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Citric Acid Assay Kit

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00:04  Introduction
00:59  Principle
02:03  Reagent Preparation
02:54   Procedure
05:47   Calculations

Citric Acid Assay Kit K-CITR Scheme
Product code: K-CITR

72 assays (manual) / 720 assays (microplate) / 840 assays (auto-analyser)

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Content: 72 assays (manual) / 720 assays (microplate) / 840 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Citric Acid
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Decrease
Linear Range: 1.0 to 100 µg of citric acid per assay
Limit of Detection: 0.491 mg/L
Reaction Time (min): ~ 5 min
Application examples: Grape juice, wine, beer, fruit juices, soft drinks, tea, dairy products (e.g. cheese), meat, processed meat, vegetable and fruit products, bakery products, paper, pharmaceuticals, cosmetics and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by MEBAK, OIV, EU, ISO2963, AOAC and IFU22 (NOTE: If the enzyme oxaloacetate decarboxylase is present in the sample, some of the oxaloacetate product is converted to pyruvate. Therefore, to ensure citric acid is measured quantitatively, D-lactate dehydrogenase (D-LDH) is employed to efficiently convert any pyruvate produced into D-lactate and NAD+).

The Citric Acid test kit is a flexible and simple method for the rapid and reliable measurement and analysis of citric acid (citrate) in foods, beverages and other materials.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

View our full list of organic acid test kits.

Scheme-K-CITR CITR Megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Reconstituted citrate lyase stable for 4 weeks at 4oC / 6 months below -10o
  • Buffer / cofactor / enzyme tablets for efficient use of kit components 
  • PVP incorporated to prevent tannin inhibition 
  • Very competitive price (cost per test) 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats
Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol Data Calculator Validation Report
Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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Physicochemical changes during controlled laboratory fermentation of cocoa (CCN-51) with the inclusion of fruits and on-farm inoculation.

Peña González, M. A., Ortiz Urgiles, J. P., Santander Pérez, F. A., Lazo Vélez, M. A. & Caroca Cáceres, R. S. (2023). Brazilian Journal of Food Technology, 26, e2023013.

Fermentation is key to developing the organoleptic characteristics of cocoa beans, as dynamic changes in metabolites have a significant impact on flavors and aromas, hence modifications of this process have been investigated. In this research, the mucilage of CCN-51 cocoa beans was replaced by a mixture of passion fruit (Passiflora edulis) and plantain (Musa paradisiaca L.) pulp, and a controlled fermentation of this mixture was carried out after its spontaneous on-farm inoculation. The physicochemical changes and correlations during the five days of fermentation were evaluated. At the end of the process, the temperature reached 47 ºC in the fermentation mass and pH 5.64 was recorded in the cotyledon. In the first 48 hours, citric acid and fructose were high but at the end of fermentation were 71% and 41.17% lower than at the start of fermentation, respectively. As glucose and fructose were consumed during fermentation, acetic acid and lactic acid levels increased from day two onward, reaching values at the end of the process of 22.48 mg/g and 16.01 mg/g, respectively. In contrast, the bromatological parameters did not show greater variability when comparing each day of fermentation. The data generated and results presented in this study will contribute to the knowledge of possible sensory improvements achieved with the inclusion of pulp fruits in the fermentation stage.

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Simultaneous saccharification and citric acid production from paper wastewater pretreated banana pseudostem: Optimization of fermentation medium formulation and kinetic assessment.

Laltha, M., Sewsynker-Sukai, Y. & Kana, E. G. (2022). Bioresource Technology, 361, 127700.

