544 JOURNAL OF FOOD SCIENCEVolume 63, No. 3, 1998

Lactobacillus acidophilus Group Lactic AcidBacteria Applied to Meat Fermentation

K. ARIHARA, H. OTA, M. ITOH, Y. KONDO, T. SAMESHIMA, H. YAMANAKA, M. AKIMOTO, S. KANAI, and T. MIKI

Authors Arihara, Ota, Itoh, and Kondo are affiliated with the School of VeterinaryMedicine & Animal Sciences, Kitasato Univ., Towada-shi 034, Japan. AuthorsSameshima, Yamanaka, Akimoto, Kanai, and Miki are with Prima Meat Packers,Ltd., Tsuchiura-shi 300, Japan. Address inquiries to Dr. K. Arihara.

ABSTRACTLactobacillus acidophilus group bacteria (L. acidophilus, L.crispatus, L. amylovorus, L. gallinarum, L. gasseri, L.johnsonii), probiotic lactic acid bacteria, were applied to meatfermentation. Of six strains, L. gasseri (predominant lacto-bacilli in human intestinal tracts) JCM1131T exhibited great-est fermentation performance in meats. This strain resistedgastric acid and bile, and would thus have no detrimentaleffects in the intestinal tract. Inoculation of the strain de-pressed the propagation of S. aureus cells and their entero-toxin production during meat fermentation. Results suggestprobiotic lactic acid bacteria could be effectively utilized formeat fermentation to produce healthy meat products.

Key Words: lactic acid bacteria, Lactobacillus acidophi-lus, Lactobacillus gasseri, meat fermentation, probiotic bac-teria

INTRODUCTIONLACTOBACILLUS ACIDOPHILUS GROUP LACTIC ACID BACTERIA(LAB) have been considered to be predominant lactobacilli in theintestinal tract of healthy humans (Arihara and Luchansky, 1994;Ray, 1996). For their therapeutic activities associated with an intes-tinal microbial balance, L. acidophilus group strains have been widelyutilized as a dairy starter culture. They are also probiotics, definedas cultures of live microorganisms that, applied to animals or hu-mans, benefit the host by improving properties of indigenous micro-flora (Havenaar and Huis int Veld, 1992). Such effects have beenattributed to biochemical, physiological, and antimicrobial effects,as well as competitive exclusion in the intestinal tract (Goldin andGorbach, 1992). The valuable properties of probiotic LAB, have notbeen utilized in the meat industry.

LAB, such as Lactobacillus plantarum and Pediococcus cerevi-siae, have been used as starter cultures for the fermentation processof meat products (Smith and Palumbo, 1983; Lcke, 1994). Thesebacteria mainly contribute to flavor development and product safetyin meats. The therapeutic activities of LAB have not been studiedfor meat products, unlike fermented dairy products (i.e. acidophilusmilk). There appears to be the possibility for the development ofhealth beneficial meat products prepared with probiotic bacteria. Ourobjective was to investigate the therapeutic utility of the L. acido-philus group LAB, which consisted of six genome clusters [species:L. acidophilus, L. crispatus, L. amylovorus, L. gallinarum, L. gasse-ri, L. johnsonii (Fujisawa et al., 1992)], for their potential applica-tion to meat fermentations.

MATERIALS & METHODS

Bacterial strainsSix type strains of Lactobacillus acidophilus group species (L.

acidophilus JCM1132T, L. crispatus JCM1185T, L. amylovorusJCM1126T, L. gallinarum JCM2011T, L. gasseri JCM1131T, L.johnsonii JCM2012T) were obtained from JCM (Japan Collection ofMicroorganisms, Wako, Japan). Four commercial starter cultures formeat fermentation (Lactobacillus plantarum MMF-101, L. caseiMMF-141, L. sake MMF-161, Pediococcus cerevisiae MMF-121)were donated (San-Ei Sucrochemical, Chita, Japan). All LAB cul-tures were maintained as frozen stocks (kept at 55C) in MRS broth(Difco Laboratories, Detroit) plus 10% glycerin, and prior to use,they were transferred at least twice in MRS broth at 37C. Viablecounts of lactobacilli were determined by plating on MRS agar. Theplates were incubated at 37C for 48h under anaerobic conditions.

Enterotoxin-producing Staphylococcus aureus strains 101 (en-terotoxin A producer), 104 (enterotoxin B producer), 105 (entero-toxin C producer), and 106 (enterotoxin D producer) which wereused were from our laboratory collection. Viable counts of S. au-reus were determined by plating on Vogel-Johnson agar (Eiken,Tokyo, Japan). The plates were incubated at 37C for 24h underaerobic conditions. Enterotoxin production was detected by reversedpassive latex agglutination method. This assay was carried out uti-lizing a commercial kit, SET-RPLA-SEIKEN (Denka Seiken, To-kyo, Japan).

