2021 Volume 27 Issue 5 Pages 759-768
Lactic acid bacteria (LAB) are known to promote deterioration of quality in meat products, such as discoloration and slime production. In this study, five types of liquid smoke, considered food additives, were assayed for potential anti-spoilage LABs from meat products. The spoilage LABs isolated from meat products were identified as Lactobacillus plantarum, Enterococcus malodoratus, Streptococcus thermophilus, or Carnobacterium maltaromaticum. The liquid smoke product (LS1), made from mixed wood, was more effective in terms of spoilage LAB isolate activities. It had the highest phenol content along with the most varied component among the tested products. In the model sausages containing 0.5% LS1, which was incubated at 30 °C for 5 d for accelerating their deterioration, the enumeration of LAB and staphylococci was significantly lower than that of the control. Therefore, LS1 was suggested to be useful for suppressing not only LAB but also the other bacteria related to spoilage of meat products.
Meat is recognized as having high nutritional value; it is a good source of protein and contains vitamins, minerals, and trace elements. The nutrient-rich environment provides chemicals and physical conditions favorable to the growth of microbes, which makes meat and meat products highly perishable, with colonization and development of a variety of microorganisms, especially bacteria (Nychas et al., 2008; Pennacchia et al., 2011; Chaillou et al., 2015). Lactic acid bacteria (LAB) are used for food processing, in manufacturing the fermented meat products (Laranjo et al., 2019). However, LAB represent a controversial cohort of microbial species as they contribute to spoilage by generating metabolites that cause the subsequent organoleptic downgrading of meat quality, promoting discoloration and slime production (Pothakos et al., 2015). Depending on the type of meat product and the storage conditions, the genera, families, and species of LAB are different and they are associated with the product quality (Schirmer et al., 2009; Pothakos et al., 2015). Thus, it is important to control LAB growth closely during meat processing.
Liquid smoke has been used extensively in food systems to impart flavor characteristics that are similar to smoked food products (Varlet et al., 2010). It may be used to preserve food quality and ensure food safety (Schubring, 2008; Martin et al., 2010). Moreover, components of liquid smoke have been suggested to be effective against various types of spoilage and pathogenic microorganisms (Milly et al., 2005). Liquid smoke is usually obtained from the condensation of wood smoke produced by smoldering wood chips or sawdust under limited oxygen conditions (Montazeri et al., 2013). The major constituents of liquid smoke are water, tar, acids, carbonyl-containing compounds, and phenol derivatives (Baltes et al., 1981); however, the chemical composition of liquid smoke depends primarily on the type and moisture content of the wood, which influences the pyrolysis temperature and duration of smoke generation (Guillen and Ibargoitia, 1999; Montazeri et al., 2013). Therefore, the differences in the liquid smoke components affect the overall organoleptic, antioxidative, and antibacterial properties of the final products (Guillen and Manzanos, 1999; Milly et al., 2005; Wei et al., 2010). These factors identify the potential of liquid smoke to be useful in the preparation of cooked meat products. However, to the best of our knowledge, few detailed studies have investigated the antimicrobial effects of liquid smoke on LAB and other spoilage bacteria in meat products.
The aim of this study was to assay LAB for their participation to the spoilage of meat products. In addition, several types of liquid smoke, which were derived from different woods, were subjected to an antimicrobial activity test with the spoilage LAB isolates from the meat products. Phenol compounds in liquid smoke was then investigated as the active substances for the anti-LAB isolate activity, and the antimicrobial effects of liquid smoke in meat products were demonstrated.
Meat product samples Four kinds of meat products (smoked sausage, uncured and non-smoked sausage, smoked boneless ham, and smoked bacon) were used to isolate LAB in this study. Smoked sausage, smoked boneless ham, and smoked bacon were prepared at Azabu University. Meat for these products were purchased from a local butcher shop (Sagamihara, Kanagawa, Japan). For the preparation of smoked sausage, domestic pork was cut into 8–10 mm pieces, and 2.0% salts (w/w), 1.0% sugar (w/w), 0.1% sodium ascorbate (w/w), 0.02% potassium nitrate (w/w), and 0.01% sodium nitrite (w/w) were mixed and cured at 4 °C for 3 d. Following this, back fat, ice, polyphosphate-agents and a spice mixture (white pepper, garlic powder, nutmeg, glutamate, onion powder, and ginger) were added. The mixture was then ground finely using a silent cutter and stuffed into a sheep casing. For the preparation of smoked boneless ham and smoked bacon, the pork round and belly were injected with a pickle solution (5.0% salt, 2.5% sugar, 0.3% sodium ascorbate, 0.3% glutamate, 0.2% polyphosphate-agents, 0.1% potassium nitrate, 0.05% sodium nitrite, and spices) and soaked in the solution for one week at 4 °C. Smoked sausage, smoked boneless ham, and smoked bacon were dried at 55 °C for 30 min, smoked with cherry wood chips at 75 °C for 1 h, and cooked to an internal temperature of 75 °C. The products were then cooled, vacuum-packed in a sanitary plastic bag, and heated at 90 °C for 1 min to sterilize in a water bath. The uncured and non-smoked sausages used in this study were purchased from a local supermarket (Sagamihara, Kanagawa, Japan).