This study optimized the simultaneous saccharification and citric acid (CA) production from banana pseudostem (BP). Thereafter, kinetic assessment of Aspergillus brasiliensis growth and CA production were determined for the optimum conditions using fresh water (SSFoptimizedFW) or dairy wastewater (SSFDWW) and compared to Sabouraud Dextrose Emmon’s medium modified with BP (SSFSDEmodified). The optimized conditions gave a CA concentration of 14.408 g/L. Kinetic assessment revealed the same maximum specific growth rates (μmax) (0.05 h−1) for all three bioprocesses, while the SSFSDEmodified process resulted in the highest maximum potential CA concentration (Pm) (13.991 g/L) in comparison to the SSFDWW (Pm = 13.095 g/L) and SSFoptimizedFW (Pm = 12.967 g/L) systems. Findings from this study facilitates the implementation of waste-based lignocellulosic bioprocesses that may eradicate the use of expensive pretreatment chemicals, fermentation medium constituents, and resources, in keeping with the water, energy and food nexus towards developing a circular bioeconomy.

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Classifying interactions in a synthetic bacterial community is hindered by inhibitory growth medium.

Dos Santos, A. R., Di Martino, R., Testa, S. E. & Mitri, S. (2022). Msystems, 7(5), e00239-22.

Predicting the fate of a microbial community and its member species relies on understanding the nature of their interactions. However, designing simple assays that distinguish between interaction types can be challenging. Here, we performed spent medium assays based on the predictions of a mathematical model to decipher the interactions among four bacterial species: Agrobacterium tumefaciens, Comamonas testosteroni, Microbacterium saperdae, and Ochrobactrum anthropi. While most experimental results matched model predictions, the behavior of C. testosteroni did not: its lag phase was reduced in the pure spent media of A. tumefaciens and M. saperdae but prolonged again when we replenished our growth medium. Further experiments showed that the growth medium actually delayed the growth of C. testosteroni, leading us to suspect that A. tumefaciens and M. saperdae could alleviate this inhibitory effect. There was, however, no evidence supporting such “cross-detoxification,” and instead, we identified metabolites secreted by A. tumefaciens and M. saperdae that were then consumed or “cross-fed” by C. testosteroni, shortening its lag phase. Our results highlight that even simple, defined growth media can have inhibitory effects on some species and that such negative effects need to be included in our models. Based on this, we present new guidelines to correctly distinguish between different interaction types such as cross-detoxification and cross-feeding.

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Molecular and Physiological Properties of Indigenous Strains of Oenococcus oeni Selected from Nero di Troia Wine (Apulia, Italy).

Cappello, M. S., Falco, V., Curcio, R., Mita, G. & Zapparoli, G. (2022). Microorganisms, 10(4), 795.

The characterization of Oenococcus oeni strains isolated from Nero di Troia wine (Apulia, Italy) sampled in two distinct production areas was carried out. The two indigenous populations, consisting of 95 and 97 isolates, displayed high genetic diversity when analyzed by amplified fragments length polymorphisms (AFLP). Based on the UPGMA dendrogram obtained by AFLP analysis, the two populations displayed similar genotypes that grouped in the same clusters with a high level of similarity (>95%). One genotype was found in only one of the two areas. Representative strains of each cluster were analyzed for their enzymatic activities (esterase, β-glucosidase, and protease), assayed in whole cells, and tested for their metabolic properties (consumption of L-malic acid, citric acid, acetaldehyde, and arginine) and growth parameters. Significant differences among strains, including the reference strain ATCC BAA-1163, were observed for all of these properties. Principal component analysis evidenced phenotypic differences among strains, and well separated some of them belonging to different genotypes. Strains exhibiting the best performances in most of these traits could be further investigated in order to select possible candidates as malolactic starters for Nero di Troia wine. This study provided insights on the population structure of O. oeni of a local winemaking area useful to the understanding of the regional diversity of this bacterium, an issue not yet completely resolved

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Type A and B bovine milks: Heat stability is driven by different physicochemical parameters.

Loveday, S. M., Weeks, M., Luo, D. & Cakebread, J. (2021). Journal of Dairy Science, 104(11), 11413-11421.

