OPTIMIZATION OF IMMOBILIZATION PROCESS ON CRAB SHELL CHITOSAN AND ITS APPLICATION IN FOOD PROCESSING

Optimization of immobilization process on crab shell chitosan was carried out. The chitosan purified from the crab shell was used as the matrix for the immobilization of α-galactosidase. The prepared matrix was activated with glutaraldehyde at different concentrations and different time intervals and coupling time was determined. Immobilization of α-galactosidase on crab shell chitosan resulted in 72% immobilization yield. The parameters like the effect of pH, temperature, thermal stability and storage stability were determined. The study revealed that immobilized enzyme shows better thermal and storage stability than the free enzyme. The performance of the free and immobilized α-galactosidase was tested in continuous stirred batch reactor to hydrolyze raffinose family oligosaccharides in soymilk. The oligosaccharide content of the soymilk was reduced by 77% in continuous reaction by immobilized α-galactosidase.

PRACTICAL APPLICATIONS

Chitosan used for the immobilization of α-galactosidase offers several advantages for enzyme immobilization and it contains all characteristic features for use as industrial material. Immobilization of one of the industrial important enzyme α-galactosidase, as it has many potential application in hydrolyzing raffinose series of oligosaccharides. The hydrolyzed soymilk after processing by immobilized α-galactosidase is free from flatus-inducing factors like raffinose and stachyose. It can be used as an alternative means for cow's milk for lactose intolerance, particularly among individuals in developing countries. As chitosan used is from the crustacean waste from the crab shell, the production and utilization of chitosan provides an economical alternative means of crustacean shell waste disposal sought worldwide.     

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy

In a return to tradition, the popularity of caprine milk is on the rise. However, particularly in countries with developed dairy industries based on bovine milk, there is the risk of adulteration with bovine milk, which is a cheaper alternative. Thus, a rapid, robust, and simple method for the detection of bovine milk added to caprine milk is necessary, and ¹H nuclear magnetic resonance spectroscopy appears to provide a solution. A matrix of 115 pure and artificially adulterated pasteurized milk samples was prepared and used to discover biomarkers of bovine milk that are independent of chemical and biological variation caused by factors such as genetics, diet, or seasonality. Principal component analysis and orthogonal projections to latent structures discriminant analysis of pure bovine milk and pure caprine milk revealed spectral features that were assigned to the resonances of 4 molecules. Of these, the peaks corresponding to protons in the N-acetylglucosamine and N-acetylgalactosamine acetyl moieties showed significant applicability for our method. Receiver operating characteristic curve analysis was used to evaluate the performance of the peak integrals as biomarkers of adulteration. This approach was able to distinguish caprine milk adulterated with 5% of bovine milk with 84.78% accuracy and with 10% of bovine milk an excellent 95.65% accuracy. This study demonstrates that N-acetyl carbohydrates could be used as biomarkers for the detection of bovine milk in caprine milk and could help in protecting caprine milk authenticity.     

Effect of bacterial galactosidase treatment on the nutritional status of soybean seeds and its milk derivative

Four field strains of Lactobacillus plantarum (LS 4, 19, 21, 133) obtained from fufu (a semi-solid product obtained by boiling fermented cassava - Manihot esculenta Crantz) and a type strain DSM 2017 were grown on different carbon sources to induce galactosidase production. LS 21 produced the highest concentration of α- and β-galactosidase with 0.28 μmol/1 and 0.28 μmol/1 respectively on lactose and galactose. Milk obtained from soybean seeds treated with the enzyme mixture for 24 h showed a 99, 98 and 96% reduction respectively in the raffinose, stachyose and sucrose content when compared with the dry soybean seed. Glucose and galactose which were not detected in the dry seeds became readily available after soaking in both enzyme mixture and distilled water. Although there was reduction in the nutritional composition of both milk samples, reduction of phytic acid and trypsin inhibitor is beneficial to the consumers. The result of the sensory evaluation showed that the milk prepared from enzyme-treated soybean seeds was rated better in terms of flavour, texture, appearance and palatability.     

Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500–4,000 Da; 4–20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).     

