Screening of aroma-producing performance of anticlostridial Lacticaseibacillus casei strains for use as adjunct cultures in cheesemaking

The cheesemaking industry is increasingly interested in using adjunct cultures with potential aromatic and anticlostridial activities. In this study, 34 Lb. paracasei and 2 Lb. rhamnosus strains were isolated from a semi-hard cheese and characterized for their proteolytic, esterase, and anticlostridial activity. Moreover, the strains were inoculated in a curd-based medium and the volatile compounds in the headspace of samples were evaluated by SPME-GC-MS analysis. Proteolytic activity was present in 30 strains, whereas only one Lb. paracasei strain showed esterase activity. All strains inhibited Cl. sporogenes, Cl. beijerinckii, and Cl. butyricum, and 18 isolates inhibited at least one Cl. tyrobutyricum strain. Principal component analysis and clustering analysis based on the volatilome grouped strains into three groups. One of these groups was characterized by high amounts of acids and esters and clustered with control samples inoculated with commercial starter cultures, suggesting similarity in the aroma pro�le. Strains belonging to this group with inhibitory effects against Cl. tyrobutyricum might be exploited as autochthonous adjunct cultures for the reduction of late-blowing defects in semi-hard cheeses.


Introduction
The volatile pro le is generally recognized as one of the most relevant aspects for evaluating cheese quality and its connection with the area of production.Microorganisms play an important role in developing cheese avor through the production of proteolytic, lipolytic, and amino acidmetabolizing enzymes, which convert milk constituents into numerous volatile compounds (Afshari et al. 2020;Pogačić et al. 2016).There are several reasons to explore the potential of microorganisms in the production of aroma compounds.Firstly, new strains are always needed to modify or intensify the avor pro le of industrial cheese (Law 2001).In addition, autochthonous starter cultures can also be selected to retain the unique avor of traditional dairy products (Innocente et al. 2016(Innocente et al. , 2023).Therefore, the cheese industry is increasingly interested in using adjunct cultures for the production of cheeses with speci c aromatic pro les (El Soda et al. 2000).The ability of microorganisms to generate volatile compounds is a strain-speci c characteristic (Alemayehu et al. 2014;Poveda et al. 2014).
Adjunct cultures can moreover counteract microbial pathogens as well as microorganisms responsible for unwanted abnormal fermentations or able to decarboxylate amino acids and produce biogenic amines (Innocente et al. 2009; Renes et al. 2014).To be an effective adjunct culture, the microorganisms should maintain a high cell density during the ripening process and positively contribute to the overall quality of the cheese (Irlinger et al. 2017).To ensure adaptation to the technological process and characteristics of the food matrix, adjunct cultures made by a single or a group of microbial strains can be directly isolated and selected from the same type of cheese (Gobbetti et al. 2015).
The Lacticaseibacillus casei group, which includes Lb. casei, Lb. paracasei, and Lb.rhamnosus, is of great relevance since all these species have been frequently found in different semi-hard and hard cheeses at late stages of ripening, suggesting the a nity of these microorganisms to the NSLAB (Non Starter Lactic Acid Bacteria) group (Bottari et al. 2018; Gatti et al. 2014).The growth of Lb. casei-group species in cheese is strongly associated with several technological factors, including curd cooking conditions, physicochemical properties of the curd and cheese, the presence of other microbial species, and the ripening time (Bottari et al. 2018; Gobbetti et al. 2015).Normally, these species are part of the micro ora of ripened products since they survive the technological stresses of the cheesemaking process and use alternative energy sources rather than lactose, determining strong adaptability in the cheese matrix.The Lb. casei-group is involved in essential biochemical activities, including the development of the characteristic volatile pro le and texture of the product during cheese ripening (Carafa et al. 2019;Wang et al. 2021).Many LAB (Lactic Acid Bacteria), including Lb. casei-group, also exert antimicrobial activity against pathogenic and spoilage microorganisms, thus helping to increase safety and reduce waste (Mani-López et al. 2022; Morandi et al. 2019).Their use might represent a valid alternative to common antimicrobial agents, such as lysozyme, in the reduction of blowing risks related to the presence of species belonging to the Clostridium genus (Rodi et al. 2020).These sporeformer species are responsible for butyric fermentation which triggers undesired late-blowing defects (LBD) in semi-hard and hard cheeses during ripening (Gómez-Torres et al. 2015).Nowadays, many studies have demonstrated the anti-clostridial activity of LAB in cheese products, with a consequent control of blowing defects (Gómez-Torres et al. 2015; Rodi et al. 2020).Multiple LAB strains exploited the production of antimicrobial substances to inhibit microorganisms responsible for blowing defects with positive effects on cheese features at the same time, which makes this biological approach a promising alternative to the current strategies (Demirbaş et al. 2022).Several mechanisms involving LAB and the production of compounds of different chemical nature were proposed in the scienti c literature to explain the bio-protection activity in cheese.Many strains of the Lb.casei group have been found to produce bacteriocins (García-Cano et al. 2019; Yang et al. 2012).In addition, LAB produce other antimicrobial substances with inhibitory effects such as organic acids (lactic and acetic acid), hydrogen peroxide, and carbonyl compounds, such as acetaldehyde, diacetyl, and acetoin (Cintas et al. 2001).
This study aimed at isolating and characterizing several indigenous strains of Lb. casei from a semi-hard Italian cheese to identify a suitable pool of autochthonous microorganisms with potential application as adjunct starter cultures in semi-hard cheeses.Semi-hard cheeses were chosen because they are often subject to blowing defects, which can have a signi cant impact on quality.Furthermore, these cheeses have a medium-long ripening period during which secondary cultures can play a very important role in developing the avor of the product.Isolated strains have been studied for their proteolytic, esterase, and anti-clostridial activity, and for their ability to contribute to the aromatic pro le of cheese.