Isolation and identification of LAB from the tested meat products To isolate LAB strains involved in the spoilage of meat products, the tested products were incubated at 30 °C for 7 d. After incubation, the meat products were cut with a sanitary knife and homogenized in 9 mL of sterilized 0.85% saline using the ULTRA-TURRAX T25 (IKA Japan, Osaka, Japan) at 8 000 rpm for 1 min on ice. To isolate LAB from the products, the modified glucose yeast peptone (GYP) agar was used (Takeda et al., 2011). Serial dilutions in 100 µL aliquots (10−1 to 10−8) were prepared and spread on modified GYP agar plates, which were incubated anaerobically at 37 °C for 3 d with anaerobic packs and jars (Mitsubishi Gas Chemical, Tokyo, Japan). The colonies were purified by streak plating on new plates of the same agar. Purified bacterial strains were suspended in 10% glycerol solution and stored at −80 °C.
For preliminary bacterial identification, the bacteria were stained by Gram-staining and tested for catalase activity; Gram-positive and catalase-negative bacteria were tentatively classified as LAB, and then identified based on the 800 base pair (bp) sequences of the 5′-end of the 16S ribosomal RNA gene. DNA was extracted from bacterial colonies using ISOFECAL for Beads Beating (Nippon Gene, Tokyo, Japan). For DNA extraction, bead beating was performed at 4 000 rpm using a Shakeman3 BMS-SMN03 (Biomedical Science, Tokyo, Japan). PCR was carried out using the Prime Taq DNA Polymerase kit (GENETBIO, Daejeon, Korea) in a thermal cycler (582BR iCrycler, Bio-Rad Laboratories, CA, USA) following the manufacturers' instructions. The primers (forward: 5′- GTTTGATCCTGGCTCA-3′, reverse: 5′-TACCAGGGTATCTAATCC-3′) were used for PCR. The cycling program consisted of an initial denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, with a final extension at 72 °C for 5 min. Amplified and purified 16S rDNA was sequenced by DNA sequence analysis at Fasmac Co., Ltd (Kanagawa, Japan). A homology search was performed using the BLAST search in the Bioinformation and DNA Data Bank of the Japan Center. The LAB species were identified using BLAST results and a score higher than 98%.
The primer p11 (5′- GTTTCGCTCC -3′) was used for randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) (Takeda et al., 2011). The reactions were performed in a 25 µL volume containing 1.0 µL of bacterial DNA, 2.0 µL of deoxyribonucleoside triphosphate mixture (GENETBIO), 2.5 µL of 10× buffer (GENETBIO), 0.2 µL of 5 units/mL Taq DNA polymerase (GENETBIO), 1.0 µL of primer (50 mmol/L), and 18.8 µL of pure water. The PCR cycling conditions were 94 °C for 4 min, followed by 40 cycles of 94 °C for 1 min, 37 °C for 1 min, and 72 °C for 2 min. The RAPD products were separated electrophoretically at 100 V on a 2.5% (w/v) agarose gel, and the products were detected using UltraPower DNA Stain (Gellex International, Tokyo, Japan) with an InGenius3 CCD imager (Gellex International).
