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The purposes of the industrial attachment programme is to nurture students to be effective and productive to their future employment organizations, as the industrial attachment programme supplements NTU's in-house practical professional training, lessen on-the-job training(s) of students, and/or instill the positive work attitude and professionalism.
Company profile overview
Henri Nestlé, a trained pharmacist, founded Nestlé Company in 1866. With the motivation of searching for a healthy and economical alternative of infant nutrition for mothers who were unable to breastfeed, he developed a baby cereal: Farine Lactée Henri Nestlé (translates to baby cereal). With this new product, Henri Nestlé hoped to achieve his ultimate goal of combating infant mortality due to malnutrition.
In the 1920s, Nestlé expanded beyond its traditional product line of milk-related products. After Chocolate--the Company's second most important product line, new products followed steadily: Milo (powdered beverage), powdered buttermilk for infants, and in 1938, Nescafé, the popular household coffee.
Focusing on convenient, nutritious, delicious foods and beverages, Nestlé is committed to becoming the very best nutrition, health and Wellness Company, and stay on at the leading edge in key scientific disciplines in order to remain at the forefront of the Food Industry.
The Company Logo
Nestlé actually means 'little nest' In German dialect. The Nestlé logo with the nest was adopted as his own coat of arms and trademark in the Food Industry. The logo, a nest containing a mother bird and her babies, stands for security, maternity and affection, nature and nourishment, family and tradition.
Brands and Products
The Nestlé Gold Collection -Nestlé Gold - the very best chocolates from Nestlé.
Have a break, have a KIT KATÂ®Created in 1935, and known as Row tree Chocolate Crisp.
MILKYBARÂ®has a distinctive taste profile and milk content up to 50% to 100% higher than for ordinary milk chocolate.
NESVITAâ„¢ NESTUMÂ® 3-in-1 Cereals NESTUMÂ® 3in1 Cereal. It is the first in the market with low in fat and less sugar.
NESTLÉ BLISS Yoghurt Drink ~ The No.1 Yoghurt drink
More Malt, More Milk MILOÂ®
Produced in Singapore, the new formula MILOÂ® boasts of more malt, more milk and is produced using the best ingredients from around the world!
MAGGIÂ® HCS Low Fat NoodleMAGGIÂ® LOW FAT NOODLES - The Ultimate Healthier Choice,
Best Enjoyed without Guilt!
MAGGIÂ® Stock -No Added MSGfor healthy and wholesome meals.
MAGGIÂ® HCS Sauces - Chilli &Tomato--The No. 1 brand of Chilli and Tomato Sauces in Singapore! Now with up to 40% LESS Salt.
Complete Liquid Nutrition for Children
The world's leading energy bar, POWERBARÂ®PERFORMANCE has fuelled more than 200 Olympic athletes.
Nestlé R&D Center (Pte) Ltd, Singapore
As one of Nestlé S.A.'s international network of 17 R&D centers, Nestle Research and Development Centers (R&D) Singapore, works in close collaboration with the Product Technology Centers to meet regional R&D requirements and provide input to local application groups. It is a vital component in delivering local taste and balance to products which support the rapidly growing Asian markets. The R&D work takes into account local tastes and preferences, in addition the cultural and ethnic considerations. These activities are by no means limited to Asia and benefits Nestlé as a whole. In other words, distinctive Asian cuisines are developed to suit the tastes of people all over the world.
There are 9 different departments working together to fulfill Nestlé's objectives and vision in this R&D Center, Singapore, they are Management Services, Culinary, Milo Hub, Noodles, Nutrition & Manufacturing Support, Engineering & Pilot Plant, Food Science & QA (Quality Assurance), Product Design, Packaging & Design, NQAC (Nestlé Quality Assurance Center) . Owing to the close cooperation of all the 9 departments, Nestlé has contributed towards the well-being of Singaporeans not only through its high quality products, but has also played a role in social development and environment conservation.