Strain isolation
Bacterial cultures (189) were isolated from nineteen samples of semi-hard cheeses from different dairies located in Friuli Venezia Giulia (North-East of Italy).Isolation was carried out from count plates (dilutions 10 − 5 -10 − 7 ) of MMV agar incubated at 30°C for 48 h under anaerobic conditions (Di Lena et al. 2015).For each sample, ve to ten morphologically unique colonies were randomly picked up and puri ed onto MRS Agar (Oxoid, Milan, I).The isolates were stored at -80°C in MRS broth with 30% glycerol (v/v).

Proteolytic and esterase activities
Proteolytic activity was evaluated by streaking a loopful of each isolate onto Skim Milk Agar plates (Oxoid).Plates were incubated at 30°C for 48 h under anaerobic conditions, and proteolytic activity was indicated by a clear zone surrounding the colonies (Pereira et al. 2001).
Esterase activity was evaluated by streaking each isolate onto Tributyrin agar plates (Sigma).Plates were incubated at 30°C for 48 h.Esterase activity was indicated by the presence of a clari cation halo around the culture (Morandi et al. 2013).

Anticlostridial activity
Anticlostridial activity was determined by the standardized agar disk diffusion method using commercially available paper disks (9 mm in diameter) (Macherey-Nagel GmbH, Duren, D) (Morandi et al. 2015).Before use, strains were propagated in MRS broth at 30°C for 24 h, while Clostridium strains were grown in Reinforced Clostridial Medium (RCM; Oxoid) at 37°C for 48 h in anaerobic conditions.Strains were spotted (20 µL) onto RCM agar plates seeded with Clostridium indicator-type strains, namely Clostridium beijerinckii DSM 791 T , Cl. butyricum DSM 10702 T , and Cl.tyrobutyricum DSM 2637 T from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig (Germany), Cl.
sporogenes ATCC 3584 T provided by the American Type Culture Collection (USA), and Cl.tyrobutyricum Coc1 and Cl.tyrobutyricum Coc2 from the Institute of Sciences of Food Production collection (ISPA-CNR, Milan, Italy) (Silvetti et al. 2018).Before use, lactobacilli were propagated in MRS broth (Oxoid) at 30°C for 24 h, while Clostridium strains were grown in RCM broth at 37°C for 48 h with an anaerobic incubation system (Anaerocult A, Merck Millipore, Darmstadt, Germany).The RCM agar plates were incubated anaerobically at 37°C for 72 h.Anticlostridial activity was expressed as the difference between the diameters of the inhibition zone and the disc used (9 mm).Anticlostridial activity was considered absent (halo < 10 mm), weak (10-20 mm), moderate (21-29 mm), or strong (> 30 mm).