Liquid smoke In this study, five types of liquid smoke products were examined. Mixed-chip LS1 (made of maple (Acer), oak (Quercus), and hickory (Carya)) and the mixed chip LS2 (made of oak (Quercus) and beech (Fagus)) were used. The LS3, LS4, and LS5 were made of hickory (Carya), maple (Acer), and sakura (wild cherry) chips, respectively (Table 1). A voucher of liquid smoke was deposited at Logos Co. (Kyoto, Japan).
| Liquid Smoke Number | Chip source | Acidity (%) | Phenols (%) | Carbonyls (%) |
|---|---|---|---|---|
| LS1 | Maple (Acer), Oak (Quercus) and Hickory (Carya) | 11.3 | 31.1 | 11.1 |
| LS2 | Oak (Quercus) and Beech (Fagus) | 10.9 | 5.1 | 2.2 |
| LS3 | Hickory (Carya) | 12.4 | 24.8 | 15.5 |
| LS4 | Maple (Acer) | 5.6 | 13.2 | 7.1 |
| LS5 | Sakura (Wild cherry) | 1.4 | 4.1 | 1.8 |
Voucher of these liquid smoke were deposited at Logos co., Kyoto, Japan.
Anti-microbial activity of liquid smoke on the LAB The liquid smoke was filtered through a 0.2 µm pore-size filter (KURABO, Osaka, Japan) prior to use. LAB isolates were grown in modified GYP broth, inoculated onto GYP 0.7% agar, and poured into Petri dishes. The paper disk was then placed on the agar, 100 µL of filtered liquid smoke was inoculated on the disk, and the agar was incubated at 30 °C for 48 h. After incubation, the diameters of the LAB inhibition zones were measured.
In addition, the colony counts of LAB isolates in the GYP broth containing liquid smoke were assayed. After the LAB isolates were grown in GYP broth, they were inoculated at 1.0% (v/v) into the GYP broth with filtered liquid smoke and/or sterilized distilled water and incubated at 30 °C for 48 h. Liquid smoke was added at 0.1% (v/v, low dose), 1.0% (v/v, middle dose) and 10.0% (v/v, high dose) in all isolates. After incubation, the colony-forming units of the LAB cultures were estimated using GYP agar plates. Briefly, the cultured GYP broth was serially diluted with sterilized 0.85% saline, and the diluent was cultured with GYP agar at 30 °C followed by counting the LAB colonies by pour plate method.
GC-MS of phenol compounds in liquid smoke Liquid smoke (0.5 mL) was diluted with 4.5 mL of distilled water, and 2.0 mL n-hexane was added to the solution. The mixture was homogenized thoroughly for 1 min and centrifuged at 3 000 rpm for 10 min. The supernatant hexane layer was collected and used for GC-MS analysis using a GC system (Agilent 6890) and a mass-selective detector (MSD) (type 5973; Agilent Technologies, Palo Alto, CA, USA). Samples were analyzed on an HP-5ms column (30 m × 0.25 mm × 0.25 µm film thickness; Agilent). The injection port was programmed to 250 °C, retained for 5 min, and then the sample was injected in split mode (1:10). The carrier gas (helium) flowed at a rate of 1.0 mL min−1. Toluene-d8 was used as an internal standard, and MSD was used for chemical identification. The ionization energy voltage was set to 70 eV, and the temperatures of the ion source, quadrupole mass filter, and transfer line were set to 230, 150, and 250 °C, respectively. The total ion current was monitored to record the chromatograms, and the scanning range was 40–450 m/z. The library search was performed using a Wiley mass spectral database (Wiley NIST 2008 MS Spectra, Agilent G1035B) to identify the compounds.
Estimation of microbial count in the model sausage For the model sausage, minced pork and 2.0% salts (w/w), 1.0% sugar (w/w), 0.1% sodium ascorbate (w/w), 0.02% potassium nitrate (w/w), and 0.01% sodium nitrite (w/w) were mixed and cured at 4 °C for 3 d. After curing, the liquid smoke (0.0%, 0.1%, and 0.5% of the meat) and spice mixture described above were added and ground finely. Sterilized water was used as a control to replace liquid smoke. The mixture was stuffed into a sheep casing, dried at 55 °C for 30 min, and cooked to an internal temperature of 75 °C. After cooling, the products were vacuum-sealed in a sanitary pack and heated at 90 °C for 1 min to sterilize in a water bath. The products were then incubated at 30 °C for 7 d for accelerating their deterioration.
