Detection of guaiacol produced by Alicyclobacillus acidoterrestris in apple juice by sensory and chromatographic analyses, and comparison with spore and vegetative cell populations

Spoilage of fruit juice by Alicyclobacillus acidoterrestris is characterized by a distinct medicinal or antiseptic off odor attributed to guaiacol, a metabolic by product of the bacterium. Detection of low populations of A. acidoterrestris that would precede sensory detection of guaiacol would enable juice processors to select appropriate processing and storage conditions that would minimize or eliminate spoilage. The objective of this study was to determine the recognition threshold of guaiacol in apple juice by sensory analysis and the population of A. acidoterrestris and incubation time at 21 and 37 degrees C necessary for chemical detection of guaiacol. Commercially sterilized apple juice (pH 3.54 +/- 0.04, 11.3 +/- 0.3 degrees Brix) was inoculated with a five-strain mixture of A. acidoterrestris spores (2.98 log10 CFU/ml) and stored at 21 or 37 degrees C for up to 61 days. Using an experienced sensory panel and the forced-choice ascending concentration method of limits, the best estimate threshold (BET) for recognition of guaiacol added to uninoculated apple juice was 2.23 ppb. Numbers of A. acidoterrestris spores and cells in inoculated juice remained constant during the 61-day storage period; however, the panel detected (P < or = 0.01) guaiacol in juice stored at 37 degrees C within 8 days. At three of four sampling times ranging from 13 to 61 days at which the sensory panel detected (P < or = 0.001) guaiacol, concentrations of 8.1 to 11.4 ppb were detected by chromatographic analysis. The panel detected (P < or = 0.1 to P < or = 0.01) guaiacol in five samples stored at 21 to 37 degrees C for 8 to 61 days in which the compound was not detected by chromatographic analyses. It appears that guaiacol content in apple juice inoculated with A. acidoterrestris is not always correlated with numbers of cells, and the limit of sensitivity of chromatographic quantitation of the compound is higher than the BET.     

Impact of Multicolored Asian Lady Beetles on the Sensory Properties of Concord and Niagara Grape Juice

ABSTRACT: The presence of Multicolored Asian Lady Beetles (MALB) in grape juice is increasingly problematic. The overall objective of this study was to determine the specific sensory impacts of MALB on Concord and Niagara grape juice. The aroma threshold for MALB-taint in both juices was determined and expressed as the best estimate threshold (BET). The aroma BET for MALB-taint in Concord grape juice was 1.8 and 0.65 MALB/L Niagara grape juice. The specific sensory attributes of the grape juices influenced by the presence of MALB were then described. In Concord grape juice, trained panelists (n = 9) found significant increases in vegetal aroma and earthy flavor as MALB concentration increased from 0.45 to 7.2 MALB/L. In Niagara grape juice, with increasing MALB concentration, trained panelists (n = 8) indicated significant decreases in honey and sweetness with corresponding increases in sourness, astringency, and vegetal and earthy aromas and flavors (P < 0.05). For both aroma and flavor, consumer rejection studies were performed using paired preference comparisons of control juice to MALB-tainted grape juice. For Concord grape juice, consumers (n = 60) rejected the aroma of MALB-tainted juice at 1.8 MALB/L and the taste/flavor of the MALB-tainted juice at 3.6 MALB/L. For Niagara juice, consumers rejected the aroma of the juice at 3.6 MALB/L. This study demonstrated the presence of MALB negatively impacts the sensory properties of Concord and Niagara grape juice. Grape juice processors may use this research for quality control measures and for establishing tolerance limits. Practical Application: The Multicolored Asian Ladybeetle (MALB), initially used for biocontrol, has been shown in previous literature to impact the sensory properties of wines. The presence of MALB in grape juice is now becoming problematic; however, there is little research describing the specific sensory impacts of MALB on grape juice. This study examined the sensory impact of MALB on both Concord and Niagara grape juice. While the threshold and trained panel evaluations from this study provide valuable sensory profile information, grape juice processors may be particularly interested in the consumer rejection of MALB-tainted grape juice as they can use this research for quality control measures and for establishing tolerance limits.     