Growth in curd-based medium
Strains having unique RAPD pro les were investigated for their ability to grow and produce volatile compounds in a curd-based medium.Samples inoculated with two commercial adjunct cultures, namely Lb. casei C1a and Lb.paracasei C1x isolated from Lyofast CPR1 (Sacco s.r.l., Cadorago CO, I), were also assessed.The cultures were reactivated in MRS broth, then streaked onto MRS agar plates, and incubated at 30°C for 48 h under anaerobic conditions.Cell suspensions were prepared with bacterial colonies in 1 mL of Maximum Recovery Diluent (MRD; Oxoid).In a preliminary step, the optical density at 600 nm and viable count onto MRS agar of such suspensions were estimated, to simplify the following inoculation of medium (Pogačić et al. 2015).
A curd-based medium was prepared from a 30-d curd of semi-hard cheese, as previously described (Pogačić et al. 2015).After removing the external part, 100 g of curd was nely chopped and added with 200 mL of a solution containing 1.2 g/L of Bacteriological Peptone (Oxoid) and 1% w/v NaCl, then stirred for one hour.The resultant suspension was then ltered with three layers of sterile gauze and portioned (5 mL) under stirring into 20 mL vials closed with a Te on septum and sealed with aluminum.The vials were sterilized at 110°C for 15 min, then, for each strain, three vials were inoculated at a nal concentration of 10 7 CFU/mL and incubated at 12°C for 30 days.After 15 d and 30 d of incubation bacterial viability and pH, and volatile compounds at 30 d were evaluated.Uninoculated vials (CT0) served as control.

Evaluation of viability
The curd-based medium was decimally diluted in MRD, and the dilutions were pour-plated onto MRS agar.Plates were incubated anaerobically at 30°C for 48 h.pH pH was measured using a pH meter Basic 20 (Crison, Barcelona, Spain) equipped with a pH electrode previously calibrated with standard solutions at pH 4.01, 7.00, and 9.21.

Analysis of volatile compounds
Volatile compounds were determined in duplicate by using a solid-phase microextraction (SPME) coupled to the gas chromatography-mass spectrometry (GC-MS) technique.For the extraction, an HT2800T autosampler (HTA s.r.l., Brescia, I) provided with a heater was employed.Samples were equilibrated at 60°C for 30 min, then a 2 cm × 50/30 µm Stable ex 24Ga divinylbenzene/carboxen/poly-dimethylsiloxane (DVB/CAR/PDMS) coated SPME ber (Supelco, Bellefonte, PA) was exposed for 30 min in the headspace for extraction.A QP2020 NX gas chromatography-mass spectrometry (GC-MS) system (Shimadzu Corporation, Kyoto, J) equipped with a DB-WAX capillary column (30 m length, 0.25 mm internal diameter, and 0.25 µm lm thickness; Agilent Technologies, CA) was used for separation and identi cation of volatile compounds.The ber was desorbed in the GC injection port at 270°C for 3 min under splitless conditions.For the analysis the following conditions were adopted: 1 mL/min Helium ow rate; interface, source, and quadrupole temperature were 240, 200, and 150°C, respectively.The temperature program was set initially at 50°C for 5 min, followed by the rst ramp at 10°C/min to 230°C, the temperature was then kept steady for 10 min, and a second ramp at 10°C/min to 240°C for 10 min.Scan mode with a mass range from 25 to 350 m/z was used for the analysis.Chromatographic pro les were evaluated using the GC-MS solution software ver.4.52 (Shimadzu Corporation, Kyoto, Japan) and compounds were identi ed by spectra comparison using the NIST/EPA/NIH 20 Mass Spectral Library (John Wiley & Sons Inc., Hoboken, NJ) and Kovat's retention index (RI) from the literature (https://webbook.nist.gov/chemistry/).Data were expressed as absolute areas of the obtained peaks measured in the headspace of each curd-based medium.

Statistical analysis
Statistical analyses were carried out using Origin Pro 9 software (OriginLab, Northampton, MA).Statistical differences between the mean values of control and inoculated samples (p < 0.05) were assessed using a t-test.Principal Component Analysis (PCA) and heatmap with hierarchical clustering analysis on volatile compounds were performed using pre-processed data, in the speci c log10[x]-transformed and scaled data, while Euclidean distances and Ward's minimum variance method were set for clustering.