Preparation of model sausageFresh pork trim (ham) was obtained from a local packer. The

meat was ground, mixed with glucose (1%), sodium chloride(2.5%), sodium nitrite (5 ppm), sodium ascorbate (550 ppm), start-er LAB (106107 CFU/g meat) and/or Staphylococcus aureus strains(106 CFU/g meat). The concentration of sodium chloride (04%) andsodium nitrite (0200 ppm) was varied to determine the resistanceof lactobacilli to these compounds. The batter (50g each) was stuffedinto commercial high density polyethylene pouches (10 m thick-ness) and incubated at 37C for 2448h.

For determining bacterium numbers in prepared sausages, each5-g portion of sausage was macerated with 45 mL of 0.1% peptonewater for 2 min by using a stomacher and diluted for plate counts.Respective agar plates for lactobacilli and staphylococci, and incu-bation conditions described above were used for plating. For deter-mining the pH of sausages, each 5-g portion of sausage was macer-ated with 5 mL distilled water for 2 min in a stomacher. The pH wasmeasured with a combination electrode and pH meter.

Acid and bile-tolerance of lactobacilliAcid and bile-tolerance were tested to estimate the resistance of

L. gasseri under conditions simulating those of the stomach and in-testines of humans. The survival of L. gasseri in acid solutions wasexamined by the addition of 0.1 mL of the cell suspension into aseries of 10-mL volumes of sterile distilled water at pH 1, 2, 3 ad-justed by HCl. The incubation mixture was maintained at 37C for0, 0.5, 1.0, 3.0, and 5.0 h. Immediately after respective incubationperiods, viable cells of lactobacilli in the mixture were enumeratedby plating on MRS agar. The plates were incubated at 37C for 48hunder anaerobic conditions.

Bile-tolerance was determined essentially by a published proce-dure (Klaenhammer and Kleeman, 1981). In brief, the cultures of L.gasseri were plated in MRS agar (pH 5, 6, 7) containing various

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concentrations (02,000 ppm) of bile powder (Wako Pure ChemicalIndustries, Osaka, Japan). The plates were incubated at 37C for 72h.

Scanning electron microscopySamples (ca. 5 5 5 mm) from prepared sausages were sub-

merged in 2.5% glutaraldehyde buffered at pH 7.0 with 50 mM sodi-um phosphate buffer, and were held for 18h at 4C to allow fixation.Particles were washed with distilled water followed by dehydrationin a series of graded concentrations (50100%) of ethanol and driedby the t-butyl alcohol freeze-drying method (Inoue and Osatake,1988). Dried specimens were fractured manually with a razor blade,coated with platinum, and observed under a Hitachi S-450 scanningelectron microscope with an accelerating voltage of 15 kV.

RESULTSTHE PH CHANGES OF THE SAUSAGES FROM THE SIX LACTOBACIL-lus acidophilus group strains (species) and four commercial startercultures were compared (Table 1). The pH drop of meats was uti-lized for estimating the growth of Lactobacillus strains. Of these 10strains, L. gasseri JCM1131T demonstrated largest pH decline causedby lactic acid production. L. johnsonii(genetically closely-relatedspecies to L. gasseri; Fujisawa et al., 1992) JCM2012T showed asimilar pH change. It is remarkable that JCM1131T and JCM2012Tgrew faster than commercial starter cultures normally utilized formeat fermentation. Since L. gasseri JCM1131T demonstrated the larg-est pH drop (fastest growth) during fermentation, that strain was usedfor further experiments.

As shown (Fig. 1), the inoculated model sausage fermented withL. gasseri JCM1131T suggested that the microorganisms were notevenly distributed in the interior of the sausage. Like the report byKatsaras and Leistner (1991), strain JCM1131T was immobilized incavities or nests within the sausage matrix (solid-state-fermenta-tion). Since intestinal LAB prefer an anaerobic condition (Kandlerand Weiss, 1986), this suggests that the density of cells would prob-ably increase toward the center of the sausage.

Since most meat products contain sodium chloride and sodium ni-trite for preservation during storage, effective starter cultures used for

conventional meat products must resist those compounds (e.g., 3.3%and 200 ppm, respectively). L. gasseri JCM1131T exhibited resis-tance (as evidenced by a pH drop after the fermentation period) againstonly 2% sodium chloride and 100 ppm sodium nitrite (Table 2).

