Hydroxycinnamic acid decarboxylase activity of Brettanomyces bruxellensis involved in volatile phenol production: relationship with cell viability.

Brettanomyces bruxellensis populations have been correlated with an increase in phenolic off-flavors in wine. The volatile phenols causing the olfactory defect result from the successive decarboxylation and reduction of hydroxycinnamic acids that are normal components of red wines. The growth of B. bruxellensis is preventable by adding sulfur dioxide (SO(2)), with variable effectiveness. Moreover, it was hypothesized that SO(2) was responsible for the entry of B. bruxellensis into a viable but non-culturable (VBNC) state. The aim of this project was to investigate the effects of SO(2) on the remaining enzyme activities of B. bruxellensis populations according to their viability and cultivability, focusing on the hydroxycinnamate decarboxylase enzyme, the first enzyme needed, rather than the metabolites produced. Enzyme activity was determined both in cell-free extracts and resting cells after various SO(2) treatments in synthetic media. After slight sulfiting (around 50 mg/L total SO(2)), the yeasts had lost part of their enzyme activity but not their cultivability. At higher doses (at least 75 mg/L total SO(2)) the majority of yeasts had lost their cultivability but still retained part of their enzyme activity. These results suggested that non culturable cells retained some enzyme activity.

Variety and variability of glycosidase activities in an Oenococcus oeni strain collection tested with synthetic and natural substrates.

AIMS: To evaluate the capacity of Oenococcus oeni strains to release aroma compounds from glycosylated precursors by measuring glycosidase activities with both synthetic and natural substrates. METHODS AND RESULTS: Five glycosidase activities were investigated in 47 O. oeni strains using synthetic substrates. This screening revealed that activity levels vary considerably, not only for each strain (depending on the substrate tested), but also between strains. Fifteen strains exhibiting different activity profiles were further analysed using natural substrates extracted from both untoasted and toasted oak. In the latter, various amounts of aromatic compounds were measured, thus confirming the specific potentials of the selected strains, but the results were different from those obtained using synthetic substrates. In addition, the use of toasted wood extracts significantly increased the release of wood aromas, which minimized differences between strains. CONCLUSIONS: The capability of O. oeni to hydrolysate glycoconjugate aroma precursors is strain-dependent and variable, depending on the substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: Instead of synthetic substrates, natural aroma precursors should be used for an adequate evaluation of the glycosidase potential of O. oeni.

Biogenic amines in fermented foods.

Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.

Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider.

A collection of 810 lactic acid bacteria (LAB) strains isolated from wine and cider was screened for potential biogenic amine (BA) producers by combining molecular and phenotypic approaches. A newly developed multiplex PCR method allowed for the simultaneous detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via either ornithine decarboxylase, odc, or agmatine deiminase, agdi) while TLC and HPLC analysis allowed for BA-production determination. One hundred and fifty-eight LAB strains were monitored by the molecular/phenotypic double approach and revealed a good correlation between genotypic and phenotypic data. Eighteen per cent of the tested strains were positive for at least one BA target gene with up to three detected simultaneously, in particular amongst Lactobacillus brevis and Lactobacillus hilgardii isolates for the tyrdc and agdi genes. The most frequent gene corresponded to the agdi gene detected in 112 strains (14% of all LAB strains) of 10 different LAB species. The tyrdc gene was detected in 67 strains represented by 7 different LAB species (8% overall), especially those isolated from wine. Lower levels of hdc(+) (2% of strains) and especially odc(+) (0.5% of strains) strains were observed. Interestingly, species that have never been described to carry BA-producing pathway genes were identified in this study. Furthermore, only one cadaverine-producer was detected and corresponded to Lactobacillus 30a, a collection strain not found in fermented beverages, although cadaverine is commonly detected in wines.

Characterization of the yeast ecosystem in grape must and wine using real-time PCR.

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.

Brettanomyces bruxellensis evolution and volatile phenols production in red wines during storage in bottles.

AIMS: The presence of Brettanomyces bruxellensis is an important issue during winemaking because of its volatile phenols production capacities. The aim of this study is to provide information on the ability of residual B. bruxellensis populations to multiply and spoil finished wines during storage in bottles. METHODS AND RESULTS: Several finished wines were studied. Brettanomyces bruxellensis populations were monitored during two and a half months, and volatile phenols as well as chemical parameters regularly determined. Variable growth and volatile phenols synthesis capacities were evidenced, in particularly when cells are in a noncultivable state. In addition, the volatile phenol production was clearly shown to be a two-step procedure that could strongly be correlated to the physiological state of the yeast population. CONCLUSIONS: This study underlines the importance of minimizing B. bruxellensis populations at the end of wine ageing to reduce volatile phenols production risk once the wine in bottle. Moreover, the physiological state of the yeast seems to have an important impact on ethyl-phenols production, hence demonstrating the importance of taking into account this parameter when analysing wine spoilage risks. SIGNIFICANCE AND IMPACT OF THE STUDY: Little data exist about the survival of B. bruxellensis once the wine in bottle. This study provides information on the alteration risks encountered during wine storage in bottle and reveals the importance of carrying on further studies to increase the knowledge on B. bruxellensis physiology.

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non-ropy wines.

AIMS: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine. METHODS AND RESULTS: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with beta-glucan formation and ropiness were grown on grape juice medium, dialysed MRS-derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol-sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf-). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. CONCLUSION: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than beta-glucan. SIGNIFICANCE AND IMPACT OF THE STUDY: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.

Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production.

The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS-PCR, PCR-DGGE and REA-PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.

Purification of an alcohol dehydrogenase involved in the conversion of methional to methionol in Oenococcus oeni IOEB 8406.