Introduction to Project involved during attachment
The objective of the project within the industrial attachment is to improve the yield of hydrolysis and inhibit growth of pathogenic micro-organism by various approaches in the production of bio-hydrolysate. During the attachment, challenge test was assigned to project the contamination of Bacillus Cereus at certain level in the beginning of Plant protein Koji hydrolysis process.
Work Nature and Scope
The work nature of this project is mainly laboratory analysis and pilot simulation.
At the beginning of the attachment, training on general microbiological techniques and the operation of laboratory equipment were conducted. After preparation for background knowledge, laboratory scale hydrolysis runs were started on the inhibition of general pathogen growth, following with microbiological analysis of the fermentation. Many other tasks were undertaken during the internship period. Nevertheless, minor tasks that are of low relevance to main scope of will not be discussed in details in this report.
The attachment lasted 22 weeks from 11th Jan 2010 to 12th Jun 2010.
REVIEW OF THEORY AND PREVIOUS WORK
Literature review on Fermentation
Fermentation, one of the oldest methods for food processing, is cardinally important to the food industry as it can be considered as a desirable microbial activity in foods. Traditionally, fermentation was used as a conservation technique for raw food material. After the fermentation, the product usually has a much longer shelf-life than the unfermented one. Fermented food products such as bread, beer, wine, soy sauce, yoghurt, and cheese have been known for a long time.
Preservation of fermented foods depends on the principles of oxidation of carbohydrates and the related derivatives to generate end-products - generally acids, alcohol and carbon dioxide. As a result of partially oxidation, the food retains sufficient energy potential to be nutritional benefit to the consumer.
During the fermentation process by food grade (lactic acid) bacteria or yeasts, food safety can be improved by the production of (lactic) acid or ethanol by these micro-organisms. The conditions generated by the fermentation are important in ensuring the microbiological safety of the products. The available carbohydrates decrease after fermentation process; they are transformed into a range of small organic molecules which exhibit antimicrobial activity, the most common being lactic, acetic, and propionic acids. In addition to the production of these inhibitory primary metabolites, many other antimicrobial components can be formed by different protective micro-organisms.
Moreover, the end-products of carbohydrate catabolism by those bacteria involved in fermentation process contribute to the flavor, aroma and texture of the products. Fermentation may also improve the nutritional quality of the food by increasing digestibility.
General introduction to Koji
Koji was the major component of the hydrolysis substrate in the hydrolysis challenge test conducted during the attachment.
The word "Koji" means mouldy grains, which is derived from the Chinese character. The preparation of Koji is regarded as an essential step in fermentation of variety of fermented foods. Generally speaking, the process is the solid substrate cultivation of moulds to produce hydrolytic enzymes on seeds, for example, wheat or other cereals. Classes of enzymes inside Koji can catalyze the degradation of solid raw materials to soluble products, providing fermentable substrates for yeast and bacteria in the following fermentation stage. Besides the enzymes, several chemicals necessary for the fermentation process are also produced during Koji making. 
The advantages of Koji have generated numerous researches leading to prospective application and technological improvement. It can be used to produce several of non-food products such as antibiotics, as well as a step to provide enzymes and chemicals for subsequent food processing.
Both whole wheat and wheat flour can be commonly used for the production of Koji, they are chosen as the raw materials due to the advantages listed below:
The less moisture content inside wheat inhibits the growth of undesirable micro-organisms, while adequate for mould growth;
Wheat assists in motivating the growth of Koji moulds;
Wheat serves as the precursors of sugars, alcohols and organic acid;
Wheat is a rich source of glutamic acid.
Usually whole wheat is first roasted and then coarsely crushed, or wheat flour which is generally steamed before use, it is to satisfy the following two contradictory factors:
To make the alpha-starch content or the enzymatic digestibility starch to a maximum level;
To make the enzymatic digestibility of protein to a maximum level.