During storage of the model sausage fermented with L. gasse-ri JCM1131T, levels of viable cells of LAB were well maintained(Table 3). This aspect is important for the utilization of probioticbacteria. Also, such strains must survive in the environment withgastric acid and bile, when viable cells go through the gastrointesti-nal tract. Several workers have reported on standards of acid andbile-tolerance required for the LAB to reach the intestinal tract (Itoh,1992; Gohran, 1994). Bacteria would contact pH values ranging from2.0 to 8.0 in the stomach, depending upon whether or not food wasbeing consumed (Hood and Zottola, 1988). Resisting at pH 3 for 2 his one standard for low pH tolerance of probiotic bacteria (Itoh, 1992).In addition, they are usually required to grow with 1,000 ppm bileacids at neutral pH (Gohran, 1994). Using these standards, L. gasse-ri JCM1131T had satisfactory resistance characteristics (Table 4, 5).

For maintaining viable cells of probiotic bacteria in final prod-ucts, it is necessary to eliminate pathogens without heat treatment.As shown (Fig. 2), during incubation without starter cultures, S. au-reus strains increased their numbers and production of respectiveenterotoxins was detected at the surface of sausages. However, whenfermentation was carried out with addition of L. gasseri JCM1131T,the propagation of S. aureus was depressed and enterotoxin produc-tion was eliminated.

DISCUSSIONBACTERIAL STRAINS FOR MEAT STARTER CULTURES HAVE BEENrequired to lower pH, produce desirable flavor, and to be nontoxicand nonpathogenic (Jessen, 1995). By using probiotic LAB, we canintroduce potential health benefits to meat products and improve theirvalue. It is generally recognized that products fermented with LABimprove nutritional properties (Goldin and Gorbach, 1992) and thegrowth of LAB in the intestinal tracts competitively excludes en-teropathogens (Tannock, 1990). In addition, more specific therapeu-tic activities of such bacteria, such as antimicrobial activities, anti-cancer properties and cholesterol assimilation, have been reported(Goldin and Gorbach, 1992; Sanders, 1993).

In our results, L. gasseri JCM1131T exhibited proper growth formeat fermentation. L. gasseri has been reported to be a predominant

Table 1pH change of model sausagesa inoculated with Lactoba-cillus acidophilus group strains and commercial starter culturesduring fermentation

Sausage fermentation24h 48h

Species/Strainsb pHc

Lactobacillus acidophilus groupL. acidophilus JCM1132T 5.6 5.2L. crispatus JCM1185T 5.6 5.2L. amylovorus JCM1126T 5.6 5.2L. gallinarum JCM2011T 5.5 5.2L. gasseri JCM1131T 5.1 4.3L. johnsonii JCM2012T 5.3 4.4

Commercial starter culturesLactobacillus

L. plantarum MMF-101 5.3 4.7L. casei MMF-141 5.6 4.9L. sake MMF-161 5.5 5.2

PediococcusP. cerevisiae MMF-121 5.3 5.0

aModel sausages were prepared from ground pork trim with glucose (1%), sodiumchloride (2.5%), sodium nitrite (5 ppm), sodium ascorbate (550 ppm) and starter lacticacid bacteria.bEach of the strains was inoculated 106 CFU/g meat for fermentation (37C, 2448h).cpH of sausages prior to fermentation was 5.9.

Fig. 1Scanning electron micrograph of the inside dissected sur-face of a model sausage fermented with L. gasseri JCM1131T.

546 JOURNAL OF FOOD SCIENCEVolume 63, No. 3, 1998

Meat Fermentation by Probiotic Bacteria . . .

Table 2Resistance of L. gasseri JCM1131T to sodium chloride andsodium nitrite in model sausagesa

Sodium Sodium Sausage fermentationchloride nitrite 24h 48h

(%) (ppm) pHb0 0 4.3 4.1

25 4.3 4.250 4.4 4.2

100 5.3 4.7200 5.9 5.7

2 0 5.0 4.425 5.1 4.550 5.2 4.7

100 5.6 5.0200 6.0 5.8

4 0 5.6 5.225 5.6 5.350 5.8 5.3

100 5.9 4.7200 6.0 5.9

aModel sausages were prepared from ground pork trim with glucose (1%), sodiumchloride (04%), sodium nitrite (0200 ppm), sodium ascorbate (550 ppm) and 106 CFUof JCM1131T/g meat. They were fermented at 37C for 24-48h.bpH of sausages prior to fermentation was 6.0.

