Lactococcus lactis ssp . lactis as Potential Functional Starter Culture

Lactic acid bacteria (LAB) are natural food preservatives that have been Generally Recognized as Safe (GRAS) by the US Food and Drug Administration (FDA). Their benefi ts have been known since ancient times, and modern science has confi rmed their positive eff ects on human intestinal microfl ora, including resistance to infectious diseases, immunomodulatory action, and digestion of lactose (1).


Introduction
Lactic acid bacteria (LAB) are natural food preservatives that have been Generally Recognized as Safe (GRAS) by the US Food and Drug Administration (FDA).Their benefi ts have been known since ancient times, and modern science has confi rmed their positive eff ects on human intestinal microfl ora, including resistance to infectious diseases, immunomodulatory action, and digestion of lactose (1).
Considering their benefi ts, LAB have a great potential for application in functional foods (2).In meat preservation and fermentation, LAB are considered technologically fundamental.They decrease pH with lactic acid, produce bacteriocins that prevent the growth of pathogenic and spoilage microorganisms, modify sensory properties (fl avour, colour and texture) of raw material, and improve the safety, stability and shelf-life of meat products (2,3).
Naturally fermented sausage products are considered to be of higher quality than sausages whose fermentation is controlled with industrial starters (4).Many authors support the view that indigenous microfl ora or microorganisms found in traditional sausages originate from raw materials or the manufacturing environment (5,6).This microbiota is commonly known as the house fl ora (7).
More and more research is focused on the isolation and identifi cation of autochthonous functional starter cultures with the aim of developing new functional meat products that will be recognised and labelled as autochthonous to the region in which they are produced (8,9).
The aim of this study is to identify and characterise microbial population, LAB in particular, naturally present in homemade horsemeat sausages from Pakrac (Slavonĳ a, eastern Croatia).We also wanted to investigate the technological and functional properties and adhesion ability of the dominant bacterial strain in the intestinal epithelial cells of mice in vitro to see its potential as a starter culture for industrial production.

Materials and Methods
All experiments were carried out on fi ve samples of homemade horsemeat sausage.The sausages had been prepared according to a traditional recipe without the use of industrial starters or any additives such as nitrites or ISSN 1330-9862 preliminary communication doi: 10.17113/ b.52.04.14.3794 ascorbic acid.The stuffi ng consisted of horsemeat (75 %), pork back fat (25 %), and seasoning (kitchen salt, pepper, paprika and garlic).The sausages were smoked for 2-3 days and then le to ripen in the ripening chamber until day 42.Samples were taken at the end of the ripening stage.

Isolation and identifi cation of microbial population
To isolate and identify the microbial population in the sausages we used classical microbiological procedures and biochemical analytical profi le index (API) methods, as well as random amplifi cation of polymorphic DNA polymerase chain reaction (RAPD PCR) analysis for genetic fi ngerprinting.A mass of 10 g of each sample was homogenised in 90 mL of sterile 0.5 % NaCl solution and serially diluted before plating on selective and non-selective media as shown in Table 1.
One hundred and fi y colonies taken at random from selective plates were identifi ed on the basis of colony morphology, Gram staining, cell morphology and catalase reaction.Genomic DNA was isolated from 1.5 mL of overnight cultures using the method described elsewhere (2,10), which is a modifi cation of the salting-out procedure described by Miller et al. (11).The extracted DNA was then used as a template in subsequent PCR reactions, which were performed according to Gardiner et al. (12).PCR amplifi cations were performed in a thermal cycler (Eppendorf Mastercycler ® Personal, Hamburg, Germany) using 0.2-mL Eppendorf tubes for a total volume of 50 µL.PCR mixtures contained 1 µL of template DNA, 1× Taq polymerase buff er (Fermentas, Pi sburgh, PA, USA), 4 mmol/L of MgCl 2 , 0.2 mmol/L of deoxynucleoside triphosphate (Fermentas), 5 U of Taq DNA polymerase (Fermentas), and two primers (5'ACGAGGCAC3' and 5'ACGC-GCCCT3'), each at a concentration of 1 µmol/L.DNA was amplifi ed in 40 cycles using the following temperature profi le: denaturation at 94 °C for 30 s, annealing at 36 °C for 30 s, and polymerisation at 72 °C for 2 min.The initial denaturation was performed at 94 °C for 5 min, and the fi nal extension step at 72 °C for 10 min.The PCR products (10 µL of each reaction mixture) were analysed on a 1.5 % (by mass per volume) agarose gel with 1× TBE buff er (89 mM Tris, 89 mM boric acid, 2 mM EDTA) and ethidium bromide staining.The DNA of bacteriophage λ cut with HindIII was used as molecular size standard.

