Since many years, the food packaging industry has been using disposable paper based materials such as boxes, bags, cups, containers, plates, sacks, and tissue papers. The biggest advantage of using such materials in place of other materials is that they do not affect the environment and are biodegradable in nature  . Due to changes in operating conditions, laws, strict international competition and increasing demand from the consumers, the role of maintaining proper hygiene and management of microbes in the paper and food packaging industry has increased  . There should not be any transfer of components from food packaging material to food in any such way that it could affect the health of human beings, bring a change in the food product in a way that is not acceptable or degrades the taste and odor of food  . During paper production, microbial contamination can occur from various sources like water which has been recycled, use of raw materials, various parts of the machine and from the environment of the factory  . In the environment of producing and processing paper, a large number of microbes are present but only few of these microbes are harmful. The microbes which produce slime or are filamentous in nature are harmful to the process, while the microbes which produce toxins and odor are harmful to the safety of end products  . Odor in the food packaging PPBs is generated due to enzymatic and non-enzymatic oxidation of fatty acids  . PPBs contain large amounts of saturated and unsaturated fatty acids  . B. subtilis and P. aeroginosa, produce enzymes like peroxidases and lipoxygenases respectively   , which act as catalysts for inserting oxygen in fatty acids which contain methylene interrupted dienes causing oxidation of fatty acids by enzyme action and hence odor generation in the PPBs food packages. Globin proteins (hemoglobin and myoglobin) release ferric ion at high temperatures, and catalyze the production of odorous volatile oxidation products of fatty acids  . This is especially undesirable in producing aseptic food packages as it lowers the hygienic quality of the product  . Pseudomonas sp. and Bacillus sp. are the pre-dominant bacteria present in agro-wastes  . The raw materials used for the purpose of PPBs are biodegradable in nature. However, the disadvantage of this fact is that microbes may grow in such a natural biodegradable raw material and cause contamination in the PPBs  . Presently, there are very few published reports on the bacteriological safety of food PPBs. Thus, the knowledge on Indian food PPBs containing bacterial species and their potential to cause food borne diseases and odor generation is also less. The purpose of this research is to gain knowledge on the bacteria present in Indian food PPBs with various contents of pulp fiber, as the first step for creating a basis for assessing the risk to packaged food.
2. Materials and Methods
2.1. Sample Collection
A total of 10 samples were collected from local nearby markets of Saharanpur in India in January 2019. The samples included paper plate, cake box, fruit tray, tissue paper, coffee cup, pastry box, sweet box, pizza box, French fries box, and paper bag. In additional testing, 3 samples of each group of food packaging PPBs were purchased from the local retail outlets.
2.2. Examination of Pulp Fiber % in the Samples
The pulp fiber % in the samples was determined by IS 5285 (1998): Fiber Analysis of Paper and Board-Methods of Test [CHD 15: Paper and its products] method.
2.3. Examination of the Total Number of Microorganisms in the Samples
The international standard method which is based on the disintegration of paper (ISO 8784-1:2005) was used to examine the total number of colony forming units (CFU) of bacteria. 1% fiber suspension was obtained by disintegrating 1 g of sample paper in cooled sterile Ringers solution. In the nutrient agar petri plates different dilutions of the fiber suspension were plated. Petri plates were then placed in an incubator at a temperature of 37˚C ± 1˚C for the duration of 48 ± 3 h. After the incubation was complete, the total number of CFUs was estimated and the concentrations of bacteria present in 1 g of paper were calculated. The average values obtained by carrying out the experiments in duplicate are presented in this study  .
2.4. Isolation of Pure Colonies by Streak Plate Method
A sterilized transfer needle was used to obtain a loop of bacteria from the grown colonies. A solidified nutrient agar plate was taken and the loop of bacteria in the sterilized transfer needle was then streaked on it to make a series of non-overlapping, parallel streaks. The plates were incubated at 35˚C, in an inverted position for the time of around 48 - 72 hours to obtain bacteria that are pure in nature i.e. bacteria which has been obtained from a single cell  .
2.5. Biochemical Tests
Morphological and Physiological tests (Gram stain, Agar slant cultural characteristics, Starch hydrolysis, Lactose, Dextrose, Sucrose, H2S production, Indole production, MR reaction, VP reaction, Citrate use, Urease activity, Catalase activity, and Oxidase activity) were carried out on the pure bacterial colonies using conventional methods. The characteristics of bacterial species were identified based on the Bergey’s Manual (Claus and Berkeley 1986).
