D-Amino Acids in Milk as Related to Heat Treatments and Bacterial Activity

The occurrence in milk of free and bound D-amino acids was investigated by chiral phase GC and related to heat treatments and bacterial activity. Significant amounts of free D-alanine (D-Ala), D-aspartic acid (D-Asp), and D-glutamic acid (D-Glu) have been found in raw cow milk but not in human milk. The amount did not increase by pasteurization, UHT treatment, and sterilization. In contrast, the content of D-Ala in raw milk samples increased during cold storage at 4°C, and it was suggested that D-Ala may be considered as an indicator of bacterial milk contamination. The presence of D-Ala, D-Asp and D-Glu detected in milk protein hydrolysates was due to hydrolysis conditions.     

Free D- and L-Amino Acid Evolution During Sourdough Fermentation and Baking

The evolution of free D- and L-amino acids in sourdoughs started with various lactic acid bacteria (LAB) and yeasts was studied. Lactobacillus brevis subsp. lindneri CB1 and Lactobacillus plantrum DC400 had high proteolytic activity. During sourdough fermentation, Saccharomyces cerevisiae 141 and Saccharomyces exiguus M14 sequentially utilized free amino acids produced by bacterial activity. Due to increased cell yeast autolysis, more S. exiguus M14 inocula caused more free amino acids which were partially utilized by LAB without causing hydrolysis of wheat flour protein. D-alanine, D-glutamic acid and traces of other D-isomers were observed in sourdoughs fermented with L. brevis subsp. lindneri CB1 and S. cerevisiae 141. Free total D- and L-amino acid content decreased by more than 44% after baking the sourdoughs. No abiotic generation of new D-amino acid isomers was detected in the baked sourdoughs.     

Limonin and Naringin Removal from Grapefruit Juice with Naringinase Entrapped in Cellulose Triacetate Fibers

Naringin and limonin are two bitter components of some citrus products such as grapefruit juice. Naringin could be removed by hydrolysis with immobilized naringinase and limonin might be removed by adsorption with cellulose monoacetate gel beads. Cellulose triacetate fibers show a similar limonin adsorption capacity as cellulose monoacetate gel beads. Thus when naringinase from Penicillium sp. was entrapped in such fibers, an enzyme column was made which could remove both bitter components simultaneously. When grapefruit juice was debittered with this enzyme column, sugar components, total organic acids and turbidity were not affected. The enzyme column could be regenerated by washing with warm water. In addition, operational stability of the enzyme column was satisfactory. Such enzyme column could be considered for industrial use.     

Present knowledge of the bacterial microflora in the extreme environment of sugar thick juice

The diversity of the bacterial population in sugar thick juice, an intermediate product in the production of beet sugar, which exhibits an extreme, osmophilic environment with a water activity value (a_w) less than 0.86, was assessed with both culture-dependent and -independent 16S ribosomal RNA (rRNA) gene-based analyses. In comparison with previous studies, the number of different thick juice bacterial species increased from 29 to 72. Remarkably, a limited, gram-positive, culturable flora, encompassing species of Bacillus, Staphylococcus and mainly Tetragenococcus dominated thick juice during storage, while a more heterogeneous and unculturable fraction of Acinetobacter, Sporolactobacillus and Thermus species could be detected in freshly produced thick juice. Notably, almost all bacteria detected in the thick juice were also detected in the air, emphasising the importance of further investigation and assessment of strategies to reduce (air) contamination during processing and storage. The discovery of the contamination source may be used for the development of management strategies for thick juice degradation resulting from microbial activity.     

Development of a DNA Array for the Simultaneous Detection and Identification of Sugar Thick Juice Bacterial Contaminants

Despite the use of generally accepted good storage practices, sugar thick juice degradation caused by microbiological contamination occasionally occurs, causing considerable financial loss. In this study, a DNA array was developed for simultaneous detection and identification of the most prominent microflora present during thick juice storage, which may cause degradation of the thick juice. Specific oligonucleotides were developed for several bacterial taxa, including the genera Bacillus, Kocuria, Staphylococcus and Tetragenococcus and the species Aerococcus viridans, Leuconostoc mesenteroides and Tetragenococcus halophilus. The DNA array was validated using both pure cultures and industrial samples. In addition, comparisons were made between the developed array, PCR assays specifically targeting the thick juice contaminants and classical microbial platings. The array was found to be reliable and sensitive enough to detect and identify the target bacteria. In addition, the array was used to monitor the target microbial populations in thick juice during long-term storage and degradation. Results are discussed in relation to DNA stability in thick juice.     