Recovery of alicyclobacillus from inhibitory fruit juice concentrates

Growth of Alicyclobacillus in low-pH fruit juices may result in off-odors and off-flavors due to the production of compounds such as guaiacol (2-methoxy phenol). An important step in preventing Alicyclobacillus contamination of fruit juices is the screening of incoming ingredients. Many fruit juice concentrates contain compounds that inhibit Alicyclobacillus growth, but beverages produced from the concentrates may not contain sufficient amounts of the active component to prevent spoilage. Therefore, accurate screening of juice concentrates is essential to prevent false-negative test results and product spoilage. The objective of this study was to evaluate isolation methods for detection of Alicyclobacillus in inhibitory juice concentrates. Recovery of Alicyclobacillus spores from inoculated and naturally contaminated concentrates was compared by using pour plate, spread plate, and filtration methods. Pour plates consistently recovered the lowest number of spores from inoculated concentrates. Spread plating was the most effective method used to recover spores from inoculated apple and pomegranate juice concentrates, while filtration resulted in the highest recovery from cranberry concentrate. When tested on naturally contaminated concentrates, the pour plate method failed to detect Alicyclobacillus in many samples. Filtration was much more effective. The filtration method increased the likelihood of detecting Alicyclobacillus contamination of fruit juice concentrates containing inhibitory compounds.     

Rapid discrimination of Alicyclobacillus strains in apple juice by Fourier transform infrared spectroscopy

Alicyclobacillus spp. are thermoacidophilic, spore-forming bacteria. Some of which cause spoilage in pasteurized and heat-treated apple juice products through the production of guaiacol. Fourier transform infrared (FT-IR) spectroscopy was used to discriminate between eight Alicyclobacillus strains (WAC, 81-2, Oly#21, 51-1, KF, 1016, 1101, and A-Gala A4) in apple juice. FT-IR vibrational combination bands reflected compositional differences in the cell membranes of Alicyclobacillus strains in the "fingerprint region" at wavenumbers between 1500 and 800 cm(-1). Distinctive segregation among spectral sample clusters of different Alicyclobacillus strains was observed using principal component analysis (PCA). Two closely related strains (1016 and 1101) of Alicyclobacillus acidoterrestris could be distinguished, suggesting that this method can be highly selective. Results of soft independent modeling of class analogy (SIMCA) demonstrated that guaiacol-producing and non-guaiacol producing Alicyclobacillus strains could be differentiated up to 89% of the time. This technique may provide a tool for fruit juice producers to detect Alicyclobacillus rapidly and to monitor and control guaiacol formation.     

不同种类果汁中脂环酸芽孢杆菌及其代谢产物污染调查

为了解不同种类果汁中脂环酸芽孢杆菌及其主要代谢产物的污染情况,随机选取6种市售果汁（共24份）进行调查。采用固相微萃取（SPME）和气相色谱（GC）-质谱（MS）技术检测果汁中愈创木酚和2,6-二溴苯酚的含量,并利用YSG培养基（p H=3.7）分离果汁中的脂环酸芽孢杆菌。在24份果汁样品中,2,6-二溴苯酚检出率为0%（0/24）;愈创木酚检出率为33.3%（8/24）,浓度最高可达26.9μg/L;脂环酸芽孢杆菌检出率为12.5%（3/24）,分离出的3株脂环酸芽孢杆菌均来自愈创木酚含量较高的橙汁样品;在未检出愈创木酚的16份果汁样品中均没有分离出脂环酸芽孢杆菌。在所检测的24份果汁样品中,部分橙汁、葡萄汁以及桃汁中检测到愈创木酚,但是只在橙汁样品中分离出了脂环酸芽孢杆菌,表明橙汁可能更容易在生产过程中被脂环酸芽孢杆菌及其代谢产物所污染。      

DEVELOPMENT OF A VOCABULARY FOR PROFILING APPLE JUICES

A protocol for selecting an apple juice lexicon and for training a company panel was developed, using a series of 21 ‘taste’ sessions. Individual assessment, round‐table discussion and consensus were used to identify reference standards for six aroma and 11 flavor attributes and 13 ‘off ‘notes. Duo‐trio tests were used to test the ability of the panelists to recognize the references and confirm their appropriateness in describing the attribute. When panelists' mean scores for the references were placed as anchors on the line scales, panel performance was improved. The development process not only served to train the panel, but was a ‘team’ building experience which benefited the company at large.     