离子色谱法测定人乳中乳糖

目的建立人乳中乳糖的离子色谱检测方法。方法采用阴离子交换柱CarboPac PA20(3 mm×150 mm)进行分离,对淋洗液梯度进行优化分离乳糖标准,建立了人乳中乳糖糖含量的自动分析方法并采用外标法进行定量分析。结果在最佳实验条件下,乳糖方法的线性范围为0.2~20 mg/L,相关系数大于0.999;方法检出限(信噪比为3)为10 mg/kg,定量限(信噪比为10)为30 mg/kg;人乳中乳糖含量日内精密度的RSD为0.72%~1.53%。结论方法优化后,排除了杂质干扰,使乳糖定量更为准确。本方法操作简单、精确、快速,可用于人乳中乳糖的定量。     

近红外光谱法检测奶粉掺假

目的建立近红外光谱法结合Adulterant Screen算法快速鉴别奶粉中大豆蛋白和尿素掺假的方法。方法采用近红外光谱仪获得奶粉未知样的光谱曲线,再用Adulterant Screen算法以及全数据库奶粉分类模型和既定类型的掺假物模型对曲线主要成分和掺假成分进行分析。结果该方法对一定浓度大豆蛋白和尿素掺假奶粉样可以实现掺假鉴别,大豆蛋白和尿素掺假奶粉样的掺假判别限分别为0.3 g/100g和0.2 g/100g,掺假物正确识别限分别为0.5 g/100g和0.8 g/100g。结论利用近红外光谱法结合Adulterant Screen算法可以快速鉴别奶粉中大豆蛋白和尿素的掺假。     

Biotransformation of isoflavone glycosides by Bifidobacterium animalis in soymilk supplemented with skim milk powder

ABSTRACT:  Two probiotic strains, Bifidobacterium animalis A and B, were used for the biotransformation of isoflavone glycosides in soymilk prepared from soy protein isolate (SPI) supplemented with skim milk powder (SMP) (SSMP). Unsupplemented soymilk (USM) and reconstituted skim milk powder (RSMP) were used as controls. The numbers of viable microorganisms in these products were enumerated. Lactose and isoflavone contents were quantified using high-performance liquid chromatography (HPLC). Our results showed that there was significantly higher biotransformation of isoflavone glycosides to aglycones in SSMP than that in USM. The levels of biotransformation were 83.96% and 85.43% for B. animalis A and B, respectively, compared to 74.30% and 72.82% for the USM. In addition, lactose utilization by both strains in SSMP was also higher than that in RSMP. At 24 h, 21.16 mg/mL of lactose was utilized in SSMP by B. animalis A compared with that of 16.88 mg/mL in RSMP. Consequently, the pH of SSMP was lower (3.80) than RSMP (4.00). However, the number of viable bacteria in SSMP was slightly lower than that in RSMP but significantly higher than that in USM. It appears that SMP enhanced the biotransformation of isoflavone glycosides to aglycones and SPI increased the lactose utilization by B. animalis A and B.     

The determination of milk powder added to whole milk

The objective was to determine the adulteration of fresh milk with reconstituted full fat milk powder. The ultra violet and visible spectra (700 to 240 nm) indicated two empirical parameters which are used to detect and quantify adulteration. Each parameter was calibrated against standards of adulteration and then tested against samples of known adulteration. One parameter, tested against two known series (5, 10, 20, 40 and 80% adulteration) gave the best correlation (coefficient of variation, 2.9%) between 20 and 60% adulteration. Correlation decreased at 80% adulteration (coefficient of variation 6.7%) and at 0 to 20% adulteration (coefficient of variation 35.9%). The second parameter, used in the range of 0 to 10% adulteration, when tested against 14 known samples gave results with an average of 12.5% below the true values. Sensitivity was 2.5% added reconstituted milk powder. The effect of low fat values (less than 2.3%) is discussed and a correction factor derived. The method when tested against 12 known samples gave the correct result in all cases within the limitations of the method; 4 commercial samples of milk were also analyzed.     

The mechanism of determining the adulteration of whole milk with milk powder by spectrophotometry

The spectrophotometric method for determination of the reconstituted milk adulteration in the raw milk has been suggested by empirical experiments. However, the mechanism is unclear. In this study, we followed and modified their methods and found that the mechanism is the turbidity difference between homogenized milk and non-homogenized milk. The equation of turbidance S=KBC˙ (d³/d⁴ + λ⁴) is offered as an explanation of this observation. There are linear relationships between transmittance (T[%]) and the amount of there constituted milk added (r=0.99) to the raw milk, non-homogenized milk, and homogenized milk. This method is recommended for detecting the addition of reconstituted milk to raw or non-homogenized milk. The result of the empirical methods showed that the detection rate of adulteration can be as accurate as 2.5%, but this method is not recommended for detecting the adulteration of homogenized milk.     

Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay

The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba.     