Storing conditions
The dominant bacterial strain (isolated from horsemeat sausages) was stored at -70 °C in the M17 broth (Difco, Detroit, MI, USA) with 30 % (by volume) glycerol and kept with the culture collection of the Department of Biochemical Engineering, Laboratory for General Microbiology and Food Microbiology, University of Zagreb, Zagreb, Croatia, until further investigation.

Eff ects of NaCl and temperature on microbial growth and proteolytic activity
The eff ect of 5 % NaCl on the viability and proteolytic activity of the dominant LAB strain at diff erent incubation temperatures (12, 18 and 22 °C) was tested according to Bonomo et al. (13) and Babić et al. (8).

Antibiotic resistance of lactobacilli
The antibiotic resistance of the dominant LAB was tested on the MRS agar (Biolife, Milan, Italy) using the agar disc diff usion method as described by Babić et al. (8).The strains were classifi ed as resistant or sensitive a er incubation at 37 °C for 18 h.The following antibiotic discs (BD-Becton, Dickinson and Company, Franklin Lakes, New Jersey, MD, USA) were used: clindamycin (2 µg); methicillin, neomycin and erythromycin (5 µg each); tobramycin, ampicillin and lincomycin (10 µg each); gentamycin, chloramphenicol, rifampicin, vancomycin and tetracycline (30 µg each).

Preparation of simulated gastric and small intestinal juice
Gastric and small intestinal juice simulations were prepared according to Kos et al. (14) with small modifi cations.
Simulated gastric juice was prepared by suspending pepsin (3 g/L) in a sterile sodium chloride solution (0.5 %) and adjusting the pH to 2 with concentrated HCl.Pepsin (from porcine stomach mucosa) was obtained from Sigma Chemical Co, St. Louis, MO, USA.Simulated small intestinal juice was prepared by suspending pancreatin (1 g/L) and bile salts (2 mg/mL of oxgall) in a sterile sodium chloride solution (0.5 %) and adjusting the pH to 8.0 with 0.1 mol/L of NaOH.Pancreatin (from hog pancreas, 165 U/mg) was obtained from Fluka Biochemica, Buchs, Switzerland, and oxgall (dehydrated fresh bile) from Difco Laboratories, Detroit, MI, USA.

Survival of the dominant LAB in gastric and small intestinal juice simulations
Washed cell suspensions of the dominant LAB (0.2 mL) were vortexed separately with gastric and small intestinal juice (1 mL) and 0.3 mL of 0.5 % NaCl.Changes in total viable count were monitored during treatment with gastric (2 h) and small intestinal juice (4 h) using the pour plate method.The plates with MRS were incubated at 37 °C for 4 h.The log CFU/mL is expressed as percentage of LAB viability.

Determination of lactic acid production by HPLC
Lactic acid produced in the samples was determined using high performance liquid chromatography (HPLC) according to Babić et al. (8), and the results were expressed as g/L.Glucose, lactic acid, acetate, and ethanol for stan-

Antibacterial activity
Antibacterial activity of the dominant LAB isolate was tested using the turbidimetric method (8)

In vitro adhesion test
The adhesion test was performed using the method of Marteau et al. (15) with some modifi cations (1).Ileal samples were collected from three-month-old Swiss albino mice obtained from the Rĳ eka University School of Medicine, Rĳ eka, Croatia.The tissues were held in phosphate--buff ered saline (PBS) at 4 °C for 30 min to loosen the surface mucus and then washed three times with PBS.The adhesion test was performed by incubating tissue samples (1 cm 2 ) in bacterial suspensions (10 9 cell/mL of PBS) at 37 °C for 30 min.Samples of ileum were fi xed in 10 % formalin, dehydrated with increasing concentrations of ethanol, and embedded in paraffi n.Serial sections (5 µm) were cut, mounted on standard microscope slides, and stained for identifi cation of Gram-positive and Gram-negative bacteria according to Frece et al. (1).The slides were examined and photographed using a Nikon Mikrophot-FXA light microscope (Nikon, Tokyo, Japan).All experimental procedures complied with the standards set out in the Guide for the Care and Use of Laboratory Animals of the National Research Council (16).The best of the three photographs is presented in the results.