Reduction of bitterness and acidity in grapefruit juice by adsorptive processes

By choosing an appropriate adsorbent, the bitter principles (naringin and limonin) and titratable acids could be removed from grapefruit juice in varying combinations. For example, Amberlite XAD-7 removed about 63% naringin, 85% limonin and 3% titratable acids and Deacidite-FFIP removed about 20% naringin, 8% limonin and 23% titratable acids when contacted for 1 h with grapefruit juice at the rate of 20 g (dry wt) of adsorbent per litre of juice. Such treatments, applied consecutively, would eliminate excessive bitterness and acidity from juices containing up to 2 g naringin and 36 mg limonin litre^?1 and with a total soluble solids:titratable acid ratio of < 6. The adsorbents are readily available, may be used as supplied and can be reactivated simply and economically; some of them have already been approved for use with foods, and such approval seems possible for the others.     

Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii

A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii. The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, while DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C. boidinii. We also showed that the three C-terminal amino acid sequence of DAO, -AKL was necessary and sufficient for the import of DAO into peroxisomes.     

Decontamination of Inoculated Beef with Sequential Spraying Treatments

Spray-washing/rinsing treatments utilizing warm/hot water and/or acetic acid solution were evaluated separately and in sequence for their efficacy in reducing microbial contamination on beef tissue inoculated with Escherichia coli. Treatments reduced the aerobic plate (APC) and E. coli counts of the samples inoculated to have 5.0-7.4 log CFU/cm² (APC) by 1.1 to 4.3 logs. Similarly, most treatments reduced APC and total coliform counts of samples inoculated to have 1.8-3.7 log CFU/cm² (APC) by 0.1 to 1.7 logs. Combinations involving 3 or 4 treatments were more effective in reducing bacterial contamination than single- or 2-treatment combinations.     

A review of limonin in grapefruit (Citrus paradisi) juice,its relationship to flavour,and efforts to reduce it

Ever since limonin was first discovered in grapefruit (Citrus paradisi Macf) juice in 1965 and found responsible for significantly contributing to bitterness in the juice, a great deal of research has been directed towards understanding its role therein. Information is presented relative to limonin's chemical determination, taste threshold and content in grapefruit juice, and to relevant legislation. Also considered are (1) several factors affecting the limonin content of grapefruit juice (2) the effect of limonin content on sensory flavour in grapefruit juice (3) the interaction between the Brix: acid ratio and limonin content in affecting grapefruit juice flavour and (4) methods for reducing limonin in citrus juices generally. Finally, recommendations for future studies on limonin in grapefruit juice are reviewed.     

Moulds and yeasts in fruit salads and fruit juices

Thirty-eight fruit salad samples including cantaloupe, citrus fruits, honeydew, pineapple, cut strawberries and mixed fruit salads, and 65 pasteurized fruit juice samples (apple, carrot, grapefruit, grape and orange juices, apple cider, and soy milk) were purchased from local supermarkets in the Washington, DC area and tested for fungal contamination. The majority of fruit salad samples (97%) were contaminated with yeasts at levels ranging from <2.0 to 9.72 log10 of colony forming units per gram (cfu/g). Frequently encountered yeasts were Pichia spp., Candida pulcherrima, C. lambica, C. sake, Rhodotorula spp., and Debaryomyces polymorphus. Low numbers of Penicillium spp. were found in pineapple salads, whereas Cladosporium spp. were present in mixed fruit and cut strawberry salads. Twenty-two per cent of the fruit juice samples tested showed fungal contamination. Yeasts were the predominant contaminants ranging from <1.0 to 6.83 log10 cfu/ml. Yeasts commonly found in fruit juices were C. lambica, C. sake, and Rhodotorula rubra. Geotrichum spp. and low numbers of Penicillium and Fusarium spp. (1.70 and 1.60 log10 cfu/ml, respectively) were present in grapefruit juice.     