高效离子色谱法检测婴幼儿配方奶粉中的功能性低聚糖

建立一种同时测定蔗果三糖、蔗果四糖、蔗果五糖、蔗果六糖、蔗果七糖、棉子糖、水苏糖、毛蕊花糖的高效离子色谱检测技术,采用CarboPac PA20色谱柱分离,以氢氧化钠-乙酸钠为流动相,梯度洗脱,使用脉冲安培检测器进行检测。结果表明,在本实验条件下8种功能性低聚糖得到了很好分离,回收率为80.0%~103%,相对标准偏差小于3%(n=6)。该方法能快速、准确、可靠地检测婴幼儿配方奶粉中功能性低聚糖的含量。     

掺假羊乳及其制品中牛乳的检测技术研究进展

羊乳具有营养价值高、蛋白质组成更接近人乳、脂肪球直径小及致敏性低等优点, 更利于人体消化吸收, 受到消费者和乳品企业的青睐。近年来我国羊乳产业发展迅速且潜力巨大, 但由于受羊乳产量和养殖规模的限制, 羊乳价格昂贵, 市场中存在羊乳及其制品掺假牛乳的现象, 且掺假手段多样, 难以辨别。为了保证消费者的健康和权益, 保障羊乳市场良性发展, 羊乳及其制品的纯正性、真实性检测已经成为热点研究方向。本文通过分析基于乳中蛋白质、脂肪和核酸差异的羊乳中牛乳掺假检测技术的研究现状, 介绍和探讨了各检测技术的基本原理及其在应用中的优缺点, 同时展望羊乳掺假检测技术的发展方向, 旨在为牛羊乳混合掺假检测技术的进一步发展提供资料参考和思路。     

基于电子舌的掺假羊奶快速定量预测模型

为实现对掺假羊奶的快速、客观辨别,模仿人体味觉感知机理研制了一套便携式电子舌检测系统,并建立了一种能够快速鉴别掺假羊奶的新方法。系统检测时,首先对样本溶液进行大幅脉冲扫描,用以获取掺假羊奶的"指纹"信息,然后利用离散小波变换(discrete wavelet transform,DWT)对"指纹"数据中的特征信息进行提取,最后在此基础上,采用主成分分析(principal component analysis,PCA)方法对不同掺假比例的羊奶进行定性辨别。采用粒子群优化极限学习机(Particle swarm optimization extreme learning machine,PSO-ELM)对不同掺假比例的羊奶进行了定量预测。通过试验数据得出,PCA对6种不同掺假比例的羊奶区分达到100%,区分效果好。PSO-ELM羊奶纯度预测模型拟合曲线非常接近实测值曲线,因此采用PSO-ELM方法建立掺假羊奶纯度定量预测模型具有较高的预测精度。     

Oligosaccharide Content of 20 Varieties of Cowpeas in Nigeria

Mature dry seeds of 20 varieties of cowpeas (Vigna unguiculata) grown in Nigeria were analyzed for their sucrose, raffinose and stachyose content. The dry seeds were ground into powder, extracted with 80% ethanolandthe extract analyzed by paper chromatography using a mixture of n-butanol, ethanol, water and ammonia solution 8:1:2:1 v/v). The results show a progressive decrease in raffinose and stachyose content with dehulling and cooking and an increase in sucrose level after cooking. The average content of the sugars in whole beans on dry weight basis was sucrose 0.8%, raffinose 2.6% and stachyose 3.3%. The average content for dehulled raw beans were sucrose 0.7%, raffinose 1.8% and stachyose 2.4% while in cooked beans it was 1.6%, 1.3%, and 1.8%, respectively.     

Metabolism of oligosaccharides and aldehydes and production of organic acids in soymilk by probiotic bifidobacteria

Four strains of Bifidobacterium were assessed for α‐galactosidase activity in MRS broth using p‐nitrophenyl‐α‐d ‐galactopyranoside as substrate. Bifidobacteria were then used to ferment soymilk prepared from soy protein isolate (SPI), with and without d ‐glucose and l ‐cysteine supplementation. Measurements of pH and the quantification of oligosaccharides, organic acids and aldehydes in soymilk were done after 0, 12, 24, 36 and 48 h of incubation. The presence of d ‐raffinose stimulated the α‐galactosidase activity of B. longum BB536 (BB536) and B. pseudolongum 20099 (BP20099). In soymilk, oligosaccharides and aldehydes were effectively metabolized by Bifidobacterium; d ‐glucose and l ‐cysteine supplementation enhanced the hydrolysis of raffinose and stachyose. BB536 and BP20099 degraded a significantly greater level of oligosaccharides and aldehydes than B. animalis Bb‐12 and B. longum 1941 (P < 0.05). Raffinose and stachyose were completely metabolized by BB536 after 48 h. In soymilk without any supplementation, hexanal and pentanal were not detected after 12 h of fermentation with BB536 and BP20099. BB536 was the highest producer of organic acids, with an average acetic acid/l (+)‐lactic acid ratio of 0.7.     

