并对研究纤维素酶的最适作用条件、纤维素酶的重复利用以及合理的发酵工艺进行了综述和分析。     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

Lactosylated casein phosphopeptides as specific indicators of heated milks

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α_s2-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.     

Determination of ammonium in milk and dairy products by ion chromatography

To control the quality and the biochemical evolution of milk and dairy products during their technological transformations, it is interesting to determine their ammonium concentrations. A chromatographic method for the determination of this compound is proposed. The method is based on the separation of ammonium by cation-exchange chromatography and its detection by suppressed conductivity. With an appropriate sample preparation, this method enabled identification and quantification of ammonium with good repeatability (relative standard deviation of about 5%). Moreover, good sensitivity (less than 0.5 mg/kg) and no interference between ammonium and other matrix components were determined. It was also shown that this method offers a very promising alternative for studying changes in ammonium concentration of milk or caseinate after their heat treatments and in different dairy products such as yoghurt and cheeses (hard cooked and mould ripened cheeses).     

Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose

This paper investigates the enzymatic hydrolysis of three main allomorphic forms of microcrystalline cellulose using different cellulases, from Trichoderma reesei and from Aspergillus niger, respectively. It was demonstrated that both the morphological and crystalline structures are important parameters that have a great influence on the course of the hydrolysis process. The efficiency of the enzymatic hydrolysis of cellulosic substrates was estimated by the amounts of reducing sugar and by the yield of the reaction. Changes in the average particle sizes of the cellulose allomorphs were determined during enzymatic hydrolysis. The accumulation of soluble sugar within the supernatant was used as a measure of the biodegradation process’s efficiency, and was established by HPLC-SEC analysis. Any modifications in the supramolecular structure of the cellulosic residues resulting from the enzymatic hydrolysis were determined by X-ray diffraction. The action of each cellulase was demonstrated by a reduction in the crystalline index and the crystallite dimensions of the corresponding allomorphic forms. The crystalline structure of allomorphic forms I and II did not suffer significant modifications, while cellulose III recorded a partial return to the crystalline structure of cellulose I. The microstructures of cellulose allomorph residues were presented using optical microscopy and scanning electron microscopy.     

Microwave Pretreatment of Substrates for Cellulase Production by Solid-State Fermentation

The agricultural residues, wheat bran and rice hulls, were used as substrates for cellulase production with Trichoderma sp 3.2942 by solid-state fermentation. Microwave irradiation was employed to pretreat the substrates in order to increase the susceptibility. Although the highest cellulase yield was obtained by the substrates pretreated by 450 W microwave for 3 min, pretreatment time and microwave power had no significant effect on cellulase production. The initial reducing sugar content (RSC) of substrates was decreased by microwave irradiation, but more reducing sugars were produced in later fermentation. Alkali pretreatment combined with microwave pretreatment (APCMP) of rice hulls could significantly increase cellulase yields and reducing sugar. The maximum filter paper activity, carboximethylcellulase (CMC)ase, and RSC were increased by 35.2%, 21.4%, and 13%, respectively, compared with those of untreated rice hulls. The fermented residues could produce more cellulase and reducing sugars than fresh rice hulls after they were treated by APCMP. The increased accessibility of the substrates by microwave pretreatment was mainly achieved by rupture of the rigid structure of rice hulls. However, for alkali pretreatment and APCMP, delignification and removal of ash played very important roles for increasing the acceptability of substrates.     

Dispersive solid-phase extraction followed by liquid chromatography–tandem mass spectrometry for the multi-residue analysis of pesticides in raw bovine milk

A fast multi-residue method based on dispersive solid-phase extraction (DSPE) followed by liquid chromatography–tandem mass spectrometry was developed for the simultaneous determination of 44 pesticides in raw bovine milk. Raw bovine milk samples did not percolate through SPE cartridges usually applied for pesticide extraction from homogenized pasteurized milk samples. Therefore, a DSPE technique was implemented and validated for the first time in this work. Graphitized non-porous carbon and C18 modified silica materials were tested both in combination with magnesium sulfate and bonded silica with ethylenediamine-N-propyl phase. The efficiency of the DSPE process was studied at several concentration levels obtaining the higher recoveries with C18 material. The method performance was also assessed and the limits of quantification reached the ng g⁻¹ level, complying with the most recent maximum residue levels. The DSPE method was also shown to be suited to both the fatty and skimmed fractions issued from raw milk. Finally, the extraction method was successfully applied to the analysis of raw milk samples collected in 23 farms of dairy cattle from NW Spain (Galicia).     

Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and β-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both β-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on β-Glucosidase activity. However, these sugars were shown to have significant inhibitory effects on cellulase activity during cellulose hydrolysis. Our study suggests that high-substrate consistency hydrolysis with supplementation of hemicellulose is likely to be a practical solution to minimizing end-product inhibition effects while producing hydrolysate with high glucose concentration.     

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.     

Determination of sugars by liquid chromatography with post-column catalytic derivatization and fluorescence detection

Summary A method for the determination of reducing sugars such as fructose and glucose and nonreducing sugar such as sucrose by high performance liquid chromatography followed by an acidic hydrolysis and a derivatization with benzamidine has been developed. After separation of sugars on a gel column packed with a polymer-based cation exchange material (Sugar-Pak I, Waters-Millipore), the sucrose is first hydrolysed in a solid phase reactor to convert it into reducing subunits. A post-column fluorigenic reaction with benzamidine under alkaline condition allows the selective determination of both natural and converted reducing carbohydrates.This procedure has proven to be selective (fluorigenic detection) and highly sensitive (allowing detection as little as picomoles amounts), reproducible and linear over a broad range of concentrations: 5×10^–4 to 1.0×10^–2 M.The applicability of this method to natural matrices such as plant extracts and beverages is also described. The sugar content of a barley extract has been determined and compared with a specific enzymatic test. The determined sugar content of natural and commercial lemon juices as well as of Cola beverages has been compared with those found by the conventional LC refractive index analytical procedure. In all cases, the results were comparable and were within the experimental errors of the methods.