Oenococcus oeni, the major lactic acid bacteria involved in malolactic fermentation (MLF) in wine, is able to produce volatile sulfur compounds from methionine. Methional reduction is the last enzymatic step of methionol synthesis in methionine catabolism. Alcohol dehydrogenase (ADH) activity was found to be present in the soluble fraction of O. oeni IOEB 8406. An NAD(P)H-dependent ADH involved in the reduction of methional was then purified to homogeneity. Sequencing of the purified enzyme and amino acid sequence comparison with the database revealed the presence of a conserved sequence motif specific to the medium-chain zinc-containing NAD(P)H-dependent ADHs. Despite the great importance of ADH activities in wine flavor modification, this is the first report of the purification of an ADH isolated from O. oeni. The purified ADH does not seem to be involved in the modification of buttery and lactic notes or to be involved in the specific formation of volatile alcohols during MLF. The enzyme was not strictly specific of methional reduction and the highest reducing activity was obtained with acetaldehyde as substrate. The function of the purified ADH remains unclear, although the role of the sulfur atom in methional molecules in the interaction between enzyme and substrate was evidenced.

In vitro anti-bacterial and anti-adherence effects of natural polyphenolic compounds on oral bacteria.

AIMS: To investigate the action of different polyphenolic compounds, extracted from red wine, grape marc and pine bark, on oral bacteria. METHODS AND RESULTS: The anti-microbial activity of extracts was examined by determining the Minimal Inhibitory Concentration and Minimal Bactericidal Concentration using the macro dilution broth technique. Their effect on the adherence was tested on growing cells of Streptococcus mutans on a glass surface and on a multi-species biofilm grown on saliva-coated hydroxyapatite discs. The effect on glucosyltransferase activity was analysed through the reductions in the overall reaction rate and the quantity of insoluble glucan (ISG) synthesized. Pine bark and grape marc extracts were the most effective inhibitors of the multi-species biofilm formation and of the ISG synthesis. CONCLUSION: The tested components showed an interesting anti-plaque activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is, to our knowledge, the first and the most complete report on the properties of wine and pine bark extracts that could be used for oral disease prevention purpose.

Effect of antiplaque compounds and mouthrinses on the activity of glucosyltransferases from Streptococcus sobrinus and insoluble glucan production.

INTRODUCTION: The development of therapeutic agents inhibiting the activity of glucosyltransferases (GTF) and their production of glucans is a potential strategy to reduce dental decay. The aim of this study was first to characterize a GTF preparation from Streptococcus sobrinus ATCC 33478 and then to evaluate the effects of select compounds and mouthrinses on insoluble glucan (ISG) formation by combined GTFs. METHODS: The purity of the crude GTF mixture was assessed by electrophoresis. The effects of pH, temperature, sucrose, and dextran T10 concentrations on GTF activity were analyzed and the chemical structure of the products was investigated. Finally, the inhibition of GTF by commercial mouthrinses used in oral hygiene and their active components (chlorhexidine, polyphenolic compounds, fluoride derivatives, polyols, cetylpyridinium chloride, and povidone iodine) was analyzed through the reductions in the overall reaction rate and the quantity of ISG synthesized. RESULTS: The S. sobrinus ATCC 33478 crude GTF preparation obtained contains a mixture of four different GTFs known for this species. For optimal adherent ISG formation, the reaction parameters were 37 degrees C, pH 6.5, sucrose 50 g/l, and dextran T10 2 g/l. Under these conditions, the most effective agents were chlorhexidine, cetylpyridinium chloride, and tannic acid. Eludril, Elmex, and Betadine were the most effective inhibitors of all the mouthrinses tested. CONCLUSION: As the formulation of commercial products considerably influences the efficiency of active components, the fast representative ISG inhibition test developed in this study should be of great interest.

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine.

AIMS: Brettanomyces/Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine. METHODS AND RESULTS: Sterile red wines were inoculated with 5 x 10(3) viable cells ml(-1) of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10(6)-10(7) colony forming units (CFU) ml(-1) and volatile phenol concentrations ranged from 500 to 4000 microg l(-1). Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C(2) to C(10)), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine. CONCLUSIONS: Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni.

AIMS: Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine. METHODS AND RESULTS: Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni. CONCLUSION: This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni. Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed. SIGNIFICANCE AND IMPACT OF THE STUDY: This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.

Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking.

Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 degrees C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods.

AIMS: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. METHODS AND RESULTS: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10(6) cells ml(-1)) during spontaneous malolactic fermentation. A minimum of 10(3) ml(-1) producing cells was required for production of more than 1 mg l(-1) of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. CONCLUSION: Methods using quantitative PCR are efficient to enumerate biogenic amines-producing cells in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.

Vanillin production from simple phenols by wine-associated lactic acid bacteria.

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level.

AIMS: In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. METHODS AND RESULTS: Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. CONCLUSIONS: Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.

In Vivo PCR-DGGE analysis of Lactobacillus plantarum and Oenococcus oeni populations in red wine.

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety "Primitivo di Puglia"), a polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30-60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking.

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.

Double-hadron leptoproduction in the nuclear medium.

The first measurements of double-hadron production in deep-inelastic scattering within the nuclear medium were made with the HERMES spectrometer at DESY HERA using a 27.6 GeV positron beam. By comparing data for deuterium, nitrogen, krypton, and xenon nuclei, the influence of the nuclear medium on the ratio of double-hadron to single-hadron yields was investigated. Nuclear effects on the additional hadron are clearly observed, but with little or no difference among nitrogen, krypton, or xenon, and with smaller magnitude than effects seen on previously measured single-hadron multiplicities. The data are compared with models based on partonic energy loss or prehadronic scattering and with a model based on a purely absorptive treatment of the final-state interactions. Thus, the double-hadron ratio provides an additional tool for studying modifications of hadronization in nuclear matter.