A duplex polymerase chain reaction for the quantitative detection of cows’ milk in goats’ milk cheese

A duplex polymerase chain reaction (PCR) has been applied for the detection of both cows’ and goats’ milk in goats’ milk cheeses using primers targeting the mitochondrial 12S rRNA genes. By means of a linear normalised calibration curve between the log of the ratio of the cows’ band intensity and the sum of cows’ and goats’ intensities vs. the log of cows’ milk percentage, it was possible to quantify cheese adulteration with cows’ milk in the range of 1–60%. The duplex PCR technique allowed the detection of 0.1% of cows’ milk in goats’ milk cheese. The proposed method was successfully applied to cheeses with defined amounts of cows’ milk and to 15 of a total of 17 commercial cheese samples. The results showed the fraudulent addition of cows’ milk in three samples labelled as pure goats’ milk cheeses and the omission of goats’ milk mentioned on the label of two cheeses containing mixed milk.     

Rapid detection and quantification of milk adulteration using infrared microspectroscopy and chemometrics analysis

The application of attenuated total reflectance mid-infrared microspectroscopy (MIR-microspectroscopy) was evaluated as a rapid method for detection and quantification of milk adulteration. Milk samples were purchased from local grocery stores (Columbus, OH, USA) and spiked at different concentrations of whey, hydrogen peroxide, synthetic urine, urea and synthetic milk. Samples were place on a 192-well microarray slide, air-dried and spectra were collected by using MIR-microspectroscopy. Pattern recognition analysis by Soft Independent Modeling of Class Analogy (SIMCA) showed tight and well-separated clusters allowing discrimination of control samples from adulterated milk. Partial Least Squares Regression (PLSR) showed standard error of prediction (SEP) ∼2.33, 0.06, 0.41, 0.30 and 0.014 g/L for estimation of levels of adulteration with whey, synthetic milk, synthetic urine, urea and hydrogen peroxide, respectively. Results showed that MIR-microspectroscopy can provide an alternative methodology to the dairy industry for screening potential fraudulent practice for economic adulteration of cow’s milk.     

高效液相色谱法分析大豆和大豆低聚糖浆中糖的组成与含量

采用高效液相色谱法(HPLC)对大豆种子及市售的大豆低聚糖浆中的可溶糖进行了分析,并利用α-半乳糖苷酶酶解大豆低聚糖浆及结合HPLC分析来推测大豆低聚糖浆的糖组成。HPLC分析条件为:Sugar-D色谱柱,75%乙腈为流动相,柱温40℃,流速为1.0ml/min,示差折光检测器。Sugar-D色谱柱的分离效果好,基线稳定,相对标准偏差1.0%～2.0%,标准回收率96.7%～100.7%。分析结果表明大豆种子和市售的大豆低聚糖浆中均含有果糖、蔗糖、蜜二糖、棉子糖、水苏糖以及二种保留时间介于蔗糖与蜜二糖之间的未知成分,而大豆低聚糖浆中还含有葡萄糖(半乳糖)和甘露三糖;大豆种子中主要含有蔗糖和水苏糖,而大豆低聚糖浆中含有较多的蔗糖、果糖、水苏糖和葡萄糖(半乳糖),功能性低聚糖主要为水苏糖和甘露三糖。     

Hydrolysis of soya milk oligosaccharides by Bifidobacterium longum CRL 849

 The reduction of soya milk oligosaccharides by Bifidobacterium longum CRL 849 was studied. The utilization of stachyose was concomitant with the use of sucrose. Maximum hydrolysis of stachyose (49.3%) occurred during the first 7 h of incubation at 37  °C, while a 79.3% decrease in the concentration of sucrose was observed after 9 h. No raffinose was detected after hydrolysis of the stachyose. Cell population decreased after 8 h of incubation because of the low pH attained (pH 4.7). l(+)-Lactate concentration was higher than acetate (molar ratio 6.7 : 1) at 6 h followed by a slow increase in acetate formation. Ethanol was detected in small amounts at the end of the incubation time (24 h). Received: 29 December 1997     

Hydrolysis of oligosaccharides in soybean products by Debaryomyces hansenii UFV-1 α-galactosidases

α-Galactosides are abundant sugars found in legumes such as soybean. Since humans and monogastric animals lack α-galactosidase in the digestive tract, they are unable to digest these sugars, which induce flatulence. The use of α-galactosidases is promising as a means to overcome this problem, and to increase the consumption of soy products. Immobilized α-galactosidase, derived from Debaryomyces hansenii UFV-1, exhibited an activity of 40 U per g of silica and an activity yield of 50%. The optimum pH of free and immobilized α-galactosidase was 5.0 and the optima temperatures were 60 and 80 °C, respectively. The soymilk stachyose was completely hydrolyzed by different enzyme forms after incubation for 4 h at 60 °C, while raffinose was reduced by 100%, 25% and 68% by free, immobilized enzymes and permeabilized cells, respectively. The soy molasses treatment with free enzyme for 6 h promoted reduction in stachyose and raffinose contents by 100% and 50%, respectively.