Discrimination of Alicyclobacillus strains using nitrocellulose membrane filter and attenuated total reflectance fourier transform infrared spectroscopy

Alicyclobacillus spp. are thermoacidophilic, spore-forming bacteria, some of which cause spoilage in pasteurized and heat-treated apple juice products through the production of guaiacol. It would be helpful if a rapid method to detect and discriminate Alicyclobacillus strains was available. A simple and rapid sample preparation method using nitrocellulose membrane filter (NMF) and a single reflection horizontal attenuated total reflection (HATR) accessory with Fourier transform infrared (FT-IR) was developed here. Fourier transform infrared (FT-IR) spectroscopy was used and tested on 8 Alicyclobacillus strains (KF, WAC, NWN-13501, NWN-12697, NWN-12654, NWN-10682, 1016, 1101). A linear discriminant analysis (LDA) was established to discriminate Alicyclobacillus strains. The sample preparation method could successfully separated strains into different groups by principal component analysis (PCA). High identification accuracy (95%) was achieved with the LDA model. PRACTICAL APPLICATION: The method developed in the paper can be used to discriminate different Alicyclobacillus strains from each other making it possible to easily determine whether the strain of Alicyclobacillus present is associated with juice spoilage.     

Sensory Characteristics of Apple Juice Evaluated by Consumer and Trained Panels

ABSTRACT: To identify sensory characteristics of unclarified apple juice and to compare unclarified and clarified types, 140 consumers and 10 trained panelists evaluated 16 commercial apple juice samples (4 clarified and 12 unclarified). Unclarified and clarified juices were clearly separated by the first principal component (PC1), whose main factor was fresh, green, and sweet aroma. It was difficult to predict consumer preference by regression models using trained panel preference and analytical attributes. Mapping consumers' overall preferences on a sensory profile made by PC1 and PC2, the consumer panel preferred apple juice with moderately increased fresh and green aroma and thoroughly decreased sour and astringent.     

分光光度计法快速检测果汁中嗜热耐酸菌

酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris)是果汁污染的主要嗜酸耐热菌。它可以将香草酸或者香兰素转化为愈创木酚,后者在过氧化物酶的作用下产生红褐色产物,从而可以快速检测该菌。根据这个原理,设计实验探究该颜色反应的最优培养条件以及实际体系中检测该菌的可行性。结果表明：在静止培养情况下,在BAM培养基中添加100μg/kg的香草酸最有利于愈创木酚的产生;不同浓度的嗜热耐酸菌芽孢液在上述条件下培养,随着浓度的降低产生高浓度愈创木酚时间依次延迟,最低浓度为3个/mL的芽孢培养21h后,愈创木酚含量用分光光度计测定的光密度值OD470为1.0以上,为肉眼可见的红褐色;模拟实际体系下的培养也出现了颜色反应,证明该方法确实可以实现果汁中嗜热耐酸菌的快速、简便、高灵敏度的检测。     

塞威氏苹果酒的研制开发

塞威氏苹果(野苹果)是新疆伊犁地区稀有天然的野生苹果,营养丰富,以其为原料经挑选、清洗、榨汁后,添加酵母进行发酵,可酿制成果香、酒香协调,酸甜适口,醇和绵柔,具有浓郁的野苹果特有果香的野苹果酒.     

品种和后成熟对NFC苹果浊汁挥发性香气成分的影响

为了探讨果实品种和后成熟度对苹果浊汁风味的影响,采用顶空-固相微萃取结合气相色谱-质谱联用技术,基于多元数据分析手段,非靶向分析了富士、嘎啦、华硕、金冠、澳洲青苹等5个品种,在3个后熟度下制成的NFC苹果浊汁挥发性香气成分差异。结果表明:醇类（2-甲基-1-丁醇、1-己醇等）、醛类（己醛、（E）-2-己烯醛等）和酯类（2-甲基乙酸丁酯等）为苹果浊汁的主要挥发性香气成分。品种和后熟度对苹果汁的特征性挥发性香气成分的含量和组成影响显著。澳洲青苹果汁挥发性香气成分总量少且含量低,醛类是其主要的挥发性香气物质;富士、嘎啦和金冠果汁则表现出相似的香气组分特征,酯类、醛类和醇类物质含量丰富,具有浓郁的香甜水果气味和青草味;华硕苹果汁的挥发性香气成分以醇类和醛类为主,整体具有清淡的香味和生青味。另外,随着不断后熟,不同品种苹果浊汁挥发性香气成分的变化呈现不同趋势。澳洲青苹和富士苹果汁中的香气成分逐渐累积,分别有15和28种挥发性化合物含量显著增加;嘎啦和金冠苹果汁香气物质含量呈现先上升后下降的趋势,在第二个后熟阶段风味最好;用采摘初期华硕苹果原料制成的苹果汁香气最浓郁,但随着不断后熟,挥发性香气含量显著降低。本研究开展的苹果汁风味特性研究,可为苹果浊汁加工的原料选择提供理论基础。     

