Tailed Pepper (Piper cubeba) L. berries extract reduced number of microbial population in tofu

The family of Piperaceae contains the species P. cubeba L., which has been used as a
spice in countries such as Malaysia, Indonesia, India, Morocco, and Europe. A previous
study has shown that crude extracts of tailed pepper (P. cubeba L.) have antimicrobial
activities against foodborne pathogens species. The excellent antimicrobial activity of P.
cubeba L. berries extract makes it suitable for use as a natural preservative or sanitizer in
the food. The aim of this study was to evaluate the effect of the P. cubeba L. berries
extract on microbial population in tofu. The tofu samples were cut into small pieces and
mixed thoroughly to ensure the homogeneity of natural microflora. Ten grams of the
samples were immersed in 20 mL varying concentrations of P. cubeba L. extract; 0.00%,
0.05%, 0.50% and 5.00% for 1, 2 and 4 hrs at room temperature (23±2°C) with the
agitation of 50 rpm. At 1, 2 and 4 hrs the numbers of total plate count (TPC), Bacillus
cereus, coliform and Escherichia coli were counted. The result shows that a reduction of
at least 3 Log10 CFU/g of TPC, Bacillus cereus., coliform and E. coli in tofu samples was
observed when the samples were treated with 0.50% extract for four hours. The result
suggested that P. cubeba L. berries extract can be used as a natural preservative to reduce
the microbial load in raw food.


Introduction
Globally, the actual challenge faced by the food industry is addressing consumers' concern regarding food safety, quality and conservation of foods. This is done by limiting the use of synthetic chemical additives (Negi, 2012). A wide variety of plant products have been evaluated to determine the feasibility of using natural antioxidants to preserve and improve the overall quality of food products, especially meat and meat by-products (Shah et al., 2014).
Natural products contain ingredients that have a promising potential of new types of therapeutic agents (Newman et al., 2003). Globally, there are about 500,000 species of plants; however, considering that phytochemical investigations have been performed on only 1% of these plant species, it is very likely that novel bioactive compounds would be discovered in the future (Palombo, 2011). Piper cubeba L. is commonly known as a condiment and is widely used in food preparation as a flavor enhancer and to impart spiciness (Alharbi et al., 2017). In the food industry, the apparent antimicrobial activities of P. cubeba L. extract could be exploited for use as a natural preservative or sanitizer (Junqueira et al., 2007;Khan and Siddiqui, 2007;Aneja et al., 2010;Parvez et al., 2010;Nahak and Sahu, 2011;Al-Tememy, 2013;Alsaid et al., 2013).
The scientific name of cubeb or tailed pepper (named on account of the attached stalks) is Piper cubeba L. The tailed pepper which a perennial climbing plant is also known in Indonesia as the Javanese pepper (Al-Tememy, 2013). The pepper normally has between four and six leaves, round branches and a climbing stem, and it is usually about half an inch long and between half an inch to two inches wide (Al-Tememy, 2013). There are over 700 species under the genus Piper which can be found in both hemispheres of the earth. The family of Piperaceae contains the species P. cubeba L., which has been used as a spice in countries such as Indonesia, India, Morocco, and Europe since the middle ages (Silva et al., 2007).
For the past decade, plants that are generally categorized as Generally Recognized as Safe (GRAS) for humans are frequently associated with food consumption (Negi, 2012). Without complementary biological aspects, the discovery of new medicinal plants/-extracts/constituents is more of pure phytochemistry (Meyer et al., 1982). Thus, plant extracts for food applications must be declared to be biologically safe prior to be consumed by humans, Therefore, the sanitizing effect of P. cubeba L. extract on tofu cake samples was assessed.