The value of milk hinges on its physicochemical functionality under processing conditions. We examined composition–functionality relationships with individual milks from 24 New Zealand dairy cows, sampled at 3 times over the season. Milks were classified into type A or B, according to the shape of 3-point heat coagulation time versus pH profiles. Milk type changed over the season for half of the cows in the study. Best subsets regression suggested that different factors controlled heat stability in the 2 milk types. Urea concentration was key for both types, but for type A milks, osmotic pressure and milk solids were the most important predictors of heat stability, whereas casein micelle size and ionic calcium predicted heat stability for type B milks. This study revealed that milk type is prone to change over the season, and the findings suggest that optimizing heat stability could be achieved by different means for type A versus type B milks.

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Above- and below-ground resource acquisition strategies determine plant species responses to nitrogen enrichment.

Zhang, D., Peng, Y., Li, F., Yang, G., Wang, J., Yu, J., Zhou, G. & Yang, Y. (2021). Annals of Botany, 20, 1-14.

Background and aims: Knowledge of plant resource acquisition strategies is crucial for understanding the mechanisms mediating the responses of ecosystems to external nitrogen (N) input. However, few studies have considered the joint effects of above-ground (light) and below-ground (nutrient) resource acquisition strategies in regulating plant species responses to N enrichment. Here, we quantified the effects of light and non-N nutrient acquisition capacities on species relative abundance in the case of extra N input. Methods: Based on an N-manipulation experiment in a Tibetan alpine steppe, we determined the responses of species relative abundances and light and nutrient acquisition capacities to N enrichment for two species with different resource acquisition strategies (the taller Stipa purpurea, which is colonized by arbuscular mycorrhizal fungi, and the shorter Carex stenophylloides, which has cluster roots). Structural equation models were developed to explore the relative effects of light and nutrient acquisition on species relative abundance along the N addition gradient. Key results: We found that the relative abundance of taller S. purpurea increased with the improved light acquisition along the N addition gradient. In contrast, the shorter C. stenophylloides, with cluster roots, excelled in acquiring phosphorus (P) so as to elevate its leaf P concentration under N enrichment by producing large amounts of carboxylate exudates that mobilized moderately labile and recalcitrant soil P forms. The increased leaf P concentration of C. stenophylloides enhanced its light use efficiency and promoted its relative abundance even in the shade of taller competitors. Conclusions: Our findings highlight that the combined effects of above-ground (light) and below-ground (nutrient) resources rather than light alone (the prevailing perspective) determine the responses of grassland community structure to N enrichment.

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Polylactose exhibits prebiotic activity and reduces adiposity and nonalcoholic fatty liver disease in rats fed a high-fat diet.

Abernathy, B. E., Schoenfuss, T. C., Bailey, A. S. & Gallaher, D. D. (2021). The Journal of Nutrition, 151(2), 352-360.

Background: Prebiotic dietary fibers change the intestinal microbiome favorably and provide a health benefit to the host. Objectives: Polylactose is a novel fiber, synthesized by extrusion of lactose. We evaluated its prebiotic activity by determining its fermentability, effect on the microbiota, and effects on adiposity and liver lipids in a diet-induced obesity animal model. Methods: Male Wistar rats (4-5 wk old) were fed normal-fat (NF, 25% fat energy) or high-fat (HF, 51% fat energy) diets containing different fibers (6% fiber of interest and 3% cellulose, by weight), including cellulose (NFC and HFC, negative and positive controls, respectively), polylactose (HFPL), lactose matched to residual lactose in the HFPL diet, and 2 established prebiotic fibers: polydextrose (HFPD) and fructooligosaccharide (HFFOS). After 10 wk of feeding, organs were harvested and cecal contents collected. Results: HFPL animals had greater cecum weight (3 times greater than HFC) and lower cecal pH (∼1 pH unit lower than HFC) than all other groups, suggesting that polylactose is more fermentable than other prebiotic fibers (HFPD, HFFOS; P < 0.05). HFPL animals also had increased taxonomic abundance of the probiotic species Bifidobacterium in the cecum relative to all other groups (P < 0.05). Epididymal fat pad weight was significantly decreased in the HFPL group (29% decrease compared with HFC) compared with all other HF groups (P < 0.05) and did not differ from the NFC group. Liver lipids and cholesterol were reduced in HFPL animals when compared with HFC animals (P < 0.05). Conclusions: Polylactose is a fermentable fiber that elicits a beneficial change in the gut microbiota as well as reducing adiposity in rats fed HF diets. These effects of polylactose were greater than those of 2 established prebiotics, fructooligosaccharide and polydextrose, suggesting that polylactose is a potent prebiotic.