The incubated model sausages were subjected to a microbial count. The model sausages were cut using a sanitized knife and thereafter homogenized. The homogenized sausage was serially diluted with sterilized 0.85% saline solution. Next the diluent was cultured and the following microbial groups were determined: The aerobic bacteria were estimated at 37 °C for 48 h using the standard method agar (Nissui Pharmaceutical, Tokyo, Japan); lactic acid bacteria were estimated at 37 °C for 48–72 h using plate count agar with BCP (Nissui Pharmaceutical) supplemented with 10 ppm sodium azide and cycloheximide; staphylococci were estimated and cultured at 37 °C for 48 h with Pearlcore mannitol salt agar (Eiken Chemical, Tokyo, Japan) supplemented with 10% sterilized egg yolk solution (v/v) (Kyokuto Pharmaceutical Industrial, Tokyo, Japan); psychrophilic bacteria were estimated at 25 °C for 48 h using CVT agar (Eiken Chemical); coliform bacteria were estimated at 37 °C for 24 h with desoxycholate agar (Nissui Pharmaceutical); and yeast and molds were estimated at 25 °C for 5 d with Pearlcore potato dextrose agar (Eiken Chemical) with chloramphenicol at a concentration of 100 mg/L. The culturing conditions were followed by the instructions of each manufacturer.
Statistical analysis The results of the LAB inhibition circle and the colony forming units of microbes in the tested cultures and meat products were analyzed by one-way ANOVA, followed by the Tukey-Kramer test for multiple comparisons. GraphPad Prism software was used for the statistical analyses. In this study, differences with p-values less than 0.05 were considered statistically significant.
Identification of LAB from meat products A total of 35 potential LAB candidates were collected for preliminary identification from the tested smoked sausage, smoked boneless ham, uncured and non-smoked sausage, and smoked sausage, and smoked bacon (Table 2). Using the 16S rRNA sequencing method, eighteen strains were isolated from the tested smoked sausage and classified into three species, Lactobacillus (L.) plantarum, Streptococcus (S.) thermophilus, and Enterococcus (E.) malodoratus with a detection rate of 61.1%, 22.2%, and 16.6% respectively. In addition, the ten isolates detected from the tested smoked boneless ham were E. malodoratus (70.0%) and L. plantarum (30.0%); six isolates detected from the uncured and non-smoked sausages were S. thermophilus (50.0%), E. malodoratus (33.3%), and Carnobacterium (C.) maltaromaticum (16.6%); and the single isolate detected from smoked bacon was L. plantarum (100.0%) (Table 2). Among these isolates, the strains identified as L. plantarum, E. malodoratus, and S. thermophilus were subjected to RAPD-PCR to investigate their homology in the same LAB species, respectively (Fig. 1).
| Meat products | LAB species | Detected number | Rate (%) |
|---|---|---|---|
| Smoked sausage | Lactobacillus plantarum | 11 | 61.1 |
| Streptococcus thermophilus | 4 | 22.2 | |
| Enterococcus malodoratus | 3 | 16.6 | |
| Total | 18 | 100 | |
| Smoked boneless ham | Enterococcus malodoratus | 7 | 70.0 |
| Lactobacillus plantarum | 3 | 30.0 | |
| Total | 10 | 100 | |
| Non-cured and non-smoked sausage | Streptococcus thermophilus | 3 | 50.0 |
| Enterococcus malodoratus | 2 | 33.3 | |
| Carnobacterium maltaromaticum | 1 | 16.6 | |
| Total | 6 | 100 | |
| Smoked bacon | Lactobacillus plantarum | 1 | 100 |
| Total | 1 | 100 |
The tested meat products were prepared in three lots, respectively.
RAPD PCR profiles of the LAB isolates from the tested meat products
(A), (B), and (C) show RAPD PCR profiles of L. plantarum (lanes 1–15), E. malodoratus (lanes 1–12), and S. thermophilus (lanes 1–7) isolates from the incubated meat products, respectively. Lane M is a marker of DNA size.
The differences in amplified DNA products were scarcely observed in all strains (lane no. 1–15 in Fig. 1A), E. malodoratus strains (no. 1–12 in Fig. 1B), and S. thermophilus strains (no. 1–7 in Fig. 1C). The differences in the DNA product patterns were confirmed by unweighted pair group method with arithmetic mean (UPGMA) cluster analysis from the amplified DNA product patterns. Based on the results, the genotypes of strains in L. plantarum, E. malodoratus, and S. thermophilus were classified under the same cluster at an allowance of 5%, indicating that they are likely to be the same strain (data not shown).
Thus, L. plantarum strain no. 15, E. malodoratus strain no. 12, and S. thermophilus strain no. 7 were used in subsequent experiments to represent each species group.