2.2.3 Koji making procedures
The procedures of Koji making is described in figure 1 below:
Koji mould seed culture 0.1-0.2% weight of that of raw materials
Moisture content 40-45%
Culturing mould (Koji making) for 48-72 hours
Moisture content of Koji 25-35%, pH=6.5-7.0
Figure - Koji making flowchart
As illustrated in the figure, once inoculated the material with a small amount of seed mould or the pure culture of Aspergillus oryzae or A. sojae, the mixture is then spread to a large perforated stainless steel plate. The raw materials treated with heat are aerated for 2-3 days with temperature and moisture controlled air, which comes up from the bottom holes through the layer of fermenting material to give the proper conditions for mould cultivation and enzyme formation. The temperature of materials is kept at around 30oC, and moisture content of the materials decreases from 40-45% in the beginning of cultivation to 25-35% after 2 or 3 days.
This process allows the mould to grow throughout the mass and provide the enzymes necessary to hydrolyze the protein, starch and other constituents of the materials. Then, Koji, the mould cultured material making is finished. 
Variety of enzymes is involved in Koji, including amylase, cellulose, invertase, lipase and protease. Among those enzymes, amylase plays an important role in degradation of starch and oligosaccharides to provide substantial fermentable sugars for the following process; the proteolytic enzyme has significance on solubilisation of protein into peptides and amino acid, resulting in an increase of the soluble nitrogen yield.
General microbiological techniques practice
Before the start of the challenge test on antibiosis of the protective organism inoculated hydrolysate, it was important to be well prepared for the experiment both in theory and practical areas. As a consequence, general micro analysis techniques were learned from microbiologists in the microbiological laboratory, serving as a prelude to the coming Plant protein Koji hydrolysis challenge test. The main techniques learned were listed below; since the operation procedures were confidential, details will not be mentioned in this report.
Revival of bacteria( protective organism and Bacillus cereus) in the frozen culture;
Preparation of starter medium, performance testing of culture media;
Enumeration of cultural micro-organisms: plate count methods;
Enteriobacteriaceae Enumeration- application of Petri-film;
Detection and enumeration of presumptive Bacillus cereus with MYP agar plates;
Utilization of selective agar plates and broth to identify sort of bacteria.
Those micro techniques were well practiced by doing the routine fermentation sample analyzing work in the microbiological laboratory.
Hydrolysis Challenge Test
The challenge tests were conducted to investigate the reaction of Bacillus cereus on protective organism inoculated Plant protein Koji hydrolysis by projecting the contamination of Bacillus cereus at an initial level of 103-104 cfu/g at the start of Plant protein Koji hydrolysis.
During the hydrolysis process, Protein plant Koji serves as the substrate for hydrolysis; as illustrated in the previous part, it is a mould-cultured material which allows the mould to grow throughout and provides the enzyme necessary to hydrolysis. The digestion of carbon hydroxide by the enzyme proved the sugar required to the protective organisms for their own use. Protective organisms were inoculated into the Koji substrate at the start of hydrolysis and then the process was carried out at a controlled temperature and pH.
The main attention was paid to 2 kinds of bacteria implicated in plant Koji hydrolysis; they were Enterobacteriaceae and Bacillus cereus.
Enterobacteriaceae is rod-shaped Gram-negative bacteria; most occur normally or pathogenically in intestines of humans and other animal. Enterobacteriaceae could be used as a hygiene indicator in rapid hydrolysis process, the initial count of which usually have to be kept at a low level, so that its growth could be suppressed during Hydrolysis by lactic acid bacteria. Therefore, it is important to confirm a good hygiene before each trial, making sure the hydrolysis tank was sallied. If not, the bacteria will grow to a high count level and may affect the process.
Bacillus cereus is aerobic endospore-forming rod-shaped Gram-positive bacteria; it is responsible for a minority of foodborne illnesses (2-5%), causing severe nausea, vomiting and diarrhea. when the consumption of food containing more than 105 cfu/g viable toxigenic B. Cereus,  it can produce preformed, heat-stable toxins (one emetic toxin (ETE) and three different enterotoxins: HBL, Nhe,and EntK) , giving rise to foodborne illness mentioned above.