培养条件和果汁种类对脂环酸芽孢杆菌生长的影响

脂环酸芽孢杆菌是造成巴氏灭菌果汁腐败的重要细菌之一。鉴于目前脂环酸芽孢杆菌的培养方法尚未统一且污染特征不明确,本研究考察了pH、7种培养基和9种果汁对其生长的影响,同时采用固相微萃取(SPME)和气相色谱(GC)-质谱(MS)技术检测了该菌的主要代谢产物愈创木酚和2,6-二溴苯酚在果汁中的释放情况。结果表明,脂环酸芽孢杆菌在pH4.0时生长情况最好,pH2.5时生长较差,pH6.0~7.0时最差。AAM培养基和BAT培养基较其他5种培养基(YSG培养基、K氏培养基、SK培养基、PDB培养基和营养肉汤培养基)更适合用来培养脂环酸芽孢杆菌。脂环酸芽孢杆菌接种到9种果汁中后生长差异明显,在葡萄汁和橙汁中生长情况最好,细菌数量分别为1.5×10⁴ CFU/mL和8.5×10³ CFU/mL;在菠萝汁和西柚汁中生长缓慢,细菌数量分别为4.4×10² CFU/mL和1.9×10² CFU/mL。此外,9种果汁接种脂环酸芽孢杆菌后均未检测出2,6-二溴苯酚;但是,除葡萄汁外的8种果汁均检测出愈创木酚,浓度在8.6~23.9 μg/L之间。     

Taste and aroma of fresh and stored mandarins

During the last decade there has been a continuous rise in consumption of fresh easy‐to‐peel mandarins. However, mandarins are much more perishable than other citrus fruit, mainly due to rapid deterioration in sensory acceptability after harvest. In the current review we discuss the biochemical components involved in forming the unique flavor of mandarins, and how postharvest storage operations influence taste and aroma and consequently consumer sensory acceptability. What we perceive as mandarin flavor is actually the combination of basic taste, aroma and mouth‐feel. The taste of mandarins is principally governed by the levels of sugars and acids in the juice sacs and the relative ratios among them, whereas the aroma of mandarins is derived from a mixture of different aroma volatiles, including alcohols, aldehydes, ketones, terpenes/hydrocarbons and esters. During postharvest storage and marketing there is a gradual decrease in mandarin sensory acceptability, which has been attributed to decreases in acidity and typical mandarin flavor, paralleling an accumulation of off‐flavor. Biochemical analysis of volatile and non‐volatile constituents in mandarin juice demonstrated that these changes in sensory acceptability were concomitant with decreases in acidity and content of terpenes and aldehydes, which provide green, piney and citrus aroma on the one hand, and increases in ethanol fermentation metabolism products and esters on the other, which are likely to cause ‘overripe’ and off‐flavors. Overall, we demonstrate the vast importance of the genetic background, maturity stage at harvest, commercial postharvest operation treatments, including curing, degreening and waxing, and storage duration on mandarin sensory quality. Copyright © 2010 Society of Chemical Industry     

DETERMINATION AND PREDICTION OF ODOR THRESHOLDS FOR ODOR ACTIVE VOLATILES IN A NEUTRAL APPLE JUICE MATRIX

ABSTRACT

Odor thresholds were determined for 10 odor active compounds (OAC) in apple juice, using three‐alternate forced choice methodology. Thresholds were determined in a neutral juice matrix by 25–30 panelists in duplicate at 22C. Individual thresholds were calculated using the best estimate threshold method. Group thresholds were determined using the geometric mean of the individual thresholds. OAC differed substantially in their concentration ranges, aroma thresholds (0.06–5.49 µL/L) and response rates (1.5–234.5% correct response/[µL/L]). Juice thresholds exceeded water thresholds by ～5–600 times. Multiple linear regressions were used to develop models to predict juice thresholds from water thresholds and physical constants, for apple juice (AJ) and published orange juice (OJ) values. The simplest most practical models utilized just one variable, the logarithm of the water threshold. Coefficients of correlation (R²) for the AJ and OJ models were 71.7 and 72.8%, respectively, and provided satisfactory estimates of juice thresholds.

PRACTICAL APPLICATIONS

This research established aroma thresholds in a juice matrix for 10 prevalent esters in apples and related them to water thresholds using log models. These thresholds allow industry to calculate more realistic odor activity values for quality control and research purposes in the apple juice industry.     

