R. Di Cagno

Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses

ABSTRACT The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg−1. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.

Latent Bioactive Peptides in Milk Proteins: Proteolytic Activation and Significance in Dairy Processing

Referee: Mr. Dick Fitzgerald, Ph.D., Life Science Department, University of Limerick, Limerick, Ireland After a brief description of the properties of bioactive peptides, the proteolytic activation of the bioactive sequences from milk protein precursors is discussed. The ability of proteolytic enzymes from various sources, especially from lactic acid bacteria, to release bioactive peptides and the physiological and biotechnological significance of these peptides in dairy products are reviewed.

Functional Microorganisms for Functional Food Quality

Functional microorganisms and health benefits represent a binomial with great potential for fermented functional foods. The health benefits of fermented functional foods are expressed either directly through the interactions of ingested live microorganisms with the host (probiotic effect) or indirectly as the result of the ingestion of microbial metabolites synthesized during fermentation (biogenic effect). Since the importance of high viability for probiotic effect, two major options are currently pursued for improving it—to enhance bacterial stress response and to use alternative products for incorporating probiotics (e.g., ice cream, cheeses, cereals, fruit juices, vegetables, and soy beans). Further, it seems that quorum sensing signal molecules released by probiotics may interact with human epithelial cells from intestine thus modulating several physiological functions. Under optimal processing conditions, functional microorganisms contribute to food functionality through their enzyme portfolio and the release of metabolites. Overproduction of free amino acids and vitamins are two classical examples. Besides, bioactive compounds (e.g., peptides, γ-amino butyric acid, and conjugated linoleic acid) may be released during food processing above the physiological threshold and they may exert various in vivo health benefits. Functional microorganisms are even more used in novel strategies for decreasing phenomenon of food intolerance (e.g., gluten intolerance) and allergy. By a critical approach, this review will aim at showing the potential of functional microorganisms for the quality of functional foods.

Functional milk beverage fortified with phenolic compounds extracted from olive vegetation water, and fermented with functional lactic acid bacteria

Functional milk beverages (FMB100 and FMB200) fortified with phenolic compounds (100 and 200mg/l) extracted from olive vegetable water, and fermented with γ-amino butyric acid (GABA)-producing (Lactobacillus plantarum C48) and autochthonous human gastro-intestinal (Lactobacillus paracasei 15N) lactic acid bacteria were manufactured. A milk beverage (MB), without addition of phenolic compounds, was used as the control. Except for a longer latency phase of FMB200, the three beverages showed an almost similar kinetic of acidification, consumption of lactose and synthesis of lactic acid. Apart from the beverage, Lb. plantarum C48 showed a decrease of ca. Log 2.52-2.24 cfu/ml during storage. The cell density of functional Lb. paracasei 15N remained always above the value of Log 8.0 cfu/ml. During fermentation, the total concentration of free amino acids markedly increased without significant (P > 0.05) differences between beverages. The concentration of GABA increased during fermentation and further storage (63.0 ± 0.6-67.0 ± 2.1mg/l) without significant (P > 0.05) differences between beverages. After fermentation, FMB100 and FMB200 showed the same phenolic composition of the phenol extract from olive vegetable water but a different ratio between 3,4-DHPEA and 3,4-DHPEA-EDA. During storage, the concentrations of 3,4-DHPEA-EDA, p-HPEA and verbascoside of both FMB100 and FMB200 decreased. Only the concentration of 3,4-DHPEA increased. As shown by SPME-GC-MS analysis, diactetyl, acetoin and, especially, acetaldehyde were the main volatile compounds found. The concentration of phenolic compounds does not interfere with the volatile composition. Sensory analyses based on triangle and paired comparison tests showed that phenolic compounds at the concentrations of 100 or 200mg/l were suitable for addition to functional milk beverages.

Interactions between sourdough lactic acid bacteria and exogenous enzymes: effects on the microbial kinetics of acidification and dough textural properties

The interactions between lactic acid bacteria and exogenous enzymes were studied to optimize the effects on the microbial kinetics of acidification, acetic acid production and textural properties during sourdough fermentation. Eleven species of lactic acid bacteria were used alone or in association with microbial glucose-oxidase, lipase, endo-xylanase, α-amylase or protease to produce sourdoughs. Data from the microbial kinetics of acidification were elaborated by the Gompertz equation and textural properties of the sourdoughs were analysed by using a Brabender extensograph, a Brabender farinograph and a Chopin alveograph. Only Leuconostoc citreum 23B, Lactococcus lactis subsp. lactis 11M and Lactobacillus hilgardii 51B were positively influenced by the enzymes added. Lactic acidification was increased and accelerated. All the enzymes affected the acidification activity of Lb. hilgardii 51B, including their addition in mixture. The addition of lipase, endo-xylanase and α-amylase increased the production of acetic acid by Lb. hilgardii 51B. Textural analyses pointed out that sourdoughs started with Lb. hilgardii 51B and individual enzymes were characterized by higher stability and softening compared to doughs with enzymes added alone.

Metabolic responses of Lactobacillus plantarum strains during fermentation and storage of vegetable and fruit juices.

