Feasibility to Produce Fiber Textural Protein by Using Depurine and Non-degradable Yeast Protein

The industrial production of fiber textural protein with high moisture is the core technology of the development of artificial meat industrialization and reflects the technological level of artificial meat. Currently used ingredients include soy protein isolates, gluten and pea protein . Pea protein are better as artificial meat ingredients) . Bean protein odor and containing phytoestrogens bring challenges to artificial meat enterprises. For example, in the preparation of food with plant protein, usually there will be bitter, astringent, etc. In addition, China is currently heavily dependent on soybean imports, with imported soybeans accounting for more than 80%. These shortcomings may limit the further development of high-end artificial meat based on soy protein.

Based on safety, nutrition, and sustainability of industrial production, microbial single-cell proteins have been considered as the ultimate alternative protein, especially Saccharomyces cerevisiae single-cell proteins. 

Yeast protein is an all-natural, nearly perfect balanced nutrition food, is an ideal source of nutrition. Yeast has the characteristics of "three low and four excellent", low fat, low sugar, low cholesterol, containing high-quality protein (all essential amino acids), complete B vitamins, 14 rare mineral elements and high-quality dietary fiber.Yeast proteins are high-quality proteins with high bioavailability and can be used as novel alternative proteins for food-grade applications.

There are scientific theory and experimental foundation to produce fiber textural protein by using yeast protein as raw material.

1. The safety shortcoming of high purine (high nucleic acid) has been overcome.

2. The new yeast whole protein avoids the technical disadvantage of protein degradation.

3.The thermal denaturation temperature of the new yeast whole protein is between 70-75 ° C, which is consistent with the denaturation temperature of meat fibrous protein, making it more suitable for processing with high moisture extrusion technology. From the perspective of thermal denaturation temperature alone, yeast whole protein is similar to beef protein . Beef protein has a thermal denaturation temperature of 78 ° C, sourced from the PPT report online by Dr. Jinchuang Zhang of the Chinese Academy of Agricultural Sciences.

4. The use of alkali extraction and acid precipitation in the production of soy protein may lead to the disadvantage of high salt content in protein products. The new manufacturing process by using depurine and non-degradable yeast protein might avoid the high salt problem of soy protein artificial meat，which adopts the physical method of heating to recover the protein, and does not use acid and alkali, thus avoiding the pr disadvantage of high salt in artificial meat.

Taken these together, it is possible to overcome the shortcomings of artificial meat in nutrition, hydrophilicity, oil-holding, taste and flavor by using depurine and non-degradation yeast whole protein as the preparation of fiber textural protein with a high humidity and artificial meat products, it is of great significance to realize the healthy and rapid development of artificial meat industry. 

Appendix Experiment and Result Analysis: Yeast Protein Depurination (i.e. Denucleic Acid) Experiment, Depurination and Non-degradation Yeast Protein SDS-PAGE Analysis, and Yeast Protein Thermal Denaturation and Protein Precipitation Experiment.

Experiment 1: Thermal denaturation precipitation experiment of depurine (nucleic acid free) yeast whole protein.  

Result: After heating at 70-75 ° C for 0.5 hours, the whole protein of depurine (nucleic acid free) yeast was placed at room temperature (25 ° C) for 0.5 hours. It can be intuitively seen that the protein precipitation of the experimental group sample is more complete. After centrifugation to recover the whole yeast protein, no protein bands were observed in the supernatant protein electrophoresis analysis (results not listed).

Method of Experiment 1: Keep the yeast protein at 70-75 °C for 0.5 hours and leave it at room temperature (25 ° C) for 0.5 hours.

1->2 from left ->right.

Method for processing yeast protein samples: 100-200g (wet yeast cell obtained by centrifugation) is suspended in 1L of suitable buffer solution, and the yeast solution is obtained by physical method. Fully suspend the bacterial solution and take 1-5ml of sample for thermal denaturation experiment. 1: Enzymatic depurine yeast breaking solution. 2: Yeast breaking solution did not use enzymatic depurification as a negative control.

Experiment 2.1: SDS-PAGE Analysis of Yeast Whole Protein

The analysis of SDS-PAGE  shows that the yeast whole protein obtained by low-temperature physical sterilization method is basically soluble.

Method for obtaining electrophoresis samples: Yeast breaking solution is the same as Experiment 1, followed by enzymatic nucleic acid removal experiment.

M:Marker. 1: Yeast whole cell protein. 2: Yeast soluble whole cell protein. 3: Yeast insoluble whole cell protein.

Experiment 2.2.  SDS-PAGE  electrophoresis analysis of commercially available yeast protein and yeast extract product.

SDS-PAGE  electrophoresis shows: Yeast protein and yeast extract were identified as yeast protease hydrolysates, and yeast extract protein degradation was more thorough. Electrophoretic analysis did not show obvious protein bands, indicating degradation of yeast protein.

Experiment 2.2 Sample processing method: Take 20g of sample and fully dissolve it in 1L of water (25 °C),Take 5-10ul of sample (without centrifugation to ensure the completeness of the protein content) for electrophoresis analysis.

From left ->right represents 1, 2, and 3. 1: Marker. 2: The Commercially Available Yeast Protein. 3: he Commercially Available Yeast Extract.

Experiment3.The Nucleic Acid Contained in Yeast Whole Protein was Degraded by SpecificNucleases  and Analyzed by Electrophoresis.  

Note: 1. DNA standard molecular weight. 2. Yeast whole protein is not treated with enzymes. 3. Enzyme 1 treatment. 4. Enzyme 2 treatment. 5. Enzyme 3 treatment.6. Enzyme 4 treatment. 

From left to right, 1 to 6.

Yeast whole protein sample processing method: 100-200g (centrifugation to obtain wet yeast body) suspended in 1L suitable buffer, physical sterilization. Take 1ml of sterilization solution and add 1ul of the corresponding nuclease, and the enzyme degrades for 1h under suitable conditions. Take 5ul of the corresponding enzymatic hydrolysate for electrophoresis analysis.

It can be seen intuitively: about 2/3 of the DNA of the 6-lane sample is removed, and the RNA is basically invisible. RNA is digested into nucleotides or oligonucleotides.

 Using the heat precipitation method, the denucleic acid yeast protein is relatively easy to fully recover.