Materials and methods
After related research and journals were reading, planning for the challenge test under the supervision of MS Or ChengCheng was initiated and approved. Several parameters related to the execution of the experiments needed to be decided, such as the process operation conditions (temperature and pH); the concentration of the plant protein Koji in the hydrolysis substrate; initial amount of micro-organisms inoculated into the hydrolysate in each trial, and so on. After discussion, the experiment parameters were decided and introduced in the following parts.
Plant protein Koji +Water
Hydrolysis Flow diagram
Figure - Flowchart of Hydrolysis
Recipe of Hydrolysis substrate was showed in table 1:
Batch size (Kg)
Plant protein weight (Kg)
Table - Recipe of Substrate
Equipment set up and Process description
2 5-Liter sterilized beakers with stirrer were applied as hydrolysis tanks; Water bath with a wide temperature range used to maintain the system at 30oC. After preparation of the hydrolysis substrate(plant protein Koji, water and protective organism), Hydrolysis was operated in 30oC water bath for 72 hours, and the pH of the hydrolysate was set at 6.0 with adjustment by the 10%-30% sodium hydroxide solution.
Bacillus cereus: 10ml broth culture in inoculation 108 to give the count of 103 -104 cfu/g of Koji substrate respectively.
Approaches --Selection of parameters
Protective organism (cfu/g)
Bacillus cereus (cfu/g)
Table -ÂÂÂÂÂÂÂÂ initial inoculation amount of micro-organisms
After decision about the experiment parameters had been made, it was turn to devise how to execute the experiment. There were mainly 2 queries presented in front: would Bacillus cereus multiply in the Hydrolysis system? If it survived and propagated in the Hydrolysis system, Could the protective organism inoculated plant Koji hydrolysate suppress the growth of Bacillus cereus? It was essential to eliminate any other factors affecting the growth of Bacillus cereus, before investigate the effect of protective organism on Bacillus cereus.
Consequently, trial 1 was conducted to check whether Bacillus cereus can survive and grow in plant protein Koji substrate or not. After making sure that this pathogen could live in the Koji substrate, the following challenge tests investigating the antimicrobial ability of the protective organism were carried on.
Trial 2 was conducted as a control, in order to compare results, while trial 3 and trial 4 were to project the contamination of Bacillus cereus to fulfill the objectives of the tests. Repeating trials were done during the attachment in order to reproduce the promising results.
Revived of bacterial-- obtaining the viable cells.
Revive of the protective organism from frozen culture to new broth;
Revive of the bacillus cereus cocktail in TS diluents, purity and count check of the revived bacteria.
Prepared inoculums for hydrolysis, the components of the inoculums consisted of wheat gluten Koji and water.
Set up equipments: sterilization of fermentors, pH calibration, set parameters of systems.
Observed the aroma and color, recorded the temperature and pH throughout the process hourly.
Sampling and Microbiological analyses
Samples for micro analysis were taken at 0hr, 3hr, 5hr, 8 hr, 24 hr, 30 hr, 48 hr, 55 hr and 72hr to check the count of bacteria. The types of analysis conducted are listed below:
Total plate count
Protective organism count
Bacillus Cereus count
Results and Discussion
Since results of this project are confidential, only one set of data will be mentioned in this report, the detailed data is provided in the appendix.
Growth of Bacillus cereus in Koji substrate:
Figure - Growth of Bacillus cereus
The figure presented how Bacillus cereus would live in plant protein Koji substrate. With an initial count of 103cfu/g, it rose up to 105cfu/g during reproduction of cells and reached a final count of 104 cfu/g. This result bears out that Bacillus cereus can propagate in the Koji substrate.
Growth of protective organism in hydrolysate:
Figure - Growth of protective organism
This figure described how the protective organism grew in Koji hydrolysate, with an initial inoculation level of 106cfu/g to 107 cfu/g protective organism into the Koji substrate, it grow to 109 cfu/g after 72 hours, and went through the lag phase, exponential phase and stationary phase of protective organism during the hydrolysis process. These results showed that protective organism could adapt well to the new environment.