Strains characterization
149 out of 189 isolated cultures were identi ed as members of the Lb.casei group.Since the isolates came from count plates with a very high dilution of the cheese sample (10 − 5 -10 − 7 ), this con rms that Lb. casei represents a predominant NSLAB group in the cheese considered in this study (Marino et al. 2003).Among isolates, 36 unique RAPD pro les were found and identi ed as Lb.paracasei (34 strains) and Lb.rhamnosus (2), which fully re ects the diffusion of the Lb.casei group in different semi-hard cheeses.Indeed, very recently in an extensive metagenomic investigation on the micro ora of 45 different types of Italian PDO raw milk cheeses, Lb. casei was detected in only 4 out of 128 samples, while Lb.paracasei and Lb.rhamnosus in 68 and 27, respectively (Fontana et al. 2023).Thus, these 36 strains were qualitatively evaluated for their proteolytic, esterase, and anti-clostridial activity.In cheesemaking, microbial proteolytic and lipolytic/esterolytic activities are required since they contribute to the formation of cheese's textural and sensory characteristics (McSweeney 2004).30 strains isolated in this study, along with the two strains coming from commercial adjunct cultures, were positive for proteolytic activity on milk proteins, whereas esterase activity was present in one Lb.paracasei strain (Table 1).Lb. paracasei and Lb.rhamnosus strains with proteolytic activity have already been isolated from cheese in previous studies (Bonomo and Salzano 2013; García-Cano et al. 2019).During cheese ripening, when most of the residual lactose has been already metabolized by starter bacteria, peptides and amino acids constitute the main energy source for NSLAB and Lb.casei-group bacteria.Generally, strains having proteolytic activity release protease and peptidase that generate amino acids and peptides, which are important precursors of chemical compounds involved in the development of cheese sensory attributes during ripening (Innocente 1997;Maifreni et al. 2002;McSweeney 2004).In the ripening of some cheeses, an important contribution is also provided by lipolysis, which causes the release of free fatty acids, mono-and diglycerides, essential for avor and aroma development.As for strains isolated in this study, only one showed to be esterolytic on a solid medium.Lipolytic and esterolytic activities are less widespread than proteolytic within the Lb.casei group and have been rarely reported in the literature (Bonomo and Salzano 2013; Meng et al. 2018).Although lipolysis plays a relevant role in the ripening of a limited number of cheeses, LAB esterases contribute to the synthesis of esters from glycerides and alcohols (Di Cagno et al. 2003).It has been hypothesized that an esterase from Lb. casei could synthesize ethyl esters as a w decreased during cheese ripening, suggesting a possible impact on cheese avor development (Fenster et al. 2003).Isolated strains were also tested for their antimicrobial ability against six different butyric acid-producing clostridia (Table 1).All strains weakly inhibited Cl. sporogenes ATCC 3584 T , while weak to strong anti-clostridial activities were found against Cl.beijerinckii DSM 791 T and Cl.
butyricum DSM 10702 T .Among butyric acid-producing clostridia, Cl. tyrobutyricum is the main responsible for LBD in semi-hard and hard cheese during ripening, signi cantly causing food waste and economic losses (Christiansen et al. 2010).For this reason, the activity of the isolated strains against Cl.tyrobutyricum was tested against three different strains.Eighteen isolates showed to weakly or moderately inhibit at least one Cl.tyrobutyricum strain, and three Lb.paracasei strains, (C70, C177, and C184) were active against all Cl.tyrobutyricum strains.The anticlostridial activity, albeit moderate, of the strains of this study along with their ability to adapt to the stressful environment of the cheese during ripening make them particularly attractive for the development of an adjunct culture.

