Eric Beuvier

Characterization of the lactic acid bacteria in artisanal dairy products

The European Union is thanked for partly financing this project under ECLAIR contract CT-91-0064.

Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese

Abstract Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are: • a partial elimination of the milk microorganisms which may grow in cheese during ripening, • a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization., • a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria). From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae , enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of α s1 - and β -caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.

Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltered milk

Abstract Experimental mini-cheeses were made from raw (Ra), microfiltered (MF), pasteurized (Pa) (72 °C, 30s) or pasteurized mixed with microfiltration retentate (PR) milk to study the influence of the indigenous microflora and pasteurization on the quality of Swiss-type cheese. To estimate biochemical transformations during cheese ripening, several methods were used: nitrogen fractionation (water-soluble fraction and phosphotungstic acid (PTA)-soluble fraction), urea-polyacrylamide gel electrophoresis of caseins, reverse phase liquid chromatography of the water-soluble fraction, lactate and volatile fatty acids. Microbial populations were also enumerated. At the end of ripening, in comparison with MF and Pa milk cheeses, Ra and PR milk cheeses exhibited higher overall aroma intensity and pungency, characteristics which correlated with higher populations of facultatively heterofermentative lactobacilli (108 cfug−1), propionibacteria (108 cfug−1), and enterococci (106cfug−1). These cheeses had high levels of PTA-soluble N and acetic, propionic and isovaleric acids. MF and Pa milk cheeses, although somewhat different from one another, were very different from the two other types of cheese. Pa milk cheese had a lower pH than MF milk cheese, and contained a higher proportion of γ-caseins due to the activation of plasmin. Moreover, Pa milk cheese was more acidic, but demonstrated a higher overall aroma intensity. The addition of raw milk flora (retentate) to Pa milk restored almost all the biochemical and sensory characteristics of Ra milk cheese measured in this study.

Origin and diversity of mesophilic lactobacilli in Comté cheese, as revealed by PCR with repetitive and species-specific primers

The objectives of this work were to describe the diversity of mesophilic lactobacilli in Comte cheese at the strain and species levels, to determine the origin(s) of this non-starter microflora, and to get a collection of well characterised strains from Comte cheeses. Strains were isolated from milks, starter cultures and eight cheeses from two factories, with four cheeses made from the same vat in each factory. Strain and species assignations were performed with a combination of two PCR-based methods, amplification with the pairs of repetitive primers ERIC1/ERIC2 and REP–1R-Dt/REP2-D, and amplification with specific primers for Lactobacillus zeae, Lactobacillus paracasei and Lactobacillus rhamnosus. The reliability and reproducibility of these methods were assessed using 49 collection strains of mesophilic lactobacilli commonly detected in cheeses. A total of 488 isolates of mesophilic lactobacilli was collected and was assigned to 44 different strains and three different species. Lactobacillus paracasei and Lactobacillus rhamnosus were the predominant species in milks, starter cultures and cheeses, and constituted 98.7% of the isolates. Strain diversity was found at both individual cheese and factory levels. Thirteen and fifteen different strains were detected throughout cheesemaking and ripening in two individual cheeses made in different factories; only 11 different strains were detected in the two corresponding mature cheeses. The data strongly suggest that most mesophilic lactobacilli strains originate from raw milk.

Use of PCR-based methods and PFGE for typing and monitoring homofermentative lactobacilli during Comté cheese ripening

This study investigated the genotypic characteristics of selected and wild homofermentative thermophilic lactobacilli strains during ripening of Comté cheeses, made into two cheese plants. Both amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) and classical biochemical tests were used to identify isolates. Diversity within homofermentative lactobacilli was not found in their species composition since the same two species Lactobacillus helveticus and L. delbrueckii susbp. lactis were isolated from cheeses. In cheeses made with natural whey starter, it appeared that the most likely sources of L. helveticus and L. delbrueckii susbp. lactis were the starter and raw milk, respectively. The examination of RAPD profiles of lactobacilli strains revealed 19 RAPD groups among 50 isolates, which were different from selected starter strains. Using RAPD, REP-PCR, and PFGE to identify selected starter strains during cheese ripening, we showed that L. helveticus decreased quickly while L. delbrueckii susbp. lactis sustained high viability during ripening. The use of selected L. delbrueckii susbp. lactis strains diminished the genetic diversity among strains isolated from cheese, probably in preventing the raw milk microflora from growing.

Milk acidification by Lactococcus lactis is improved by decreasing the level of dissolved oxygen rather than decreasing redox potential in the milk prior to inoculation

Although redox potential is very rarely taken into account in food fermentation it could be as influential as pH on bacterial activities. Lactococcus lactis is already known to exhibit a powerful reducing activity in milk but its reduction activity was shown to occur prior to its acidification activity with a potential interaction between these two lactococcal activities. Therefore, acidification lag-type phase could be shortened by decreasing the redox potential of milk before inoculation. As the redox potential is highly dependent on the dissolved oxygen level, our objective was to study their separate and combined influences on acidification and growth kinetics of pure L. lactis strains in milk. Results showed that high level of dissolved oxygen is significantly more influential on growth, and even more on acidification kinetics, than initial decreased redox potential of milk. Reduction of milk was drastic and mostly due to bacterial activity. The redox potential of milk only dropped when dissolved oxygen was entirely consumed. When there was no dissolved oxygen from the beginning, L. lactis immediately decreased the redox potential of milk and acidified afterwards. When the level of dissolved oxygen was initially high, acidification and reduction of milk occurred at the same time. Acidification kinetics was then biphasic with a slower rate during the aerobic stage and a faster rate during the anaerobic stage. The seven strains tested demonstrated diversity in both their acidification kinetics and their adaptation to high level of dissolved oxygen, independent of their growth kinetics. To conclude, we have shown that the level of dissolved oxygen in milk has a dramatic influence on acidification kinetics and could be used to control acidification kinetics in dairy industries.