Growth of Enterobacteriaceae in protective organism inoculated hydrolysate
Figure - Growth of EB in protective organism inoculated Hydrolysate
As showed in this graph, Enterobacteriaceae grew at beginning of the process but its growth was suppressed after 30 hours. Enterobacteriaceae was used as a hygiene indicator of the process, and it doesn't endure to a low pH environment. Because of the organic acid produced during Rapid hydrolysis, the growth of Enterobacteriaceae will be restrained. In general, with a low initial count, and the inoculation of protective organism, the count of Enterobacteriaceae was controlled under the effect of organic acid, for the count was under the safety level which is 105 cfu/g.
Growth of Bacillus Cereus in protective organism inoculated hydrolysate
Figure - Growth of BC in protective organism inoculated hydrolysate
This figure showed that Bacillus Cereus didn't proliferate during the Plant Koji hydrolysis at 30oC, pH 6.0; in addition, its visible count dropped to less than 100cfu/g after 30 hours. The potential cause leading to this result might be the organic acid or other molecules generated by protective organism in the course of hydrolysis.
Other observations: Aroma and color of the hydrolysate
Throughout the hydrolysis, it was observed that the aroma and color of the hydrolysate was different between well covered batches and roughly covered ones. Hypothesis was made that, oxygen level in the hydrolysis process had close relationship with the metabolisms of microorganism participating in hydrolysis, leading to an effect on the aroma and color. Further trials were needed to be carried out as to study on this point.
Other Activities Performed
The challenge tests were not running all the time, when there was no special tasks assigned, I was mainly doing the microbiological analysis of fermentation samples and prepared the mother starter culture for the pilot plant trials. I also helped in the day-to-day activities in the pilot plant and assisted in the analyses carried out in the microbiological laboratories.
Running back over the Professional Placement period in Nestlé, I have gained not only in the social and personal skill, but also the technical skills. In the beginning of the attachment, the extensive readings on journals and books related to the project provided a basic foundation for my trials carried out later on, enabled me to understand the behind mechanisms, and helped me avoid making much mistakes throughout conducting the challenge tests, hence it was really essential to do the literature research part.
The second part of my attachment mainly consists of running the laboratory scale rapid hydrolysis and the micro analysis work. Operation of the fermentors has enabled me to realize the conflicts of theoretical knowledge and practical operation limitations. Furthermore, beyond understanding the mechanism behind the process, I have also learnt to cultivate a sense of responsibility for myself by constantly practicing safety and hygiene in the laboratories.
My supervisor emphasizes on independent learning and encourages me to be motivated in conducting my own research. She never fails to explain to me whenever I had doubts. The frequent discussions with the fermentation team has widened my perspectives, also improved my analytical and communication skills.
In the course of my attachment, I have received much assistance from the staff in Nestlé. Their tolerance and patience on clarifying my doubts enabled me to enhance skills and knowledge.
These 22 weeks of my attachment programme in Nestlé R&D Center has provided me with much insight into the commercial food industry where theory meets practicality. Being able to witness the transformation of planned trials on paper to actual pilot plant trials makes my outlook brighter. Aiming to validate the antimicrobial ability of the protective organism inoculated Hydrolysate, the project I have done in Nestlé was a step forward in the never-ending pursue towards the optimization of the industrial process. Moreover, the exposure to laboratory work proved to be truly enriching. Besides the wealth of knowledge acquired, I had gained valuable experience working in a multi-cultural environment.
In summary, 5 month work experience in Nestlé is valuable and interesting; it has boosted my interest in the food science and technology area while at the same time providing me the chance to apply knowledge learned in lectures to the practical situations.
Nestlé, Retrieved 13 May 2010.
Brian J.B. (1985) Wood, Microbiology of Fermented Foods Volume 1&2, Elsevier Applied science publishers Ltd.
C. Outwehand and S. Versterland. (2004). Antimicrobial Components From Lactic Acid Bacteria . Marcel Dekker, Inc.
Kathleen T.R. & Reginald W.B, (2003). Bacillus cereus. Marcel Dekker, Inc.