Tofu samples
The tofu samples used in the present study were purchased from the local market near Universiti Putra Malaysia (UPM), Sri Kembangan, Selangor, on August 2018 and taken to the laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia (UPM), where immediate microbiological analysis was performed. B. cereus, Escherichia coli and coliform bacteria were analyzed according to the protocols described in the Bacteriological Analytical Manual, Food and Drug Administration. Lee et al. (2017) recommended homogenizing 10 g of tofu with 90 mL of 0.85% sterile saline (8.5 g/L of sodium chloride; Sigma Aldrich) for two mins using a stomacher (Laboratory Blender Stomacher 400; Seward, London, UK). Then, 1 mL of the homogenate was serially diluted tenfold using 9 mL of 0.85% sterile saline (8.5 g/L of sodium chloride; Sigma Aldrich), and 1 mL of the diluted solution spread on each selective media. Mannitol egg yolk polymyxin agar base (Oxoid) supplemented with egg yolk emulsion was used as the selective media for B. cereus. The plates were incubated at 30°C-37°C for 24-48 hrs and the colonies of B. cereus, E. coli, and coliforms were counted.

P. cubeba L. berries samples and extraction
The dried P. cubeba L. (Piperaceae) berries employed in this study was obtained from a market selling traditional herbs in Pasar Baru Bandung, Indonesia. The P. cubeba L. was collected in April 2015 in a plantation in Jatiroto, Temanggung, Central Java, Indonesia. The Department of Biology, Institut Teknologi Bandung (Indonesia) authenticated the berries on the basis of Flora of Java (Backer and Van de Brink, 1968). A voucher specimen (HBG10PC01) was stored at the Herbarium Bandungense. The procured material was air-dried and put in storage at the Laboratory of Natural Products, Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM). A powerful blender (Waring, model 32 BL 80, New Hartford, USA) was used to pulverized the dried berries into fine power. The powdered P. cubeba L. sample was stored in an airtight polyethylene plastic bag and put in storage in a -80°C fridge.
The extraction of Piper cubeba L. was done utilizing the soaked method illustrated by Rukayadi et al. (2008). The organic solvent used in the extraction of P. cubeba L. is absolute methanol (R&M Chemicals, 99.8%). Dried P. cubeba L. berries of 100 g were ground to obtain as a powder. Sample extraction was performed using 400 mL room-temperature solvent and 48 hrs of conventional shaking. Filtration of the plant extract was done using Whatman filter paper size No. 2 (Whatman International Ltd., Middlesex, England). Following this, the extracts were concentrated using a rotary vacuum evaporator (Heidolph VV2011, Schwabach, Germany) at 40°C for 3 -4 hrs to obtain a methanol extract of the dried P. cubeba L. berries. The temperature of the rotary evaporator was increased to 85°C for 2 × 30 s at the end of the extraction process to ensure that the extract is methanol-free (Madiha et al., 2017). Finally, the extracts were freezedried for 48 hrs to eliminate water.
The stock extract of methanol was primed by dispersing crude extract of P. cubeba L. in 100% dimethyl sulfoxide (DMSO) (Fisher Scientific, Leicestershire, United Kingdom) to obtain a 100 mg/mL concentration. The stock extract was put in storage at 4°C up to the time it is ready for use. Further dilution of the solution was made using sterile deionized water, ddH2O to obtain three different concentrations of 0.05%, 0.50% FULL PAPER eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources and 5.00% P. cubeba L. extract which were then used in the treatment of tofu samples (Rukayadi et al., 2013).

Preparation of selective media agar
Different selective media of three types were used for the enumeration of specific microflora in tofu samples. Plate count agar was used for the enumeration of total bacteria colonies. The selection of media was based on Microbiology Manual (12 th ed, Merck, Darmstadt, Germany). Table 1 shows the preparations of selective media and the appearance of bacterial colonies on the selective media.

Treatment of tofu samples with P. cubeba L. extract solution
The tofu samples were cut into small pieces and mixed thoroughly to ensure the homogeneity of natural microflora. Samples of 10 g were immersed in 20 mL of tap water and varying concentrations of P. cubeba L. extract (0.00%, 0.05%, 0.50% and 5.00%) for 1, 2 and 4 hrs at room temperature (23±2°C) and were agitated. A 10 g of the sample without immersion in any sample solvent served as the control sample. Treatments were conducted under aseptic condition. A total of 10 g the sample was homogenized with 90 mL of 0.85% sterile saline (8.5 g/L of sodium chloride; SigmaeAldrich) for two minutes using a stomacher. An aliquot (1 mL) of the homogenate was then serially diluted tenfold with 9 mL of 0.85% sterile saline (8.5 g/L of sodium chloride; SigmaeAldrich). The sample was homogenized using a stomacher machine (BagMixer 400-P, Interscience, France). The enumeration of microorganisms was performed using the spread plate method. Fifty micrograms of the dilution was spread on the selective agar plates in duplicates. The colony forming unit per ml (CFU/g) was counted after 24 hrs of incubation at 37°C (Lee et al., 2017).