Growth in curd-based medium
After qualitative characterization, the selected strains, together with the two commercial cultures (C1a C1x) were incubated for 30 days at 12°C in a curd-based medium to check the ability to grow and contribute to a volatile pro le in a simulated cheese environment.
All strains showed to grow after 15 d of incubation (Table 2), due to their ability to gain energy from residual lactose and/or the nitrogen fraction (amino acids and peptides) present in the curd-based medium.Similar results have been already reported for the Lb.casei-group (Pogačić et al. 2015(Pogačić et al. , 2016)).The initial pH of the curd-based medium was 5.77.As expected, all strains showed acidifying ability, with a pH of the medium after 15 d ranging from 4.25 to 4.71 (Table 2).As for 30 d of incubation, results highlighted the different abilities of strains under study to proliferate due to different microbial metabolisms and adaptive capacities.Most strains, including the commercial ones, remained at levels similar to 15 days, probably having already reached the stationary phase.After 30 days only slight variations in pH were observed.Only four strains (C217, C286, C369, and C1x) showed a decrease in pH values at 30 days of incubation.Eight strains showed a decreased viability after 30 d, which could be due to autolysis phenomena.Autolysis is a general property of LAB, present in the Lb.casei group, and plays a fundamental role in cheese; indeed, it has been shown that the intracellular enzymes released by lysis are mainly involved in avor formation (Bancalari et al. 2017).
Principal Component Analysis (PCA) was carried out to highlight the differences in the production of volatile compounds among selected strains at 30 d of incubation in the curd-based medium (Fig. 1).In addition, a clustering analysis based on the chemical classes of volatile compounds was performed and the results were shown on heatmaps (Fig. 2).The rst two components of PCA described 56.1% of the total variance (Fig. 1).PC1 accounted for 32.8% of the variability while PC2 accounted for 23.2%.The rst component was positively associated with all volatile compound classes, except for "others".PC2 was instead positively associated with acids and esters, and negatively correlated with sulfur compounds, ketones, and alcohols (Fig. 1A).The uninoculated curd-based medium (CT0, control medium at 0 d) was in the left negative quadrant, far from the other samples, indicating that the production of avors was signi cantly different from those of all strains at 30 days of incubation (Fig. 1B).CT0 contained signi cant amounts of aldehydes, such as hexanal and benzaldehyde, ethanol, acetoin, and acetic acid (Fig. 2B).These volatiles either come from raw milk or can also derive from the metabolic activity of starter lactic acid bacteria (SLAB) inoculated to the formation of the curd that was used to prepare the cheese-based medium (Pogačić et al. 2016).Ethanol, acetoin, and acetic acid are principally produced from lactose metabolism, while aldehydes are normally generated from the catabolism of amino acids (McSweeney and Sousa 2000).
Hierarchical clustering separated strains into three distinct groups (Fig. 2A).Group I contained strains C54 (a proteolytic strain), C85, and C245 (not proteolytic), which were characterized by a high production of ethanol (Fig. 2B).Ethanol can originate from the lactate metabolism via acetaldehyde dehydrogenase activity, which many LAB including Lb. paracasei activate under limiting nutritional conditions, or from the fermentation of pentoses (e.g., ribose) released by SLAB lysis.Moreover, ethanol can be produced within the catabolism of amino acids, which produces acetaldehyde that can be further reduced to ethanol by alcohol dehydrogenase (McSweeney and Sousa 2000).A signi cant increase in the relative percentage of ethanol in a cheese medium fermented by speci c Lb. casei strains has been already reported, which may suggest a speci c strain dependency in ethanol production (Sgarbi et al. 2013).In addition, group I was also characterized by a remarkable formation of dimethyl disul de and ketones, in particular, diacetyl, acetoin, and 2-heptanone, which are relevant avor contributors in several cheese varieties (Smit et al. 2005).Dimethyl disul de originates from the catabolism of methionine, while alkan-2-ones are commonly produced by the βoxidation of saturated fatty acids, and diacetyl results from lactose and citrate metabolism (McSweeney 2004).
Group II was characterized by a low to moderate ability to produce volatiles, with the only class "others" as representative of this group (Fig. 1B).
Indeed, samples in this group are located in the same quadrant as the non-inoculated control sample (CT0) (Fig. 1B).Benzaldehyde and 2-furanmethanol were the principal compounds, arising probably from the proteolytic activity and following amino acids degradation and phenylalanine catabolism (McSweeney 2004).Recently, benzaldehyde has been detected in curd-based media after inoculation and growth of Lb. casei group strains (Picon et al. 2019).
Group III clustered 21 isolated Lb. casei-group strains and commercial strains C1a and C1x, highlighting that 58% of the strains isolated in this study were similar to commercial controls in terms of volatile production at 30 d of incubation.Acids, esters, and ketones were the predominant classes representative of this group of strains (Figs.1B and 2A).Acetic, butanoic, hexanoic, and octanoic acids were detected as prevalent fatty acids (FAs).In a medium like the one used in this study, short-and medium-chain FAs can either derive from the catabolism of amino acids or by oxidation of ketones, esters, and aldehydes.A signi cant amount of these FAs was previously detected in semi-hard cheeses at the late stages of ripening (Innocente et al. 2013), where Lb. casei group strains are dominant (Innocente and Biasutti 2013; Marino et al. 2003).In cheese, short and medium-chain FAs are key odorant components due to their low perception thresholds (Curioni and Bosset 2002).Butyl acetate was the most abundant ester, as the result of esteri cation reactions between acetic acid and 1-butanol.Regarding ketones, once more diacetyl, acetoin, methyl isobutyl ketone, and 2-heptanone showed the highest absolute area (Fig. 2b).Although C217 was the only strain showing esterolytic

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Table 2
Microbial growth in curd-based medium of isolated strains with unique pro le and controls, and pH values of the medium after 15 d and 30 days of incubation at 12°C.