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Validation of Method in Microbial Limit Tests for Two Types of Health Foods

Article  in  Annual Review of Food Science and Technology · May 2015

DOI: 10.19026/ajfst.7.2524

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Advance Journal of Food Science and Technology 7(11): 872-874, 2015
ISSN: 2042-4868; e-ISSN: 2042-4876
© Maxwell Scientific Organization, 2015
Submitted: November ‎3, ‎2014 Accepted: December ‎18, ‎2014 Published: April 10, 2015

Validation of Method in Microbial Limit Tests for Two Types of Health Foods
1
Xihong Zhao, 1Tao Song, 2Jian Li, 3Nanjing Zhong and 4Weicheng Hu
1
Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering
and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, Hubei,
2
Department of Parasitology, Hubei University of Medicine, Shiyan, Hubei, P.R.,
3
Nanjing Zhong, School of Food Science, Guangdong Pharmaceutical University, Zhongshan 528458,
4
Jiangsu Key Laboratory for EcoAgricultural Biotechnology Around Hongze Lake, School of Life
Sciences, Huaiyin Normal University, Huaian 223300, China

Abstract: In order to discuss that whether the methods of total aerobic microbial count and absence bacterial test
were appropriate in microbial limit tests for health foods hygiene. As the requirement of microorganism limits test
and validation on the Chinese Pharmacopoeia 2010, the microbial examination method of two kinds of health foods
has been validated. It is found that vitamin B was antibacterial for Bacillus subtilis and Aspergillus niger. The
results indicated the method in foods microbial limit test GB/T4789-2010 were not suitable for the microbial limit
tests of health foods. It is suggested that setting more reasonable method by validation as well as referring to the
Chinese Pharmacopoeia 2010.

Keywords: Health foods, microbiological examination, validation

INTRODUCTION necessity and feasibility of the health food

microbiological testing methods based on the validation
With the development of economy and society, of Chinese Pharmacopoeia 2010 method.
consumer demands in the field of food production have
changed considerably. Consumers more and more MATERIALS AND METHODS
believe that foods contribute directly to their health.
Today foods are not intended to only satisfy hunger and Media and reagents: Lactose bile enrichment broth,
to provide necessary nutrients for humans but also to sodium tellurite broth enrichment medium, rose red
prevent nutrition-related diseases and improve physical sodium agar, nutrient agar, nutrient broth, improved
and mental well-being of the consumers (Jones and Martin agar, improved Martin agar broth, blood nutrient
Jew, 2007). Generally, a health (functional) food can be agar, eosin methylene blue agar, 4-Methylumbelliferyl-
defined as in addition to their basic nutritive value and Glucosidase (MUG), Salmonella agar and Shigella agar
natural being, will contain the proper balance of were offered by the China pharmaceutical and
ingredients which will help us to function better and biological products.
more effectively in many aspects of our lives, including
helping us directly in the prevention and treatment of Samples: Protein powder and vitamin B were
illness and disease (Goldberg, 1994). In this regard, purchased from the local supermarket.
functional foods play an outstanding role. The
increasing demand on such foods can be explained by Strain: Escherichia coli [CMCC B44102], Bacillus
the increasing cost of healthcare, the steady increase in subtilis [CMCC63501], Staphylococcus aureus [CMCC
life expectancy and the desire of older people for 26003], Streptococcus hemolytic-β [CMCC32210],
improved quality of their later years (Siro et al., 2008). Aspergillus niger [CMCC98003], Shigella flexneri
Microbiology testing is an important issue for their [CMCC51572] and Salmonella [CMCC50094] were
security of functional food. Nowadays, the food from Guangdong provincial institute of microbial
microbiology test method is mainly in accordance with culture collection center.
GB4789-2010 (GB, 2010). However, the Chinese
Pharmacopoeia 2010 is considered the validation of this Preparation of bacteria for bactericidal assay: A 1-
microbial limit test method, which is different of mL sample of a broth culture of a strain of Escherichia
GB4789-2010 (Committee, 2010). In this study, several coli, Bacillus subtilis, Staphylococcus aureus,
types of health food were used to investigate the Streptococcus hemolytic-β, Aspergillus niger, Shigella

Corresponding Author: Jian Li, Department of Parasitology, Hubei University of Medicine, Shiyan, Hubei, P.R. China
872
 Adv. J. Food Sci. Technol., 7(11): 872-874, 2015