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The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in Aspergillus carbonarius.

Barda, O., Maor, U., Sadhasivam, S., Bi, Y., Zakin, V., Prusky, D. & Sionov, E. (2020). Frontiers in Microbiology, 11, 210.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC esulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

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Deletion of Voltage-Dependent Anion Channel 1 knocks mitochondria down triggering metabolic rewiring in yeast.

Magri, A., Di Rosa, M. C., Orlandi, I., Guarino, F., Reina, S., Guarnaccia, M., Morello, G., Spampinato, A., Cavallaro, S., Messina, A., Vai, M. & De Pinto, V. (2020). Cellular and Molecular Life Sciences, 77(16), 3195-3213.

The Voltage-Dependent Anion-selective Channel (VDAC) is the pore-forming protein of mitochondrial outer membrane, allowing metabolites and ions exchanges. In Saccharomyces cerevisiae, inactivation of POR1, encoding VDAC1, produces defective growth in the presence of non-fermentable carbon source. Here, we characterized the whole-genome expression pattern of a VDAC1-null strain (Δpor1) by microarray analysis, discovering that the expression of mitochondrial genes was completely abolished, as consequence of the dramatic reduction of mtDNA. To overcome organelle dysfunction, Δpor1 cells do not activate the rescue signaling retrograde response, as ρ0 cells, and rather carry out complete metabolic rewiring. The TCA cycle works in a “branched” fashion, shunting intermediates towards mitochondrial pyruvate generation via malic enzyme, and the glycolysis-derived pyruvate is pushed towards cytosolic utilization by PDH bypass rather than the canonical mitochondrial uptake. Overall, Δpor1 cells enhance phospholipid biosynthesis, accumulate lipid droplets, increase vacuoles and cell size, overproduce and excrete inositol. Such unexpected re-arrangement of whole metabolism suggests a regulatory role of VDAC1 in cell bioenergetics.

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Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger.

Steiger, M. G., Rassinger, A., Mattanovich, D. & Sauer, M. (2019). Metabolic Engineering, 52, 224-231.

Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H+ antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109 g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain.

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Systems analysis of metabolism in platelet concentrates during storage in platelet additive solution.

Jóhannsson, F., Guðmundsson, S., Paglia, G., Guðmundsson, S., Palsson, B., Sigurjónsson, Ó. E. & Rolfsson, Ó. (2018). Biochemical Journal, BCJ20170921.

Platelets deteriorate over time when stored within blood banks through a biological process known as platelet storage lesion (PSL). Here we describe the refinement of biochemical network of platelet metabolism iAT-PLT-636 and its application to describe and investigate changes in metabolism during platelet storage. Changes to extracellular acetate and citrate were measured in buffy coat and apheresis platelet units over 10 days of storage in the platelet additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1-3), intermediate (days 4-6), and expired (days 7-10) platelets. Changes to metabolism was studied by comparing metabolic model flux predictions of iAT-PLT-636 between stages and between collection methods. Extracellular acetate and glucose contribute most to central carbon metabolism in platelets. The anticoagulant citrate is metabolized in apheresis stored platelets and is converted to aconitate and, to a lesser degree, malate. The consumption of nutrients changes during storage and reflects altered platelet activation profiles following their collection. Irrespective of collection method, a slowdown in oxidative phosphorylation takes place, consistent with mitochondrial dysfunction during PSL. Finally, the main contributors to intracellular ammonium and NADPH are highlighted. Future optimization of flux through these pathways provides opportunities to address intracellular pH changes and reactive oxygen species which are both of importance to PSL. The metabolic models provide descriptions of platelet metabolism at steady state and represent a platform for future platelet metabolic research.