Viability of the dominant LAB strain during lyophilisation and storage
The strain was cultivated in MRS broth overnight.The cells were collected by centrifugation (8000×g for 20 min), washed, resuspended in skimmed milk (10 %, by mass per volume), and frozen at -20 °C overnight, then lyophilised in a bench top freeze dryer (model Christ Alpha 1-4, B. Braun Biotech International, Melsungen, Germany).The lyophilised cells were stored at -20 °C for six months.The recovery of viable cells was determined by colony formation on MRS agar using the standard pour plating method.The log CFU/mL is expressed as percentage of viability (17).

Statistical analysis
All experiments were carried out in triplicate.The results are expressed as mean value±standard deviation (S.D.).Apart from descriptive statistics, no other analysis was done, as it was not necessary for the scope and objective of our study.

Results and Discussion
Our microbiological analysis showed that the dominant microfl ora in the horsemeat sausage samples were LAB, followed by micrococci and yeasts (Table 2).Like LAB, micrococci contribute to the colour and fl avour of the sausage, but unlike LAB, they are not directly involved in fermentation.
Our fi nding of the yeasts Candida zeylanoides and Geotrichum candidum is in agreement with the fi ndings of Nielsen et al. (18).Yeasts also play an important role in the maturation of sausages, since they contribute to the development of sensory characteristics of fermented sausages due to their lipolytic and proteolytic activity (19,20).
With API 50 CHL identifi cation kits (bioMérieux, Lyon, France) we were able to classify 100 colonies randomly taken from the MRS agar into four main bacterial species and 16 strains (the API test precision was 97-99.9%), and the dominant microfl ora were LAB, which were then confi rmed by the RAPD PCR analysis.
Altogether four species were identifi ed: Lactococcus lactis ssp.lactis, Lactobacillus plantarum, Lactobacillus delbrueckii and Lactobacillus acidophilus, and each consisted of four strains.Each of the four strains of a species had identical RAPD fi ngerprints (Fig. 1).The most dominant LAB species was Lactococcus lactis ssp.lactis, as it was isolated from all fi ve sausage samples, followed by Lactobacillus plantarum, isolated from four samples (Table 3).
The dominance of Lactococcus lactis ssp.lactis in fermented sausage has rarely been reported so far.The metabolic properties of the L. lactis species have a direct or indirect eff ect on the organoleptic, nutritional and hygienic quality of fermented products (21).As in a preliminary research (disc diff usion method, data not shown) all Lactococcus isolates showed good antimicrobial properties, and our dominant isolate, Lactococcus lactis ssp.lactis excelled among them, we decided to further characterise it as a starter culture.Table 4 shows its technological and functional properties.Its outstanding ability to grow in the presence of NaCl at a range of concentrations and temperatures used in meat processing industry has already been reported by Kim et al. (22) and Itoi et al. (23).In order to act as a probiotic in the gastrointestinal tract, the bacteria must be able to survive the acidic conditions of the stomach and resist the bile acids at the beginning of the small intestine (1,2,24,25).The viability of our L. lactis isolate in simulated gastric juice (pH=2, salt content 0.5 %) was 51.24 % (Table 4) a er 2 h of exposure.Our results are in agreement with the report by Kimoto-Nira et al. (26), who found that L. lactis ssp.lactis was resistant to low pH and bile salts.In small intestinal juice, the viability of the isolate was 71.23 % (Table 4), which is essential for its probiotic eff ects.
The production of lactic acid, which results in a decrease in pH, contributes to the texture, colour and acidic taste, prevents the growth of pathogenic and spoilage microorganisms and improves the safety and stability of meat products (27).Our study has shown that L. lactis ssp.lactis is homofermentative.The concentration of lactic acid produced by homolactic fermentation of glucose was 13.83 g/L, and the medium was acidifi ed to pH=4.16 (28).In contrast, heterofermentative LAB are not suitable for sausage production because they produce large amounts of carbon dioxide that creates holes in the product.In addition, these LAB produce acetic acid, which causes a pungent off -fl avour (27).
L. lactis ssp.lactis isolated in our study was catalase--positive and showed good proteolytic activity (Table 4), which is important in preventing rancidity and preserving meat colour.Another essential property for the probiotic activity of a functional starter culture is the competition with pathogens (29).Our L. lactis ssp.lactis isolate inhibited the growth of all test pathogens over 72 h of cultivation, as follows (in %): E. coli 73.4-85.4,S. aureus 51.5-87.0,Salmonella spp.62.7-88.6 and L. monocytogenes 61.0-82.4(28).Antimicrobial properties of L. lactis ssp.lactis are of great importance for the application of this strain as functional starter culture in fermented meat products.One of the selection criteria for probiotic strains is the ability to adhere to the intestinal tract of the host.Good adhesion means that a probiotic strain will exert its properties be er (1,2).In this respect, our L. lactis ssp.lactis isolate showed very good adhesion (Fig. 2).
In industrial application, bacteria are subjected to stress that can lower their viability.Viability can be aff ected by harsh heat treatment, mechanical processing, storage at room temperature, food matrix, and chemical microenvironment (25).Furthermore, a range of processes has been used to obtain dry formulations with prolonged shelf-life, the most common being freeze drying, vacuum drying, and spray drying.These processes do osmotic damage to probiotic cells, and cell death usually correlates with cell membrane damage (17).The membrane is usually protected by cryoprotectants (against freezing) and lyoprotectants (against lyophilisation).These are small molecules or polymers with osmotic behaviour, which promote the formation of amorphous or glassy solids and reduce ice formation that can be cell-damaging (17).We protected our isolate cells with skimmed milk as lyoprotectant, and, judging by the high survival, they withstood well the stress associated with lyophilisation (Table 4).