In this study, the isolates from four kinds of meat products, incubated at 30 °C were classified as L. plantarum, S. thermophilus, E. malodoratus, and C. maltaromaticum, indicating that they were potentially associated with spoilage of tested meat products. LAB is often reported to be the major spoilage bacteria in meat products, particularly L. plantarum and C. maltaromaticum (Chenoll et al., 2007; Laursen et al., 2005; Mills et al., 2018). The detection number and rate of LAB species varied in the meat products used in this study. The prevalent bacterial species may differ depending on the meat product item and/or the processing conditions, such as curing and storage, as was reported previously (Miller et al., 2015). In addition, according to the RAPD analysis, since the isolates identified as L. plantarum, S. thermophilus, and E. malodoratus from each product were suggested to be the same strains, they could be derived from the same origin, such as a certain ingredient, or the laboratory environment.
Anti-LAB isolate activity of liquid smoke The anti-LAB isolate activity of liquid smoke was investigated by a paper disk assay. All tested liquid smoke exhibited zones of inhibition against the tested LAB isolates (Fig. 2). Inhibition zone of LS1, which was made from wood tips of Maple (Acer), Oak (Quercus), and Hickory (Carya) was significantly larger than that of other liquid smokes in all tested LAB (p < 0.05), except for LS3. Inhibition zone of LS3, which was made from the Hickory (Carya) tip, was significantly larger than that of LS2 in L. plantarum strain no.15 and E. malodoratus strain no. 12 (p < 0.05).
Paper disc assay for anti-LAB isolates activities of liquid smoke
Values represent the mean ± SD of three independent experiments. Values with different small letters indicate significant differences for each LAB strain (p < 0.05).
In the colony-count assay, all tested liquid smokes demonstrated the remarkable suppression against each LAB isolate in the high dose group (10% liquid smoke) (Fig. 3A). Although the colony forming of L. plantarum strain no.15 and E. malodoratus strain no. 12 was observed in LS2, it was significantly lower than those of the control (p < 0.05). LS1 and LS3 significantly suppressed the colony counts of all tested LAB isolates compared with the control and other liquid smoke in the middle dose group (1% liquid smoke) (Fig. 3B) (p < 0.05). It was also notable that the colony counts of L. plantarum strain no.15 and E. malodoratus strain no. 12 in LS1 middle dose were clearly lower than those counts in LS2 high dose. In the low dose group (0.1% liquid smoke), LS1 significantly decreased the colony forming numbers of all tested LAB isolates compared with those of the other liquid smokes, except for the L. plantarum strain no.15 (Fig. 3C). However, LS3 could not suppress the LAB colony-forming units in the low dose group. Thus, LS1 had the highest anti-LAB activity among all the tested liquid smoke samples.
Colony-count assay for anti-LAB isolates activities of liquid smoke
Liquid smoke was added to the broth at 10% (v/v, high dose), 1.0% (v/v, middle dose) (B) and 0.1% (v/v, low dose) (C). Values represent the mean ± SD of three independent experiments. Values with different small letters indicate significant differences for each LAB strain (p < 0.05).
In this study, the liquid smoke of LS1 showed remarkably antimicrobial activity against all tested LAB strains in a paper disk assay and colony-count assay (Fig. 2 and Fig. 3). Even though LS2, LS3, LS4, and LS5 demonstrated anti-LAB activities in their high dose group of colony-count assay and paper disk diffusion assays, no significant reduction was observed in the colony-count assay of all the tested LAB strains in the middle and low dose groups of LS2, LS4, and LS5 (Fig. 2 and Fig. 3). Also, LS3 was demonstrated anti-LAB activity in the middle dose, but not in the low dose group. Thus, the anti-LAB activity of tested each liquid smoke was suggested to vary, and it would be caused by the difference of components of the liquid smoke products. In addition, the colony forming of L. plantarum strain no.15 and E. malodoratus strain no. 12 appeared in LS2 high-dose group, which being significantly suppressed (Fig. 3A). In contrast, S. thermophiles strain no.7 and C. maltaromaticum strain no.1 were not detected in this group. The antimicrobial activity of smoke or liquid smoke was reported to be related to the presence of phenol compounds, aldehydes, and organic acids that alter the permeability of the microorganism membranes, causing damage to the point of intracellular medium leakage, especially in Gram-positive bacteria (Milly et al., 2005; Holley and Patel, 2005; Guilbaud et al., 2008). Thus, the susceptibility of LAB strains to liquid smoke might vary with the character of cell construction of each strain.