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An accurate description of Aspergillus niger organic acid batch fermentation through dynamic metabolic modelling.

Upton, D. J., McQueen-Mason, S. J. & Wood, A. J. (2017). Biotechnology for Biofuels, 10(1), 258.

Background: Aspergillus niger fermentation has provided the chief source of industrial citric acid for over 50 years. Traditional strain development of this organism was achieved through random mutagenesis, but advances in genomics have enabled the development of genome-scale metabolic modelling that can be used to make predictive improvements in fermentation performance. The parent citric acid-producing strain of A. niger, ATCC 1015, has been described previously by a genome-scale metabolic model that encapsulates its response to ambient pH. Here, we report the development of a novel double optimisation modelling approach that generates time-dependent citric acid fermentation using dynamic flux balance analysis. Results: The output from this model shows a good match with empirical fermentation data. Our studies suggest that citric acid production commences upon a switch to phosphate-limited growth and this is validated by fitting to empirical data, which confirms the diauxic growth behaviour and the role of phosphate storage as polyphosphate. Conclusions: The calibrated time-course model reflects observed metabolic events and generates reliable in silico data for industrially relevant fermentative time series, and for the behaviour of engineered strains suggesting that our approach can be used as a powerful tool for predictive metabolic engineering.

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Compromised Lactobacillus helveticus starter activity in the presence of facultative heterofermentative Lactobacillus casei DPC6987 results in atypical eye formation in Swiss-type cheese.

O’Sullivan, D. J., McSweeney, P. L. H., Cotter, P. D., Giblin, L. & Sheehan, J. J. (2016). Journal of dairy science, 99(4), 2625-2640.

Nonstarter lactic acid bacteria are commonly implicated in undesirable gas formation in several varieties, including Cheddar, Dutch-, and Swiss-type cheeses, primarily due to their ability to ferment a wide variety of substrates. This effect can be magnified due to factors that detrimentally affect the composition or activity of starter bacteria, resulting in the presence of greater than normal amounts of fermentable carbohydrates and citrate. The objective of this study was to determine the potential for a facultatively heterofermentative Lactobacillus (Lactobacillus casei DPC6987) isolated from a cheese plant environment to promote gas defects in the event of compromised starter activity. A Swiss-type cheese was manufactured, at pilot scale and in triplicate, containing a typical starter culture (Streptococcus thermophilus and Lactobacillus helveticus) together with propionic acid bacteria. Lactobacillus helveticus populations were omitted in certain vats to mimic starter failure. Lactobacillus casei DPC6987 was added to each experimental vat at 4 log cfu/g. Cheese compositional analysis and X-ray computed tomography revealed that the failure of starter bacteria, in this case L. helveticus, coupled with the presence of a faculatively heterofermentative Lactobacillus (L. casei) led to excessive eye formation during ripening. The availability of excess amounts of lactose, galactose, and citrate during the initial ripening stages likely provided the heterofermentative L. casei with sufficient substrates for gas formation. The accrual of these fermentable substrates was notable in cheeses lacking the L. helveticus starter population. The results of this study are commercially relevant, as they demonstrate the importance of viability of starter populations and the control of specific nonstarter lactic acid bacteria to ensure appropriate eye formation in Swiss-type cheese.

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Safety Information
Symbol : GHS07
Signal Word : Warning
Hazard Statements : H302, H319, H412
Precautionary Statements : P264, P270, P273, P280, P301+P312, P305+P351+P338, P330, P337+P313, P501
Safety Data Sheet
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