Statistical analysis
The experiment was performed in two replications with three repetitions each time (n = 2 × 3). The data were expressed as a mean ± standard deviation. Excel (v. 2010), Graph Pad Prism version 6.00 for Windows (v. 6.00, Graph Pad Software, San Diego, CA, USA) was employed to perform the statistical analysis. Results are given as a mean of three replicates ± SD. The significant difference at P<0.05 was established by performing ANOVA.

Extraction yield
Dried Piper cubeba L. berries were extracted using absolute methanol 99.9% (v/v) and the yields of the crude extract were presented in Table 2. The yield of herbal extracts are influenced by the types of soaking solvent, the ratio of soaking solvent, type of extraction technique, and soaking period (Sultana et al., 2009;Abdullah et al., 2015).
The first and important step in utilizing herbal plant is extraction. Extraction generally yields the desired chemical compounds/components which are then subjected to further separation, purification and characterization. The basic steps involved in extraction are pre-washing of plants/parts of medicinal plants, drying, grinding, and combining the samples with extraction solvents, filtration, and finally evaporation of the solvent prior to use/storage. Samples have to be pulverized to ensure their homogeneity. Moreover, this step would produce a much higher surface area of the sample, thereby enhancing the kinetics of analytic extraction. The extraction must be done following appropriate steps to prevent loss of potential active constituents and to ensure that the components are not destroyed or distorted (Sasidharan et al., 2011).
In this study, the crude extract was obtained using absolute methanol and the yield of the crude extract is 19.90%. There is no previous report regarding the yield of P. cubeba L. crude extract that uses methanol as a solvent. It is important to emphasise that the use of methanol as a solvent for food applications is not recommended. Even though the present study used methanol for extraction, precaution was taken to ensure that the final crude extract did not contain methanol residue. As the extraction process was almost complete, the temperature of the rotary evaporator was increased to 85°C for 2 × 30 s (Madiha et al., 2017). Since the boiling temperature of methanol is 50°C, the methanol is expected to completely evaporate at this temperature. This confirms that the P. cubeba L. crude extract obtained in this study is a methanol-free extract.

Effect of P. cubeba L. extract on microbial population in tofu
Plant products in the form of extracts, essential oils, and others with protuberant antimicrobial activities have remarkably emphasized for their possible applications in food production. This corresponds with the increasing awareness of the need to explore novel natural antimicrobial agents in food microbiology in an effort to inhibit and kill bacteria and fungal growth (Belguith et al., 2009).
Tofu is a popular soy-based food in East Asian countries and is a cheap source of high-quality protein.
Tofu contains approximately 6.0%-8.4% protein and 79%-87% water and has an almost neutral pH (5.2-6.2). The particularly high protein and moisture content of tofu make it a very good medium for microorganisms (Ashraf et al., 1999). Microorganisms have been reported to easily contaminate tofu even when the tofu is refrigerated. Spoiled tofu has a sour taste and an unpleasant smell due to bacterial growth. Hence, compared to other products, tofu is a delicate product with a very short shelf life. The preservation of tofu is influenced by environmental factors such as bacterial load, storage temperature, air composition, and manufacturing process (Lee et al., 2017). Table 3 shows the TPC bacterial count in tofu samples with filtered tap water and P. cubeba L. extract at varying concentrations of 0.00%, 0.05%, 0.50% and 5.00% (v/v) as a possible natural preservative. The initial TPC on the tofu samples were significantly reduced for 1 Log 10 CFU/g, from 6.77±0.71 to 5.44±0.43 Log 10 CFU/g when exposed to 5.00% extract for two hours ( Table 3). The count was further reduced to 4.34±0.08 Log10 CFU/ g when exposed to 5.00% extract for four hours. The tofu exposed to tap water and 0.00% extract did not show a significant reduction in the TPC at 1, 2 and 4 hours of treatment time. The total plate count for exposure to 5.00% (v/v) extract for one hour is not significantly different than those exposed to 0.50%, 0.05%, tap water and 0.00%.

