生物论文代写 Nestl Expanded Beyond Its Traditional Product

Objectives

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.

Background

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, [4] 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

Protective organism

Hydrolysis

30oC/ pH=6.0

72 hours

Figure – Flowchart of Hydrolysis

Recipe of Hydrolysis substrate was showed in table 1:

Trial 1

Trial 2

Trial 3

Trial 4

Batch size (Kg)

0.2

Plant protein weight (Kg)

0.024

1.14

Water (Kg)

0.176

3.86

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.

Challenged micro-organisms

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

Trial 1

Trial 2

Trial 3

Trial 4

Protective organism (cfu/g)

106

Bacillus cereus (cfu/g)

103

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.

Experiment operations

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

Enterobacteriaceae 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.

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