Phenolic compounds present in liquid smoke The phenolic compounds included in liquid smoke were assayed with GC/MS, and the results are presented in Table 3. The total relative area for phenol in LS1 was the highest in the tested liquid smoke similar to the ingredient data in Table 1. In addition, there are most kinds of phenol components in LS1 among the tested liquid smokes (Table 1). On the other hand, the total relative area and the number of phenols in LS2 were the lowest among the tested products, respectively. All detected phenol compounds in LS1 were higher than those of the other products, except for ‘4-ethyl-2-methoxy-phenol’. In addition, 1,2-benzenediol (catechol), 2,6-dimethoxy-phenol, and 1-(4-hydroxy-3,5-dimethoxy phenyl)-ethanone were the three most abundant phenol compounds in LS1.
| Component | Retention time (min) | LS1 | LS2 | LS3 | LS4 | LS5 |
|---|---|---|---|---|---|---|
| Phenol | 7.027 | 152.4 | 53.4 | 197.5 | 100.1 | 33.9 |
| 2-methyl-phenol | 8.219 | 147.0 | — | 119.8 | 66.1 | 16.8 |
| 2-methoxy-phenol | 8.822 | 339.9 | 24.9 | 182.0 | 109.1 | 47.9 |
| 2,4-dimethyl-phenol | 9.653 | 85.4 | 5.4 | 52.5 | 40.8 | — |
| 4-ethyl-2-methoxy-phenol | 10.36 | 205.1 | — | 480.4 | 269.0 | 89.5 |
| 1,2-benzenediol (catechol) | 10.392 | 766.7 | 32.7 | — | — | — |
| 3-methyl-1,2-benzenediol | 11.309 | 260.4 | 11.8 | 139.0 | 95.9 | 26.6 |
| 3-methoxy-1,2-benzenediol | 11.395 | 209.0 | 26.3 | 42.0 | 46.2 | 50.2 |
| 4-ethyl-2-methoxy-phenol | 11.594 | 116.1 | — | 59.9 | 45.6 | 21.5 |
| 4-methyl-catechol (4-methyl-1,2-benzenediol) | 11.668 | 344.5 | — | 107.7 | 84.3 | 20.4 |
| 2,6-dimethoxy-phenol | 12.652 | 584.1 | 419.8 | 447.4 | 313.4 | 256.8 |
| 2-methoxy-4-phenol | 12.739 | 77.4 | — | 16.0 | — | — |
| 4-ethyl-1,3-benzenediol | 12.801 | 209.6 | — | 92.9 | 60.3 | — |
| 1-(4-hydroxy-3,5-dimethoxy phenyl)-ethanone | 17.318 | 581.9 | — | 156.8 | 94.7 | 76.3 |
| Total | 10762.1 | 1282.0 | 5793.4 | 3449.8 | 1957.5 |
The data were analyzed, based on the internal standard (Toluene-d8).
The values express the average in triplicate. The crossbars mean an undetected.
The component was searched from the database in a Wiley mass spectral database.
Milly et al. (2005) reported that the carbonyls in liquid smoke appear to be the driving factor behind its antimicrobial efficacy against microorganisms including LAB. This study initially observed the ingredient data to investigate the compounds responsible for the antimicrobial activity of liquid smoke. As shown in Table 1, LS1 demonstrating notable anti-LAB activity had the highest concentration of phenol among all the tested products of liquid smoke. Phenols were thereafter used as candidates for anti-LAB activity in this study. From the GC/MS results, the detected phenolic compound in LS1 was most various, and almost all the phenolic compounds were detected in a higher quantity than the other products (Table 3). According to Fig. 3, some of the anti-LAB activities of LS1 middle dose were observed to be larger than the activities of LS2 high dose. Thus, the phenolic various components as well as higher level in LS1 could contribute to suppress the inhibitory zones and colony-forming units of the tested LAB. In a previous study, 2,6-dimethoxy-phenol, 2-methoxy-phenol, and 1,2-benzenediol were the most abundant phenolic compounds in full-strength liquid smoke (Montazeri et al., 2013). As shown in Table 3, these compounds were also detected in LS1, and 1,2-benzenediol and 2,6-dimethoxy-phenol had particularly high levels compared with the other phenols. In addition, a 2-methyl- phenol, which is an important contributor to palatable smoke-curing, was also present in LS1 at a higher concentration among all the tested products (Kostyra and Baryłko-Pikielna, 2006). Thus, LS1 is thought to be the most palatable liquid smoke as well as anti-LAB activity in the tested products.