The potential of NIR spectroscopy for the detection of the adulteration of orange juice

Near-infrared spectroscopy was used to investigate the adulteration of 65 authentic concentrated orange juice samples obtained from Brazil and Israel. These samples were adulterated with 100 g kg^?1 additions (ie 100 g added to 900 g) of (1) orange pulpwash, (2) grapefruit juice, and (3) a synthetic sugar/acid mixture and with 50 g kg^?1 additions (ie 50 g added to 950 g) of (4) orange pulpwash, and (5) grapefruit juice. All samples were scanned on the NIR systems 6500 spectrophotometer over the 1100-2498 nm wavelength range. Principal component analysis was used to reduce each spectrum to 20 principal components. Factorial discriminant analysis was used to distinguish between the different sample groups. Using orange juice and orange juice adulterated at the 100 g kg^?1 level, accurate classification rates of 94–95% were obtained. To classify samples adulterated at the 50 g kg^?1 level, the calibration development sample set had to be augmented by the inclusion of samples adulterated at this lower level—after this augmentation, an accurate classification rate of 94% was obtained. The results demonstrated that the application of principal component and factorial discriminate analysis to NIR reflectance spectra can detect the adulteration of orange juice with an average accuracy of 90%. Furthermore, not one adulterated sample was predicted as being an authentic orange juice throughout the entire test regime.     

Alicyclobacillus acidoterrestris: an increasing threat to the fruit juice industry?

The quality of food can be defined in various ways with processors and consumers considering flavour, odour and appearance as well as extended shelf life among the most important of its attributes. Spoilage of food by bacterial contamination may occur at any point during the processing, altering any one, or all, of these characteristics, rendering the product unusable. Alicyclobacillus spp. and Alicyclobacillus acidoterrestris, in particular, are emerging food spoilage organisms in the fruit juice and fruit juice products industry. Spores of the latter are able to survive heat treatments presently used in the industry and their elimination from products may be used as a measure of the effectiveness of any processing protocol to remove potential spoilage. This paper reviews the history, methods of detection, both traditional and rapid and the protocols that may be effective in controlling the growth of the organism and hence spoilage in the finished product.     

Volatile flavour components of grapefruit juice (Citrus paradisi Macfadyen)

The qualitative analysis of volatile flavour components in grapefruit juice (Citrus paradisi Macfadyen) was performed using a gas chromatography/mass spectro-metry/computer system which allowed the identification of 58 components, 25 of them being reported for the first time. The aroma concentrates were obtained by an efficient simultaneous steam distillation/solvent extraction method which made it possible to identify many trace aroma components. Only quantitative differences were found in the aroma composition of fresh grapefruit juice extracted from different fruit batches of the same geographic origin during one season.     

Influence of Pulsed Electric Field and Thermal Treatments on the Quality of Blueberry Juice

Fresh blueberries were selected as trial materials. Two blueberry juice samples were sterilized using pulsed electric field and thermal treatments respectively. Their qualities were analyzed and compared by evaluating the microorganisms in the samples. The changes in the quality of fresh blueberry juice samples during storage were also assessed. The results showed that the inactivation of Escherichia coli increased as electric field strength and treatment time increased. Pulsed electric field treatments at 35 kV/cm and 82 μs reduced E. coli by 5.12 logarithms. Pulsed electric field had less effect on juice quality, but after heat treatment, the vitamin C of blueberry juice sample was reduced by 14.78%, whereas its anthocyanin content was reduced by 3.64%. After sterilization, no significant change was observed in the relative reducing sugar, total acid, phenol contents, and Brix value. The vitamin C and anthocyanins contents of the fresh blueberry juice samples treated with pulsed electric field were higher than those treated with heat sterilization.     