The Microbiology of Traditional Hard and Semihard Cooked Mountain Cheeses.

Traditional cheeses originate from complex systems that confer on them specific sensory characteristics. These characteristics are linked to various factors of biodiversity such as animal feed, the use of raw milk and its indigenous microflora, the cheese technology, and the ripening conditions, all in conjunction with the knowledge of the cheesemaker and affineur. In Europe, particularly in France, the preservation of traditional cheesemaking processes, some of which have protected designation of origin, is vital for the farming and food industry in certain regions. Among these cheeses, some are made in the Alps or Jura Mountains, including Comté, Beaufort, Abondance, and Emmental, which are made from raw milk. The principle of hard or semihard cooked cheese, produced in the Alps and Jura Mountains, was to make a product during the summer-a period during which the animals feed more and milk production is high-with a shelf life of several months that could be consumed in winter. Today, these traditional cheeses are produced according to a specific approach combining science and tradition in order to better understand and preserve the elements that contribute to the distinctiveness of these cheeses. To address this complex problem, a global approach to the role of the raw milk microflora in the final quality of cheeses was initially chosen. The modifications resulting from the elimination of the raw milk microflora, either by pasteurization or by microfiltration, to the biochemistry of the ripening process and ultimately the sensory quality of the cheeses were evaluated. This approach was achieved mainly with experimental hard cooked cheeses. Other types of traditional cheese made with raw and pasteurized milk are also considered when necessary. Besides the native raw milk microflora, traditional lactic starters (natural or wild starters) also participate in the development of the characteristics of traditional hard and semihard cooked mountain cheeses. After an initial description, their roles are described, mainly for Comté.

Genetic parameters for cheese-making properties and milk composition predicted from mid-infrared spectra in a large data set of Montbéliarde cows

Cheese-making properties of pressed cooked cheeses (PCC) and soft cheeses (SC) were predicted from mid-infrared (MIR) spectra. The traits that were best predicted by MIR spectra (as determined by comparison with reference measurements) were 3 measures of laboratory cheese yield, 5 coagulation traits, and 1 acidification trait for PCC (initial pH; pH0PPC). Coefficients of determination of these traits ranged between 0.54 and 0.89. These 9 traits as well as milk composition traits (fatty acid, protein, mineral, lactose, and citrate content) were then predicted from 1,100,238 MIR spectra from 126,873 primiparous Montbéliarde cows. Using this data set, we estimated the corresponding genetic parameters of these traits by REML procedures. A univariate or bivariate repeatability animal model was used that included the fixed effects of herd × test day × spectrometer, stage of lactation, and year × month of calving as well as the random additive genetic, permanent environmental, and residual effects. Heritability estimates varied between 0.37 and 0.48 for the 9 cheese-making property traits analyzed. Coagulation traits were the ones with the highest heritability (0.42 to 0.48), whereas cheese yields and pH0 PPC had the lowest heritability (0.37 to 0.39). Strong favorable genetic correlations, with absolute values between 0.64 and 0.97, were found between different measures of cheese yield, between coagulation traits, between cheese yields and coagulation traits, and between coagulation traits measured for PCC and SC. In contrast, the genetic correlations between milk pH0 PPC and CY or coagulation traits were weak (-0.08 to 0.09). The genetic relationships between cheese-making property traits and milk composition were moderate to high. In particular, high levels of proteins, fatty acids, Ca, P, and Mg in milk were associated with better cheese yields and improved coagulation. Proteins in milk were strongly genetically correlated with coagulation traits and, to a lesser extent, with cheese yields, whereas fatty acids in milk were more genetically correlated with cheese yields than with coagulation traits. This study, carried out on a large scale in Montbéliarde cows, shows that MIR predictions of cheese yields and milk coagulation properties are sufficiently accurate to be used for genetic analyses. Cheese-making traits, as predicted from MIR spectra, are moderately heritable and could be integrated into breeding objectives without additional phenotyping cost, thus creating an opportunity for efficient improvement via selection.

A simple micro-batch ion-exchange resin extraction method coupled with reverse-phase HPLC (MBRE-HPLC) to quantify lactoferrin in raw and heat-treated bovine milk

Lactoferrin is an iron-binding cationic glycoprotein (pI = 8.7) beneficial for mammal health, especially udder and milk preservation. A new simple two-step method of quantification was developed. Lactoferrin in 1 mL of bovine skim milk was first adsorbed onto 100 mg of macroporous sulfonated-resin at pH 6.8 by rotary stirring for 90 min at 20-25 °C. After washing the resin, lactoferrin was desorbed using 1 mL of 2 M NaCl containing phenylalanine as a dilution marker, then fully resolved and quantified by RP-HPLC at 220 nm using a wide-bore C4 silica column. This robust, inexpensive and flexible method improves selectivity (no protein interference) and sensitivity compared to previous HPLC methods. In-laboratory validation demonstrated its linearity (25 to 514 µg Lf mL-1), accuracy (110 to 98% recovery), and precision (<4%), which were comparable to immuno-based methods. The results for individual raw cows milk were strongly correlated with results using an ELISA test.