Table 3Changes in viable cells of L. gasseri JCM1131T in modelsausagesa during fermentation and storage

Fermentation Storage at 4C (days)Fermentation Before After 7 14

Log10 CFU/g sausageb and (pH)24 7.0 9.7 9.0 8.5

(5.8) (5.1) (5.3) (5.5)48 7.0 10.3 9.4 9.2

(5.8) (4.3) (4.3) (4.4)aModel sausages were prepared from ground pork trim with glucose (1%), sodiumchloride (2.5%), sodium nitrite (5 ppm), sodium ascorbate (550 ppm) and 107 CFU ofJCM1131T/g meat. They were fermented at 37C for 2448h.bViable cells of lactobacilli in sausages were determined by plating on MRS agar (37C,48h).

Table 4Survival of L. gasseri JCM1131T suspended in low pH so-lutions adjusted with HCl

Incubation time (h)pH 0 0.5 1.0 3.0 5.0

Viable countsa (log 10 CFU/mL1 7.0 3.7 2 2 22 7.0 6.4 5.1 2 23 7.0 6.9 6.6 6.2 6.0

aViable cells of lactobacilli in low pH solutions were determined by plating on MRS agar(37C, 48h).

Table 5Bile-tolerance of L. gasseri JCM1131T in MRS agar plates

Bile concentration (ppm)pH 0 125 250 500 1000 2000

Bacterial growtha

5 6 7

aSymbols: 2, no growth; , , , degree of growth.

species among the lactobacilli in human intestinal tracts (Benno etal., 1989). Bacterial adhesion to the intestinal surface is important toenhance long-term survival in the intestinal tract. The ability of bac-teria to adhere to intestinal epithelial cells is probably to a large ex-tent species specific (Goldin and Gorbach, 1992). Therefore, L. gas-seri including JCM1131T strain would be an appropriate probioticspecies for meat fermentation.

The microstructure of fermented sausages influences the ripen-ing process and survival of pathogenic bacteria, and is an importanthurdle to help maintain quality of fermented meat products (Leist-ner, 1994). Inoculated cells of pathogenic bacteria (S. aureus) in sau-sages were inhibited by L. gasseri JCM1131T during fermentation.From the electron microscopic observation, the density of JCM1131Tcells in sausages seemed to be lower near the surface as compared tothe inside. Since most of the intestinal lactobacilli prefer anaerobicconditions for growth, it is desirable to promote their growth at thesurface which has an anaerobic atmosphere or to eliminate surfacepathogenic bacteria by appropriate treatment. Such surface treatmentsfor meat fermentation often utilize smoking and/or mold starter cul-tures (Lcke, 1994).

Although strain JCM1131T shows a potential for the develop-ment of healthy meat products, this strain was relatively sensitive tosodium chloride and sodium nitrite (Table 2). However, it could beutilized for low-salt, low-nitrite healthy meat products. Since cer-tain Lactobacillus strains can effectively convert meat color withoutnitrite (Arihara et al., 1993) and inhibit the growth of pathogenicbacteria (Daeschel, 1993), combinations of bacterial strains with sev-eral functions probably would be utilized for replacing nitrite. Fur-ther efforts are needed to develop mutants of JCM1131T or to screenmore suitable probiotic bacteria strains for industrial applications.Jessen (1995) described the possibility of the development of healthymeat products by using Lactobacillus or Bifidobacterium in con-junction with low-fat, low-salt meat recipes in a review article.

CONCLUSIONSIT IS POSSIBLE TO USE PROBIOTIC LAB FOR MEAT FERMENTA-tions. The potential is great that meat products with such bacteriamay develop new markets in the near future. Interest directed to ba-sic research and applications in this area are expected to increase.

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Fig. 2Inhibition of growth of S. aureus and enterotoxin produc-tion in model sausages by L. gasseri JCM1131T. Model sausageswere prepared from ground pork trim with glucose (1%), sodiumchloride (2.5%), sodium nitrite (5 ppm), sodium ascorbate (550 ppm).They were inoculated with each of the S. aureus strains (106 CFU/g meat) and were incubated at 37C for 48h with/without L. gasseriJCM1131T (107 CFU/g meat). After incubation, samples were takenfrom the surface of sausages and were counted for S. aureus cellsand detected for enterotoxin production.

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Ms received 10/2/97; revised 11/20/97; accepted 12/29/97.

This work was supported in part by a Grant-in-Aid for Scientific Research (No. 09760245) from theMinistry of Education, Science and Culture of Japan, the School of Veterinary Medicine and AnimalSciences, Kitasato Univ., and Prima Meat Packers, Ltd.Portions of this research were presented at the 42nd International Congress of Meat Science andTechnology, Lillehammer, Norway, September 2, 1996.