FERMENTATION OF CASHEW (ANACARDIUM OCCIDENTALE L.) "APPLE" INTO WINE

Cashew apple is considered as a waste in cashew nut processing industry. An undistilled alcoholic beverage (wine) was prepared by fermenting cashew apple juice with wine yeast, Saccharomyces cerevisiae var. bayanus. The wine was slightly yellowish, acidic in taste (titratable acidity [1.21 ± 0.0 g tartaric acid/100 mL]), high in tannin (1.9 ± 0.22 mg/100 mL) and low in alcohol (7%) concentration. Although sensory evaluation rated the cashew apple wine as quite acceptable as an alcoholic beverage, significant differences (P < 0.01) exist between the cashew wine and the commercial grape wine particularly in taste, aroma, flavor and aftertaste because of probably high tannin content in the cashew wine.     

Isolation and characterization of thermo-acidophilic endospore-forming bacteria from the concentrated apple juice-processing environment

Forty-five thermo-acidophilic, spore-forming bacteria were isolated from a concentrated apple juice-processing environment. All of them were Gram-positive, rod shaped, and strictly aerobic that most likely belong to the genus of Alicyclobacillus. A fast identification method-16S rDNA PCR-RFLP was used to identify them. The results indicated that at the similarity level of 87%, apple juice isolates strains of 1-4 and 1-2-4 clustered with the reference strain of A. acidoterrstris DSM 3922T, and 4-2-1, S-22 and 5-1 with A. cycloheptanicus DSM 4006T, respectively. The other tested strains were different from all the reference strains in this study and may be new species of Alicyclobacillus genus or the other. In order to confirm this conclusion, we selected 7 16S rDNA PCR-RFLP identified strains and 5 type strains of Alicyclobacillus genus, carried on 51 kinds of phenotypic characteristics and analysis the data by unweighted pair group method with arithmetic mean (UPGMA). The results showed that the similarity degree between every two strains was lower than 80%. It also suggested that they may be different from each other and the unidentified strains may be new species. In addition, spoilage effects of them on 12 Brix apple juice were also studied. The result suggested that all 19 tested bacterial strains caused apple juice to become turbid, form a precipitate and off odor at varying rates when incubated at 37 degrees C up to 12 days. It suggested that these bacteria are associated with the spoilage of apple juice during storage.     

微波提取苹果汁中脂环酸芽孢杆菌DNA的PCR检测方法研究

根据Genbank公布的酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris DSM 3922T)鲨烯环化酶序列自行设计一对引物,利用微波技术直接从果汁样品中提取目标菌DNA,对引物特异性、微波法提取DNA的扩增效果及检测灵敏度进行探讨。结果表明：微波功率1000W、处理时间30s、经5000r/min离心2min即可获得酸土脂环酸芽孢杆菌基因组DNA,所得模板质量符合聚合酶链式反应检测要求,目的条带清晰,检测时间仅为2h,检测限为200CFU/mL,有望真正应用于苹果汁生产的在线检测。     

EFFECT OF ULTRASONIC TREATMENTS ON THERMOACIDOPHILIC ALICYCLOBACILLUS ACIDOTERRESTRIS IN APPLE JUICE

The effect of ultrasonic treatments on Alicyclobacillus acidoterrestris in apple juice was investigated. In general, inactivation of the cells was more pronounced at an elevated power level and as the processing time increased. Approximately 60% of the cells were inactivated after treating the apple juice with 300-W ultrasound for 30 min. The reduction reached more than 80% when the juice was processed for 60 min. The linear inactivation rates ( D values) of ultrasound on A. acidoterrestris were both process- and strain-dependent. The lowest D value at 36.18 min was found when using 600-W ultrasound to treat the A30 strain isolated from the air of a commercial apple juice processing plant, whereas the strain isolated from apple juice concentrate was found most resistant against ultrasound. Changes of sugar content, acidity, haze and juice browning were noted after ultrasonic treatments but did not adversely alter the juice quality.

PRACTICAL APPLICATIONS

Characterization of the effects of ultrasonic treatments on thermoacidophilic Alicyclobacillus acidoterrestris provides better understanding of how ultrasound technology could assist in alleviating the hard-to-detect spoilage caused by this spore former. Although ultrasonic technology might not be suitable as a standalone unit operation to inactivate high cell loads, the process could be integrated with other thermal or nonthermal technologies to enhance processing efficiencies, consequently preventing microbial spoilage caused by A. acidoterrestris .     

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.