flexneri and Salmonella was added to a 1.9-mL Table 1: Microbial limit tests for recovery (conventional method %)
microfuge tube and the bacteria were pelleted by Sample Test times B. subtilis A. niger
Protein powder 1 82.4 88.00
centrifugation in a microfuge at 12, 000 rpm for 30s. 2 73.0 105.9
After the removal of the supernatant, 1 mL of sterile 3 79.0 86.40
PBS was added to the pellet and the pellet was Vitamin B 1 0.00 0.000
resuspended by gentle aspiration in and out of a transfer 2 0.00 0.000
3 0.00 0.000
pipette. Then, 10-fold dilutions by PBS buffer to final
30-200 CFU/mL dilution grade (Friedman et al., 2002). Table 2: Recovery of dilution method (0.2 mL per plate, %)
Sample Test times B. subtilis A. niger
Validation of method: The microbial limit test method Vitamin B 1 78.5 100.0
referring to the Chinese Pharmacopoeia 2010 were 2 84.1 88.20
3 71.8 98.20
performed three times independently in parallel
experiments. The experimental based on GB4789-2010
method. (EPA) personnel, a written study plan should be
prepared for each step of the validation process and
RESULTS AND DISCUSSION subjected to appropriate review prior to
implementation. In addition, it is expected that all
Conventional method: B. subtilis and A. niger on laboratories involved in each step of a validation
behalf of gram-positive bacteria and fungus process will have a quality system or Quality Assurance
respectively, were tested by the method GB4789-2010 (QA) program in place to ensure standardization of
for recoveries for each bacteria. As shown in Table 1, laboratory operations, as well as adequate Quality
the protein powder plate method using B. subtilis and A. Control (QC) activities (Parshionikar et al., 2009).
niger recoveries were up more than 70%. According to In this study, two different types of health food
the Chinese Pharmacopoeia 2010 not less than 70% of were investigated for inhibitory effect. The results
the provisions of the above, protein powder can be showed that vitamin B had the strong inhibitory effect
considered there is no significant inhibitory effect for on gram-positive bacteria and fungus. Therefore, it can
gram-positive bacteria and fungus. However, the conclude that vitamin B is a major cause of these health
recovery of vitamin B was obvious less than 70%, it food ingredients antibacterial. The protein powder
meant that it was necessary to eliminate or reduce the showed no significant inhibitory effect. The health
antibacterial activity. foods usually contain extracts or chemical additives, the
experimental results showed these ingredients
Medium dilution method: The per dish was added themselves may affect microbial growth, according to
trail solution 0.2 mL, inoculated B. subtilis and A. the national standard GB4789-2010 microbiological
niger, then added medium 15-20 mL for incubation 18- examination of food hygiene methods for the health
24 h, finally counts observation and measured food limitations. As this reason, it is very necessary to
recoveries. As shown in Table 2, the recovery of these eliminate or reduce the antibacterial activity by using
two positive bacteria were able to more than 70% by medium dilution method or membrane filtration method
using the medium dilution method (per dish 0.2 mL), and other methods, in order to make the test results
this results indicated that the use of the medium dilution more scientific, reasonable and reliable.
method can effectively eliminate its antibacterial Validation is a process that determines the fitness
activity and increase the accuracy on total number of of an assay, which has been properly developed,
colonies test. optimized and standardized, for an intended purpose.
All diagnostic assays (laboratory and on-site assays)
Method validation of control bacterias: When should be validated for the species in which they will be
developing a microbiological test for foods or drugs, it used (Jacobson, 1998). Method validation is the process
should find a way to validate the methods used to check of determining if a method is suitable for its intended
the foods or drug suitable for the control of bacteria. purpose. Based on the issue of EPA guidance,
From the Table 3, the trail team detected each validation can be classified as primary validation and
bacterial growing normally and the negative control secondary validation according to its purpose. Primary
detected none growing. It indicated that there was no validation is an exploratory process for establishing the
inhibitory effect on the control bacterial using the operational limits and performance characteristics of a
medium dilution method for vitamin B. It was specific new, modified or otherwise inadequately characterized
and could meet the microbiological testing method. Secondary validation on the other hand, is the
requirements for verification purpose. process of gathering evidence that a laboratory can
It should be noted that successful validation of any meet the specifications established in primary
analytical method is very difficult without thorough and validation (Parshionikar et al., 2009). In this
systematic planning and preparation. According to the experiment, two types of health food of bacteria, mold
guidance to U.S. Environmental Protection Agency and yeast counting method, coliform bacteria and
873
 Adv. J. Food Sci. Technol., 7(11): 872-874, 2015

Table 3: Results of absence bacteria test

E. coli Salmonella Shigella flexneri S. aureus Streptococcus hemolytic-β
Vitamin B + + + + +
Negative control - - - - -
“+”: Indicated growing normally; “-”: Indicated growing none

testing methods were methodological study established. GB, 2010. National Food Safety Standard Food
This study may provide a reference as primary Microbiological Examination: Aerobic Plate Count
validation for subsequent microbiological testing and [S]. Ministry of Health of the People’s Republic of
validation of health food. China.
Goldberg, I., 1994. Functional Foods: Designer Foods,
ACKNOWLEDGMENT Pharmafoods, Nutraceuticals. Chapman and Hall,
New York.
This study was supported by a collegial research Jacobson, R., 1998. Principles of validation of
start-up grant (contract/grant number 2013QDJZR04 to diagnostic assays XA9848640 for infectious
Jian Li), a key discipline construction project diseases. Diagnosis and Epidemiology of Animal
(contract/grant number 2014XKJSXJ04 to Guangyu Diseases in Latin America, pp: 15.
Zhang) and a basic research exploration project Jones, P.J. and S. Jew, 2007. Functional food
(contract/grant number 2014XKJSTS01 to Jian Li) development: Concept to reality. Trends Food Sci.
funded by the Hubei University of Medicine. Tech., 18(7): 387-390.
Parshionikar, S., R.A. Haugland, O. Shanks, M.E. Hunt,
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