Microbe count of sausage with added liquid smoke LS1 Model sausages with 0.25% and 0.5% LS1 were prepared, along with sausage without LS1 as a control. The concentration of 0.5% LS1 would be used as the acceptable maximum level for product flavor in the preparation. When the products were incubated at 30 °C for 7 d, the aerobic bacterial count increased. On day 5 of incubation, the products without LS1 reached counts of 7.68 ± 0.46 log10 CFU/g, which was the upper level. The aerobic bacterial count on day 7 of incubation was 6.99 ± 0.95 log10 CFU/g. The microbial counts on day 5 of incubation are shown in Table 4. The aerobic bacterial count of sausages containing 0.5% LS1 showed a decreasing trend compared with those without LS1, but no significant differences were observed. The estimation of LAB and staphylococci, both positive and negative egg yolk reactions, in sausages containing 0.5% LS1 were significantly lower than those without LS1 and with 0.25% LS1 (p < 0.05). In addition, psychrophilic bacteria, coliform bacteria, yeast, and mold did not appear in any sausages.
| Microbe | Number of microbe (Log10 CFU/g) | ||
|---|---|---|---|
| 0.0% LS1 | 0.25% LS1 | 0.5% LS1 | |
| Aerobic bacteria | 7.68 ± 0.46 | 7.06 ± 0.52 | 6.94 ± 0.71 |
| Lactic acid bacteria | 7.63 ± 0.54a | 6.69 ± 0.52a | 6.05 ± 0.47b |
| Staphylococci | |||
| egg yolk reaction positive | 7.94 ± 0.43a | 6.69 ± 0.79a | 5.91 ± 0.57b |
| egg yolk reaction negative | 6.35 ± 0.50a | 6.17 ± 1.87a | 4.31 ± 0.59b |
| Psychrophilic bacteria | ND | ND | ND |
| Coliform bacteria | ND | ND | ND |
| Yeast and Mold | ND | ND | ND |
Values represent as the mean ± SD in the three independent results.
ND means not detected.
The different small letters show the significant differences in each microbe group.
In this study, the aerobic bacterial counts of products without LS1 reached 7.68 ± 0.46 log10 CFU/g on day 5 of incubation. It is generally recognized that microbial spoilage of meat occurs when counts reach the level of 7.0–8.0 log10 CFU/g (Reid et al., 2017; Cauchie et al., 2020); therefore, spoilage of the incubated sausage in this study was related to the growth of microbes during incubation. As shown in Table 4, a significant reduction in viable counts of LAB, and positive and negative staphylococci egg yolk reactions in the incubated sausages containing 0.5% LS1 were observed compared with the levels in the sausages without LS1, although the number of aerobic bacteria was not significant. Generally, the spoilage-associated organisms in meat are in the Pseudomonadaceae, Listeriaceae, Enterobacteriaceae, Staphylococcaceae, Shewanellaceae, Aeromonadaceae, Moraxellaceae, and Lactobacillaceae families (Zhang et al., 2012; Cesare et al., 2018; Cauchie et al., 2020). LS1 was suggested to suppress the growth of Staphylococcaceae and LAB related to the spoilage of sausages (Table 4).
In conclusion, this study demonstrated the isolation of LAB related to spoilage of meat products, and that liquid smoke LS1 demonstrated the highest antimicrobial activity against these LAB isolates. The relative amount of phenol compounds and their varieties included in the LS1 was greater than that of the other liquid smoke in the GC/MS analysis. In the accelerating deterioration test, the bacterial numbers of Staphylococci and LAB in the model sausage with 0.5% LS1 were significantly lower than that of the control. Therefore, liquid smoke, particularly LS1, was suggested to be a useful additive to suppress not only LAB but also the other bacteria related to the spoilage of meat products. The antimicrobial activity of liquid smoke would be concerned with the higher level of various phenolic components.
Acknowledgements We thank Ms. Mizuki Hagita, Ms. Yui Kato, and Ms. Chiharu Takagi (Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University) for their technical assistance in this study. This study was supported by the JSPS KAKENHI Grant-in-Aid for Young Scientists Number 18K13024 and Scientific Research (B) Number 19H03109, and the Ito Foundation Research Grant (2019).
Conflict of interest There are no conflicts of interest to declare.