3. Results and Discussions
The bacterial contamination of Indian food packaging PPBs with various content of pulp fiber was investigated. Our findings showed that from the 10 tested food packaging PPBs, bacteria was present in all the samples. Table 1 summarizes the results obtained from the bacterial analysis of the total numbers of bacteria in the food packaging PPBs. Total number of bacteria ranged from 1.3 × 102 to 6.1 × 103 cfu/g. Our results are comparable with Mohammadzadeh findings which showed the range of 0.2 × 103 to > 1 × 105 cfu/g bacterial contamination in food packaging paperboards  . According to FDA declaration, the
Table 1. Total number of bacteria identified in the examined samples.
limit value recommendations for packaging materials is 2.5 × 102 bacterial cfu/g   . In our study, samples of tissue paper contained less bacteria than the permitted concentration, while all the other samples contained bacteria in more than the permitted concentration. The minimum bacteria number was found in tissue paper (1.3 × 102) and the maximum bacteria number was found in fruit tray (6.1 × 103). A study by Guzi?ska et al. also shows that the number of microbes that were estimated in paperboard are much higher than in paper as shown by our results too  .
Based on the colony morphology, gram staining and biochemical tests, four different bacterial strains were identified which included B. cereus, B. subtilis, S. aureus and P. aeroginosa (Figure 1).
Out of 10 samples which were tested, three samples were contaminated with B. subtilis, four samples with B. cereus, two samples with S. aureus and one sample with P. aeroginisa (Table 2).
The various % of pulp fibers in the samples are shown in Figure 2. In comparison to chemical pulping, the mechanical pulping causes the disintegration of wood into fibers to yield pulp having different characteristics. The pulp still contains the lignin due to which although the yield is higher, but the strength is low. A paper mill is generally equipped with the production of mechanical pulp  . In chemical pulping kraft process is the most commonly used process for a variety of end uses  . The maximum % of agro-residues, softwood mechanical pulp, softwood chemical pulp and hardwood chemical pulp was present in cake box, pizza box, coffee cup and tissue paper respectively, while the minimum % of agro-residues, softwood mechanical pulp, softwood chemical pulp and hardwood chemical pulp was present in coffee cup, paper plate, french fries box and paper bag respectively.
During 1998-2008, FBD outbreak survillenace system showed one out of ten FBD were caused due to B. cereus and S. aureus. Thus, the presence of these
(a) (b) (c) (d)
Figure 1. Microscopic images of identified bacteria (a) B. cereus, (b) B. subtilis, (c) S. aureus, (d) P. aeroginosa.
microbes in food packaging PPBs imposes potential hazard for consumers   . In our study, B. cereus has been identified in cake box, pastry box, sweet box, and pizza box while, S. aureus has been identified in coffee cup and french fries box. The outcome of McCusky Gendron et al. showed that the samples they tested also contained microorganisms especially of Bacillus species  . It is believed that forming heat-resistant spores which describe its survival during the drying phase of paper board machine operation can be the main reason for finding Bacillus genera  . The aerobic bacteria genera mainly found in the paper industry include Baciilus and Pseudomonas  . P. aeroginosa was found in fruit tray samples while B. subtilis was present in paper plate, tissue paper, and paper bag. This is in agreement with Ibrahim and Sobeih finding who evaluated the effect of packaging containers on the bacteriological profile of cheese, the bacterial isolates in their study also included B. subtilis  . B. subtilis is a good source for the production of peroxidase enzyme  and a novel and thermostable lipoxygenase enzyme has been identified and characterized in P. aerogiosa  , these enzymes act as catalysts to insert oxygen into fatty acid with methylene interrupted dienes contributing to oxidation of fatty acids by enzymes and hence odor generation in food packaging PPBs. Similar to these enzymes, globin proteins (hemoglobin and myoglobin) release ferric ion at high
Table 2. Biochemical tests.
+ = positive, − = negative, +, − = variable reaction, a = acid.
temperatures, and catalyze the production of odorous volatile oxidation products of fatty acids  .
The Association of Food and Drug Officials (AFDO), states that if the simple process of packaging or repackaging operations does not occur under strict aseptic conditions and environment then it can result in contamination with pathogens. Testing for these organisms at particular control points prepares the best means of quality control. Contamination can be prevented by following the best techniques of continuous surveillance and good manufacturing practices  .
It is essential to monitor the level of bacterial exposure to consumers through food PPBs, especially when the food comes in contact with the packages. The results inferred by the present study give an idea about the number and type of bacteria present in the Indian food PPBs that may cause health hazards, generate odors, etc. The source of bacteria Bacillus and Pseudomonas has been identified in agro-residues present in the raw material. The percentage of other pulp fibers like softwood mechanical pulp, softwood chemical pulp, and hardwood chemical pulp in the samples has also been determined. In summary, this is a comprehensive research on the occurrence of bacteria in various samples of Indian food packaging PPBs. The findings of this study will aid in understanding
(a) (b) (c) (d)
Figure 2. % of pulp fiber in food PPB samples.
of the potential risk of rejection of food packages due to odorous compounds generation, which are catalyzed by enzymes produced by bacteria.
The authors are grateful to Central Instrumentation Facility, IIT Roorkee (SRE Campus) and Ministry of Human Resource Development (MHRD), India; fellowship contingency for carrying out this work.
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