Effect of P. cubeba L. extract on total plate count (TPC) population in tofu
Total Plate Count (TPC) is commonly used to measure the level of hygiene in food processing plants and the overall degree of bacterial contamination (Edris et al., 2015). The TPC in tofu sample is 7.69±0.11 Log 10 CFU/g. The initial count for tofu samples with B. cereus, coliform and E. coli are 4.87±0.18, 5.88±0.14 and 6.72±0.07 Log 10 CFU/g, respectively. Previous studies have reported identifying the presence of pathogenic bacteria in tofu, such as coliform bacteria, Pseudomonas spp., E. coli, Enterococcus spp., lactic acid bacteria, B. cereus, Staphylococcus spp., Salmonella spp., Yersinia sp., and Cronobacter sakazakii (Ananchaipattana et al., 2012). There is also a report of Enterobacteriaceae, Enterococci, Staphylococci, and yeast being detected in tofu, where the total aerobic mesophilic count is 10 5   (Lee et al., 2017).

Effect of P. cubeba L. extract on Bacillus cereus population in tofu
A 5.00% (v/v) extract of B. cereus with an initial load of 4.87±0.18 Log 10 CFU/g was able to reduce bacteria count to zero after treatment for 2 and 4 hours. The count was significantly reduced to 3.33±0.37 Log 10 CFU/g when the tofu was exposed to a 0.50% (v/v) extract for two hours and 3.09±0.17 counts after four hours of exposure. Treatment with tap water and 0.00% extract did not result in any significant reduction of B. cereus count (Table 4). According to the Food Standards Australia New Zealand, the permitted level of B. cereus in food is below 10 2 CFU/g. A count greater than or equal to 10 4 CFU/g is considered unsafe and may result in immediate recall of foods for further inspection. However, in the UK the level is only regarded as hazardous if it is greater than 10 5 CFU/g. The allowable limit in the UK is below 10 3 CFU/g (Bilung et al., 2016).
Foodborne bacteria produce endospores that are resistant to heat. They are able to grow at temperatures of between 10 -48°C, and the optimum temperature for growth range from 28 to 35°C. Treatments for disinfection are high temperature (for canning) and lowering pH. The pathogenesis of B. cereus depends primarily on the dose consumed and the site of the bacteria that produce the toxin in either food or intestine. The bacterial infection is manifested as emesis and diarrhoea (Bilung et al., 2016).

Effect of P. cubeba L. extract on E. coli population in tofu
The initial count of E. coli in the tofu samples is 6.72±0.07 Log 10 CFU/g. TPC was significantly reduced to 4.63±0.34, 5.25±0.27 and 4.95±0.05 Log 10 CFU/g when the tofu was exposed to 5.00% and 0.50% (v/v) extract for 1, 2 and 4 hours, respectively (Table 5). A significant reduction of about 2.09 Log 10 CFU/g was observed after one hour of treatment with 5.00% extract. Exposing the E. coli to 5.00% extracts of P. cubeba L. for two and four hours resulted in the death of all E. coli. However, exposing the E. coli to 0.05% extracts for one and two hours produced similar results as exposing the bacteria to tap water and 0.00% extract.

