Biogenic amine content and proteolysis in Manchego cheese manufactured with Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

Two different autochthonous strain starter cultures, in which the acidifying starter was composed of strains of Lactococcus lactis, were used for the manufacture of pasteurised milk Manchego cheese. Proteolysis parameters, biogenic amines and sensory characteristics were evaluated and compared with those of commercial starter Manchego cheese and raw milk Manchego cheese manufactured without starter. Autochthonous starter cheeses, and especially those including Lactobacillus paracasei subsp. paracasei as adjunct, presented higher levels of proteolysis than in commercial starter cheese. The concentrations of total biogenic amines in autochthonous starter cheeses were much lower than in raw milk cheese and even lower than in commercial starter cheese. Cheese manufactured with the adjunct strain gave the best results for both flavour intensity and flavour quality, and was the most preferred by panellists. The results suggest that the culture containing Lb. paracasei subsp. paracasei as adjunct could be used for the manufacture of industrial Manchego cheese.     

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions.     

Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese

Thirty-three Lactobacillus strains were tested as adjuncts in a cheese model system. Eighteen strains originated from cheese (nine Lactobacillus spp. and nine Lb. paracasei/casei) and 15 from human intestinal mucosa (11 Lb. rhamnosus; three Lb. paracasei; one Lb. plantarum). Model cheeses weighing 120 g were made of cheese grains from full-scale production of washed curd semi-hard cheese (Herrg?rd). The model system was reproducible and similar to full-scale production with respect to moisture, salt content, pH and microbial flora. The model cheeses were sampled for aerobic and anaerobic plate count and viable counts of Lactobacillus and Lactococcus. The presence of adjuncts in the model cheeses was confirmed by typing isolates with Randomly Amplified Polymorphic DNA (RAPD). The sensory properties of model cheeses were described. In a first trial 23 of the 33 adjuncts were re-isolated from the corresponding model cheeses after 9 or 13 weeks. Adjuncts of Lb. paracasei were re-isolated more frequently than adjuncts of Lb. rhamnosus. Nine strains were selected, on the basis of their ability to grow and be a dominating part of the microflora of model cheese with interesting sensory properties. These strains were further studied together with two commercial cultures. The sensory influences on model cheeses of six of the adjuncts were confirmed, and flavour scores were in the range of 2.9-7.1 for model cheeses with different adjuncts while the control had a flavour score of 5.6 (0-10 scale). Survival and growth of seven out of the nine strains correlated with the results of the first trial. Growth and influence on flavour of four adjunct cultures were confirmed in experimental cheese manufactured in a 400-1 open vat.     

Use of wild Lactobacillus strains in an adjunct culture for a Roncal-type cheese

The effect of an added adjunct culture consisting of facultatively heterofermentative lactobacilli (FHL) on the volatile compounds and sensory characteristics of a Spanish ewes'-milk cheese was examined. Three cheese batches were prepared using a commercial starter, one from raw milk, another from pasteurized milk, and a third from pasteurized milk with an added culture of wild Lactobacillus. paracasei+Lb. plantarum. Analysis of the volatile compounds was carried out by the purge and trap method and gas chromatography with a mass spectrometer and disclosed a total of 86 compounds belonging to the chemical families hydrocarbons, fatty acids, esters, ketones, aldehydes, and alcohols. After ageing for 120 and 240 days, the cheese samples underwent sensory analysis by a panel of expert assessors. The attributes evaluated were characteristic odour and odour intensity and characteristic aroma and aroma intensity. Pasteurization of the milk had an effect on the formation of certain volatile compounds, adversely affecting the characteristic flavour of the cheese. Use of the adjunct culture in addition to the commercial starter improved the flavour of the cheese made from the pasteurized milk, which earned sensory scores similar to those awarded to the cheese made from the raw milk. Use of adjunct cultures consisting of indigenous FHL strains could help to conserve the traditional characteristics of Roncal cheese made from pasteurized milk, although some technical adjustments to the Regulations would be needed.     

The design of a three strain starter system for Cheddar cheese manufacture exploiting bacteriocin-induced starter lysis

A three strain starter system was developed for increasing the level of starter cell lysis during cheese manufacture and ripening. The composition of this starter combination includes a bacteriocin (lactococcin A, B and M) producer which causes the lysis of a second strain (sensitive to bacteriocin activity) during cheese manufacture, and a third strain resistant to bacteriocin activity. The latter strain plays an essential role in ensuring acid production during cheese manufacture. Cheeses manufactured at pilot-scale (450 L vats), with the three strain starter combination were assessed for levels of the intracellular enzyme, lactate dehydrogenase, released into the cheese matrix during ripening. Experimental cheeses, manufactured with the bacteriocin-producing adjunct, exhibited higher levels of free amino acids and greater release of intracellular LDH than control cheeses manufactured in its absence. Cheese was subject to sensory analysis which revealed that experimental cheese showed a decrease in bitterness over cheeses manufactured without the bacteriocin-producing adjunct. Thus, this three strain system offers manufacturers a reliable starter system exhibiting increased lysis with concomitant improvements in cheese flavour.     

Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter

Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter.     

Aroma development in semi-hard reduced-fat cheese inoculated with Lactobacillus paracasei strains with different aminotransferase profiles

Reduced-fat, semi-hard round-eyed cheese was made from bovine milk with a mesophilic DL-starter and three Lactobacillus paracasei subsp. paracasei single-strain adjuncts with different aminotransferase (AT) activity profiles. The pilot-plant procedure was not influenced by the adjuncts, and similar cheese was made from all vats. The growth of each adjunct to dominate the cheese was confirmed by using DNA fingerprinting. Flavour profiles were different for cheeses made with the different Lactobacillus strains. Use of the adjunct CHCC 4256 significantly increased the content of flavour compounds that were produced from the branched-chain amino acids (BcAAs: Leu, Ile and Val) and Asp. These cheeses also had superior sensory characteristics as they tasted aromatic and sweet without bitterness. The adjunct CHCC 4256 did not have glutamate dehydrogenase activity, or the highest activity on BcAA, but showed typical AT activity, with a similar activity on Leu, Phe and Asp substrates.     

Influence of a defined-strain starter and Lactobacillus plantarum as adjunct culture on volatile compounds and sensory characteristics of Manchego cheese

Three batches of Manchego cheese were manufactured using one of the following starter culture systems: (1) a defined strain starter culture comprising Lactococcus lactis subsp. lactis and Leuconostoc mesenteroides subsp. dextranicum; (2) the above-defined strain starter culture and an adjunct culture (Lactobacillus plantarum), all these strains being isolated from high-quality Manchego cheeses and (3) a commercial starter consisting of two strains of Lactococcus lactis. Differences in volatile profile and the sensory characteristics of these cheeses were studied. After 4 months of ripening, the two batches of cheese made with the defined strain starter cultures obtained the highest scores for sensory attributes and for the overall impression. Additionally, Purge & Trap and SDE analysis showed a more complex volatile profile in these cheeses than in those made with the commercial starter. Extending the maturation time to 8 months for cheeses made with the defined starter cultures led to significant higher levels of free fatty acids and ethyl esters in those cheeses made without adjunct culture. However, panelists did not find significant differences among the sensory characteristics of the two cheeses.     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

Autolysis of Lactococcus lactis

During cheese making, autolysis of Lactococcus lactis starter bacteria affects cheese flavour development through release of intracellular enzymes. The gene for the major autolysin in L. lactis, N-acetyl muramidase (AcmA), has been cloned and sequenced. The activity of AcmA alone, however, does not explain the huge variation in the extent of autolysis found in commercial L. lactis starter strains. Many such strains have multiple cell wall hydrolases that can be seen as different sized clearance bands in zymograms. In addition, the recently completed L. lactis subsp. lactis IL1403 genome sequence shows the presence of several open reading frames that putatively encode cell wall hydrolases having up to 42% predicted amino acid identity to AcmA. These enzymes could have roles in the autolysis of L. lactis. In this paper, we review the literature on autolysis of L. lactis and provide experimental evidence, based on Western blot and zymogram analysis, that commercial L. lactis starter strains express varying levels of AcmA and contain other cell wall hydrolases.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Enhancement of 2-methylbutanal formation in cheese by using a fluorescently tagged Lacticin 3147 producing Lactococcus lactis strain

The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese. In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese. The Lacticin 3147 producing strain Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from Streptococcus pneumoniae, and used as starter in cheese manufacture. Starter adjuncts were the bacteriocin-sensitive strains L. lactis T1 and L. lactis IFPL730, showing branched chain amino acid aminotransferase and alpha-keto acid decarboxylase activity, respectively. Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese. The non-bacteriocin-producing strain L. lactis IFPL359-GFP was included as starter in the control batch. Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy. The bacteriocin produced by L. lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal. This led to an enhancement of the cheese aroma detected by a sensory panel. The improvement of cheese flavour and aroma may be of significant importance for the dairy industry.     

Volatile composition and improvement of the aroma of industrial Manchego cheese by using Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

The use of several autochthonous strains of lactic acid bacteria, including Lactobacillus paracasei subsp. paracasei as adjunct of the starter in the manufacture of Manchego cheese, was evaluated in an attempt to improve the aroma of the industrial Manchego cheese. Volatile composition and odour characteristics were evaluated and compared to those in Manchego cheese manufactured with a commercial starter (CS) culture and with raw milk cheese manufactured without starter. Manchego cheeses manufactured with two autochthonous strains of Lactococcus lactis subsp. lactis displayed a similar volatile profile and odour characteristics to the cheese made with the CS. The use of the strain Lactobacillus paracasei subsp. paracasei CECT 7882 as adjunct of the Lactococcus strains produced cheeses with higher amounts of some free fatty acids and alcohols, acetoin, lactones, phenylacetaldehyde, 2-phenylethanol and linalool, and higher scores of the odour intensity, odour quality, and ewe’s milk odour than the CS cheeses. It resulted in an intensification and improvement of industrial Manchego cheese aroma.     