Effect of Thermal Treatment and Storage Conditions on the Physical and Sensory Properties of Grapefruit Juice

Physical parameters, such as particle size distribution, flow behavior, density, turbidity, and color, were measured and sensory evaluation was carried out to compare the properties of freshly squeezed grapefruit juice with those of juice that has been pasteurized by microwave or by following a conventional heating method. Samples were either frozen-stored or refrigerated. In general, the physical parameters of grapefruit juice were significantly affected by heat treatment, especially in the case of the conventional process. However, from a sensory point of view, pasteurized samples were similar to fresh ones. When frozen, turbidity, particle size distribution, density, flow behavior, and color were stable throughout the studied period, regardless of the pasteurization treatment. During refrigerated storage, the turbidity, particle size distribution, and consistency index decrease. This occurs in a more pronounced way in the case of juice which has not been submitted to a heating treatment, probably due to residual pectin methyl esterase activity. Furthermore, the association between the carboxyl groups of pectin chains and Ca²⁺ could be responsible for both the subsequent increase in the turbidity of the juice and also the decrease in its density. Throughout the period under study, the smallest color change was experienced by microwave-pasteurized juice. For these reasons, and also due to the reduction in the process time, microwave treatment can be recommended as a method for the pasteurization of grapefruit juice.     

D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity

D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.     

Amino acid racemases: functions and mechanisms

L-Amino acids are predominant in living organisms, but D-amino acids such as D-alanine and D-glutamate also occur in all eubacterial cell walls. Moreover, even mammals contain endogenous D-amino acids: D-serine functions as a signaling molecule in mammalian brains, and D-aspartate acts as a mediator in endocrine systems. Various other D-amino acids have been demonstrated in archaea, yeasts, fungi, plants, insects, mollusks and other eucaryotic organisms. These D-amino acids are mostly endogenous and produced in most cases by racemization from their corresponding antipodes by the action of racemases. Therefore, amino acid racemases play a central role in D-amino acid metabolism. Most amino acid racemases require pyridoxal 5'-phosphate (PLP) as a coenzyme, but several others require no coenzymes. Recently, the structures and functions of these two classes of amino acid racemases were clarified on a molecular basis. We here describe recent advances in studies of the functions and mechanisms of PLP-dependent and -independent amino acid racemases.     

Phenolic acids, flavonoids, vitamin C and antioxidant capacity of strawberry juices processed by high-intensity pulsed electric fields or heat treatments

The effect of high-intensity pulsed electric fields (HIPEF) processing (35 kV/cm for 1,700 μs in bipolar 4-μs pulses at 100 Hz) on individual phenolic compounds (phenolic acids and flavonoids), vitamin C and antioxidant capacity of strawberry juice was evaluated and compared to heat (90 °C for 60 or 30 s) and fresh juice as a reference. Although strawberry juice underwent a substantial depletion of health-related compounds with storage time irrespective of the treatment conducted, ellagic acid was enhanced. HIPEF-treated strawberry juice maintained higher amounts of phenolic acids (ellagic and p-coumaric acid) and total anthocyanins than the thermally treated juices during the storage period. Regarding the antioxidant capacity, similar DPPH and ABTS values were obtained so that differences among pasteurized juices were non significant. HIPEF processing may be a technology as effective as thermal treatments not only to achieve safe and stable juices, but also to obtain juices with a high content of antioxidant compounds.     

Effect of thermal treatment and storage on the stability of organic acids and the functional value of grapefruit juice

The effect of conventional and microwave pasteurisation on the main bioactive compounds of grapefruit juice and their stability during 2 months’ refrigerated and frozen storage was evaluated. Ascorbic acid (AA), vitamin C and organic acids were analysed by HPLC, whereas total phenols and antioxidant capacity (%DPPH) were measured by spectrophotometry. The results showed that conventional treatment led to a significant decrease in citric acid (from 1538 to 1478 mg/100 g) and AA (from 36 to 34.3 mg/100 g), whilst microwave pasteurisation preserved these compounds. Frozen storage maintained AA and vitamin C, especially in treated samples. Frozen non-treated samples and conventional pasteurised ones preserved about a 75% and 20% of the total phenols and antioxidant capacity, respectively, whilst in frozen microwave pasteurised juices this preservation was of 82% and 33%. From these results, the use of microwave energy may be proposed as an alternative to traditional heat pasteurisation in order to preserve the natural organoleptic characteristics and essential thermolabile nutrients of grapefruit juice.