ABSTRACT Strains of Lactobacillus plantarum were grown and stored in cherry (ChJ), pineapple (PJ), carrot (CJ), and tomato (TJ) juices to mimic the chemical composition of the respective matrices. Wheat flour hydrolysate (WFH), whey milk (W), and MRS broth were also used as representatives of other ecosystems. The growth rates and cell densities of L. plantarum strains during fermentation (24 h at 30°C) and storage (21 days at 4°C) differed only in part, being mainly influenced by the matrix. ChJ and PJ were the most stressful juices for growth and survival. Overall, the growth in juices was negatively correlated with the initial concentration of malic acid and carbohydrates. The consumption of malic acid was noticeable for all juices, but mainly during fermentation and storage of ChJ. Decreases of branched-chain amino acids (BCAA)—with the concomitant increase of their respective branched alcohols—and His and increases of Glu and gamma-aminobutyric acid (GABA) were the main traits of the catabolism of free amino acids (FAA), which were mainly evident under less acidic conditions (CJ and TJ). The increase of Tyr was found only during storage of ChJ. Some aldehydes (e.g., 3-methyl-butanal) were reduced to the corresponding alcohols (e.g., 3-methyl-1-butanol). After both fermentation and storage, acetic acid increased in all fermented juices, which implied the activation of the acetate kinase route. Diacetyl was the ketone found at the highest level, and butyric acid increased in almost all fermented juices. Data were processed through multidimensional statistical analyses. Except for CJ, the juices (mainly ChJ) seemed to induce specific metabolic traits, which differed in part among the strains. This study provided more in-depth knowledge on the metabolic mechanisms of growth and maintenance of L. plantarum in vegetable and fruit habitats, which also provided helpful information to select the most suitable starters for fermentation of targeted matrices.

Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits.

The diversity of 72 isolates of Lactobacillus plantarum, previously identified from different raw vegetables and fruits, was studied based on phenotypic (Biolog System) and genotypic (randomly amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) approaches. A marked phenotypic and genotypic variability was found. Eight clusters were formed at the similarity level of 92% based on Biolog System analysis. The most numerous clusters grouped isolates apart from the original habitat. Almost all isolates fermented maltose, D,L-lactic acid, N-acetyl-D-mannosamine and dextrin, and other typical carbon sources which are prevalent in raw vegetables and fruits. None of the isolates fermented lactose and free amino acids. At high values of linkage distance, two main clusters were obtained from both UPGMA (unweighted pair group with arithmetic average) dendrograms of RAPD-PCR and AFLP analyses. The two clusters mainly separated isolates from tomatoes and carrots from those isolated from pineapples. At 2.5 linkage distance, a high polymorphism was found and several sub-clusters were formed with both analyses. In particular, AFLP allowed the differentiation of 55 of the 72 isolates of L. plantarum. The discriminatory power of each technique used was calculated through the Simpsons index of diversity (D). The values of the D index were 0.65, 0.92 and 0.99 for Biolog System, RAPD-PCR and AFLP analyses, respectively.

Genotypic and phenotypic diversity of Lactobacillus rossiae strains isolated from sourdough

Aim: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae.

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45 d did not differ from those of the control Caciocavallo Pugliese cheese at 60 d of ripening. In contrast, the cheese made with AC at 45 d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.

Use of microparticulated whey protein concentrate, exopolysaccharide-producing Streptococcus thermophilus, and adjunct cultures for making low-fat Italian Caciotta-type cheese.

Low-fat Caciotta-type cheeses were manufactured with partially skim milk (fat content of ~0.3%) alone (LFC); with the supplementation of 0.5% (wt/vol) microparticulated whey protein concentrate (MWPC) (LFC-MWPC); with MWPC and exopolysaccharides (EPS)-producing Streptococcus thermophilus ST446 (LFC-MWPC-EPS); and with MWPC, EPS-producing strain ST446, and Lactobacillus plantarum LP and Lactobacillus rhamnosus LRA as adjunct cultures (LFC-MWPC-EPS-A). The non-EPS-producing isogenic variant Streptococcus thermophilus ST042 was used for making full-fat Caciotta-type cheese (FFC), LFC, and LFC-MWPC. Cheeses were characterized based on compositional, microbiological, biochemical, texture, volatile components (purge and trap, and solid-phase microextraction coupled with gas chromatography-mass spectrometry), and sensory analyses. Compared with FFC and LFC (51.6 ± 0.7 to 53.0 ± 0.9%), the other cheese variants retained higher levels of moisture (60.5 ± 1.1 to 67.5 ± 0.5%). The MWPC mainly contributed to moisture retention. Overall, all LFC had approximately one-fourth (22.6 ± 0.8%) of the fat of FFC. Hardness of cheeses slightly varied over 7d of ripening. Microbial EPS positively affected cheese texture, and the texture of LFC without MWPC or microbial EPS was excessively firm. Free amino acids were at the highest levels in LFC treatments (2,705.8 ± 122 to 3,070.4 ± 123 mg/kg) due to the addition of MWPC and the peptidase activity of adjunct cultures. Aldehydes, alcohols, ketones, sulfur compounds, and short- to medium-chain carboxylic acids differentiated LFC variants and FFC. The sensory attributes pleasant to taste, intensity of flavor, overall acceptability, and pleasant to chew variously described LFC-MWPC-EPS and LFC-MWPC-EPS-A. Based on the technology options used, low-fat Caciotta-type cheese (especially ripened for 14 d) has promising features to be further exploited as a suitable alternative to the full-fat variant.