Effect of P. cubeba L. extract on coliform bacteria population in tofu
The presence of coliforms is a general indication of sanitary condition or water contamination in the food processing environment (Edris et al., 2015). The initial coliform count was found to be greater than B. cereus count in tofu, which are 4.87±0.18 and 5.88±0.14 Log 10 FULL PAPER Bacteria Bacillus cereus (Log 10 CFU/g)  CFU/g, respectively. The coliform count was significantly reduced to 5.18±0.26 Log 10 CFU/g and 4.79±0.25, respectively after two and four hours of treatment with a 0.50% extract. The coliform count is zero when the samples were exposed to a 5.00% extract for two and four hours. Exposure to 0.50% extract exposed for one hour did not result in any significant difference in coliform counts in contrast to exposure to tap water and 0.05% and 0.00% extracts (Table 6).
Preventing microbial spoilage of tofu is considered an important research topic in an attempt to ensure food safety (Serrazanetti et al., 2013;Rossi et al., 2016). According to Ananchaipattana et al. (2012), the sour taste of tofu is associated and characterized by its spoilage and bacterial growth. Information on the major bacteria responsible for the putrefaction of tofu is very scarce even though Acinetobacter calcoaceticus, Bacillus cereus, Klebsiella pneumoniae and Xenorhabdus luminescens have been reported to be the main causes of tofu spoilage. Likewise, coliform bacteria have been frequently identified in fresh tofu, and high levels of Escherichia coli have been identified in soybean curd being transported from the producers to retail stores (Tuttemwong and Fung, 1991;Ashraf et al., 1999).
In addition, numerous Yersiniosis outbreaks have been associated with tofu, and one case has been reported in Seattle and Washington State between December 1981 and February 1982. Several occurrences of tofu contamination with Listeria monocytogenes have also been reported (Ananchaipattana et al., 2012). In a 2010/2011 study carried out in Thailand, B. cereus was isolated from 40% of unpackaged tofu obtained from open markets (n=74) and 41% of packaged tofu (n=59) from supermarkets. Of the 54 B. cereus isolates from the tofu, four of the strains have been identified to produce diarrhoeal enterotoxin (Ananchaipattana et al., 2012).
In terms of public health, tofu is classified as a ready -to-eat and potentially hazardous food. Tofu has been implicated in many outbreaks of foodborne illness. In a Yersiniosis outbreak in Seattle, the water used to process tofu was identified as a major source of contamination. An outbreak of shigellosis has also been related to contaminated tofu. Yet another incident of Listeria monocytogenes contamination has resulted in a product recall. Several studies have identified the presence of coliform bacteria in tofu (Ashraf et al., 1999). The degree of contamination by coliform bacteria, E. coli, Salmonella sp. and Enterococcus sp. in unpackaged tofu samples is considerably higher than those of packed tofu (Ananchaipattana et al., 2012).
The values reported in the present study are higher than those recommended by "The Soy Food Association of America" for soy milk, which states that mesophilic bacteria counts should not exceed 2 x 10 4 CFU/mL (TSAA, 1996;Ribeiro et al., 2017). Rossi et al. (2016) reported the presence of mesophilic bacteria in all samples obtained from fresh tofu, and that the values are one to three Log higher than those for soybean samples obtained from the same industry. These findings showed that the processing of tofu in manufacturing industries create conditions (addition of water, grinding, coagulation, molding, etc.) that favor natural enrichment of microbial load. Any failure in the process would encourage the proliferation of unacceptable microorganisms.
Results of bacterial count on the tofu which have been exposed to different concentrations of P. cubeba L. for varying durations vary widely. In general, a significant reduction in bacterial population was observed at P. cubeba L. extract of 0.05% regardless of the duration of exposure. Interestingly, a bacterial count reduction of 3 Log 10 CFU/g and greater were observed at 0.50% and 5.00% concentration of the extract. This shows that the reduction in the bacterial count is strongly correlated with a higher concentration of extract and treatment time.

Conclusion
In conclusion, the reduction in the bacterial count is strongly correlated with the utilization of higher  Table 6. Effects of filtered tap water and different concentrations of P. cubeba L. extract on coliform bacteria in tofu samples after exposure for 1, 2 and 4 hours at room temperature (28±2°C).
Values are expressed as mean±SD. Different superscript lowercase letters in the same column are significantly different (P<0.05) while different superscript uppercase letters in the same row are significantly different (P<0.05).
eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources concentration of P. cubeba L. extract and longer treatment time. Overall, a significant (P<0.05) bacterial load reduction was observed when the tofu samples were treated with a minimum concentration of 0.05% (v/v). An extract concentration of 5.00% (v/v) seems to be the ideal antimicrobial concentration considering in that it resulted in a 3 Log 10 reduction of bacterial load. Extract with a concentration of 0.50% (v/v) resulted in a reduction of bacterial load of at least 3 Log 10 . In conclusion, on the basis of exposure time and concentration of extract used to treat the tofu samples, the relative effective combinations which result in sanitizing/antimicrobial activity is 0.50% (v/v) P. cubeba L. extract for four hours.