Conclusions
The dominant LAB, Lactococcus lactis ssp.lactis, isolated from the horsemeat sausage in our study has shown good technological and functional properties important for starter cultures.It has also shown good survival in simulated gastrointestinal environment in vitro, desirable adhesive properties in intestinal epithelial cells of mice, and suffi cient antimicrobial activity against pathogen microorganisms, which are all important properties for the selection of a probiotic starter culture.We therefore believe that it could be used for controlled fermentation of meat products.This work can be considered preliminary, as it was conducted to characterise an autochthonous functional start er culture.Implementation of this culture in small-to medium-sized enterprises and industrial production should improve the quality, uniformity and safety of the fi nal product.Hopefully, the product will meet all marketing regulations yet remain autochthonous and recognisable.Before that happens, further investigation is needed to confi rm good adhesive properties and resilience to gastrointestinal environment in vivo.Our study is a contribution to an eff ort to create a bank of traditional and autochthonous functional starter cultures for wider and controlled use in the future.

Fig. 2 .
Fig. 2. Adhesion of Lactococcus lactis ssp.lactis to the large intestine of the mouse

Table 1 .
Classical microbiological methods for the isolation and identifi cation of natural microbial populations by comparing the growth of test microorganisms in nutrient broth (beef extract 3 %, by mass, peptone 5 %, by mass; Biolife, Milano, Italy) at 37 °C over 72 h with and without the LAB isolate.The results were expressed as growth inhibition of the following test microorganisms: Staphylococcus aureus 3048, Escherichia coli 3014, Salmonella sp.3064 and Listeria monocytogenes ATCC 23074 (Collection of Microorganisms of the Laboratory for General Microbiology and Food Microbiology, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia).

Table 2 .
Microbiological and biochemical identifi cation of natural microbial populations in horsemeat sausage samples 5 1 Results are the mean value±S.D. of triplicate assays

Table 3 .
Lactic acid bacteria (LAB) isolates in samples of home-

Table 4 .
Technological and functional characteristics of Lactococcus lactis ssp.lactis