Contribution of lactic acid bacteria to flavour compound formation in dairy products

Cheese flavour cannot be produced without starter bacteria. Lactic acid bacteria convert lactose to lactic acid and this together with their production of diacetyl and acetaldehyde are their main contributions to the flavour of cultured milks and fresh cheeses. In matured cheeses, the starter bacteria die out quickly and the rate at which they lyse and release their enzymes into the system has an influence on the rate at which free amino acids are formed. Rennet alone is mainly responsible for the formation of large, medium and small peptides but, without interaction with other enzymes, is capable of producing only methionine, histidine, glycine, serine and glutamic acid at quantifiable levels. Free amino acids in Cheddar cheese are mainly the result of microbial peptidase activity. These amino acids, together with the products of glycolysis, form substrates for secondary flora, the nature of which, in many cases, determines the cheese variety. They also form substrates for enzymes from the milk, e.g. the production of H₂S appears to be dependent on milk enzymes. Methionine, which is released by rennet, is further metabolized by starter enzymes with the production of methanethiol which plays a major role in cheese flavour possibly as a potentiator for other flavours. —Dicarbonyls, particularly methylglyoxal and diacetyl, and bacteria which can produce them, appear to play a crucial role in the formation of cheese flavour, both the desirable flavour of full-fat cheese and the meaty-brothy off-flavour of low-fat cheese. Although, theoretically, there are many compounds in cheese which could react purely chemically to form flavour compounds, these reactions are also mediated by enzymes in the cheese system and it seems unlikely that straight out chemical reactions play a major role in the production of cheese flavour. The role of the secondary flora is likely to be much more important than that of chemical reactions. Particularly in Cheddar and Emmental it has been shown that good quality cheeses have a low oxidation-reduction potential. This is more likely to be an indicator for the establishment of the anaerobic conditions required for the flavour forming reactions to proceed than an active causal agent of flavour formation. The function of glutathione is more likely to be as some sort of facilitator in enzyme reactions than as an agent for the reduction of oxidation-reduction potential. The ability of bacteria to accumulate glutathione from their media is likely to be one of the indicators of flavour generating capacity. Suitable selected strains of adjunct bacteria increase the rate and intensity of formation of Cheddar cheese flavour but unsuitable adjuncts can also cause off-flavours.     

Sensory properties of low fat cheddar cheese:effect of salt content and adjunct culture

The influence of salt concentration and of direct-to-vat culture adjuncts (DVCA) on the sensory properties of low fat hard cheese of the cheddar type has been investigated. Salt content has a major influence, and, at a level of 1·8%, low fat cheese with high levels of cheddar flavour and low bitter scores was produced. In addition, further enhancement of flavour was achieved by use of DVCA. Using the methodology described in this report, low fat cheese was manufactured which corresponded in flavour level and balance to that of commercial mature full fat cheese. Nevertheless, textural differences between low fat and full fat product remain to be resolved.     

Influence of adjuncts as a debittering aids in encountering the bitterness developed in cheese slurry during accelerated ripening

An attempt was made to accelerate the flavour development in cheese base with the help of exogenous proteolytic and lipolytic enzymes (1:1 proportion, each at the rate of 0.025% by weight of cheese‐base) and ripening at elevated temperatures (i.e. 20 ± 1 °C) for up to 12 days. To counter the bitterness developed, adjunct cultures were used: viable or attenuated (freeze‐shocked or heat shocked). Study of biochemical characteristics, electrophoretic pattern and sensory evaluation of the product were carried out. An acceptable enzyme‐modified, lightly salted cheese base was obtained using 0.025% each of proteolytic and lipolytic enzymes, along with 5% starter culture and adjuncts followed by ripening up to 12 days. Freeze‐shocked adjunct Lactobacillus helveticus produced enzyme‐modified cheese base with no detectable bitterness. The usage of exogenous enzymes, temperature of ripening, ripening period and interactions amongst these parameters had significant (P < 0.01) influence on all of the biochemical characteristics monitored.     

Effect of a hygienized rennet paste and a defined strain starter on proteolysis,texture and sensory properties of semi-hard goat cheese

A hygienized rennet paste (HRP) and a defined strain starter culture, including Lactobacillus casei subsp. casei IFPL as adjunct, were considered for manufacturing Majorero cheese, a Spanish traditional variety made from goat milk. Influence of both factors on physicochemical characteristics, proteolysis, rheological and sensory properties, was evaluated throughout the ripening. Cheeses produced either industrially (IL) or in artisanal manner (AL) were compared with the experimental lot (EL), which included HRP and IFPL starter in its manufacture. Results showed a low level of primary proteolysis, expressed by a low content of non-casein nitrogen (NCN), in experimental cheeses. Despite the slightly poor texture (hard and crumbly) related to the high TS and salt contents, a good general acceptability was attained for EL, with the best scores for aroma and flavour intensities achieved at 30 ripening days. In fact, the sensory panel detected the “piquant” flavour (typical of the artisanal cheese variety) in EL after 15 days of ripening.