TWI472298B - Peptide product for enhancing calcium absorption and fabrication method thereof - Google Patents

Description

Peptide product for promoting calcium absorption and preparation method thereof

The present invention relates to peptide products, and in particular to peptide products which promote calcium absorption and methods for their manufacture.

According to the National Nutrition Survey of the Republic of China, the average calcium intake of men in Taiwan is 520 mg, while that of women is 542 mg, while the average intake of developing adolescents aged 13-19 is only 51-56% of the recommended amount. Compared to the US RDA (1200 mg) or RDI (1300 mg) standards, it is less than half of its recommended intake. The "Reference intake of dietary nutrients for Chinese people", which was just revised by the Department of Health in 2002, has increased from 600 mg per day to 800-1000 mg for adults and 1200 mg for adolescents.

The more common sources of calcium are provided by dairy products, but many people have lactose intolerance. In addition, in Taiwan, the per capita consumption of dairy products is much lower than in Western countries, which is also the reason for reducing the total calcium intake.

Studies have found that casein in dairy products, after decomposition by proteolytic enzymes in the stomach and intestines, produces a peptide component that inhibits the precipitation of calcium ions in an alkaline environment, called Caseinophosphopeptide (CPP). ). Calcium ion absorption in the small intestine, except for a small part (15-20% by weight) in the duodenum part of the front end requires the active transport of vitamin D, most of the calcium ions enter the human circulatory system, relying on the jejunum and ileum Passive transport (60-65% by weight) (Bronner, F. 1998. Calcium Absorption-A Paradigm for Mineral Absorption J. Nutr. 128: 917-920; Bronner, F. and Pansu, D. 1999. Nutritional Aspects of Calcium Absorption. J. Nutr. 129: 9-12). Since the pH of the posterior segment of the small intestine is weakly alkaline, calcium can be maintained in the presence of a CPP peptide fragment containing the SerP-SerP-SerP-Glu-Glu sequence. The dissolved state of ions and passive transport through the intestinal cell space (Ferraretto, A.. Signorile, A Gravaghi, C. Fiorilli, A. and Tettamanti, G. 2001. Casein Phosphopeptides Influence Calcium Uptake by Cultured Human Intestinal HT-29 Tumor Cells J. Nutr. 131: 1655-1661). In order to avoid the occurrence of diseases such as osteoporosis caused by the lack of calcium, in addition to increasing calcium intake, it is also a good method to increase the absorption rate of calcium. However, patients with lactose intolerance are not suitable for using casein in dairy products to promote calcium absorption.

In the past, scholars have proposed to use cheese containing casein phosphopeptide as a method to promote calcium absorption (Kato, K., Takada, Y., Matsuyama, H., Kawasaki, Y., Aoe, S., Yano, H). Toba, Y. 2002. Milk calcium taken with cheese increased bone mineral density and bone strength in growing rats. Biosci Biotechnol Biochem. 66:2342-2346), this method is caused when it is supplied to patients with lactose intolerance. The problem of physical discomfort. In addition, some scholars have proposed a way to produce a single calcium-promoting peptide by chemical synthesis (Galzigna, L., Domergue, N., Previero, A. 1998. A synthetic calcium-chelating L-glutamyl-L-serine phosphate (Glu) -Ser.P) enhances calcium absorption in the rat. J Pept Res. 52(1): 15-18), but its cost is quite high and cannot be practically applied to the industry. In addition, some scholars use squid bone meal to increase calcium intake. However, this method will decrease the absorption rate with the increase of calcium amount (Lai Aiji. 1996. Comparison of calcium utilization effect of tibia powder and calcium carbonate as calcium source) Journal of the Taiwan Nutrition Society. Vol. 21, No. 2, pp. 121-131). The mainland patent CN1101471C discloses liquid fermentation using soybean as a raw material to obtain a soy protein peptide product. In addition, Taiwan Patent TW I298622: a two-stage fermentation method for improving the nutritional efficiency of soy protein, wherein the specification discloses a two-stage fermentation method for improving the nutritional efficiency of soybean protein, which is a pre-fermentation process of soybean raw material (fermentation fermentation) After the treatment, the obtained product is further mixed with the soybean raw material, and after adjusting the water, the lactic acid bacteria are inoculated, and the lactic acid bacteria fermentation and the enzyme hydrolysis project are continued, and finally, the lactic acid bacteria fermentation and the enzyme hydrolysis project can be directly used or dried to be pulverized, and the above method is required. After cooking, and the use of lactic acid bacteria for subsequent fermentation, the steps are more complicated and costly. In addition, in the second stage of fermentation, the moisture is controlled at 35-50%, which basically assumes a solid state, so The hydrolysis reaction is not sufficiently carried out, so it is difficult to obtain a soy protein hydrolyzate of a small fragment peptide.

In summary, there is a need for a product that has a low manufacturing cost and can increase the absorption rate of calcium that can be consumed by patients with lactose intolerance.

The invention provides a method for preparing a peptide product for promoting calcium absorption, comprising: mixing a raw material containing soy protein with water to form a mixture having a water content of about 45%-65% by weight; Inoculation in the above mixture to obtain a culture, wherein the bacillus flora comprises: Aspergillus oryzae, Aspergillus sojae, or a mixture thereof, and the inoculation amount of the sputum bacteria is about 0.006% to 0.05% by weight of the soybean protein material. The above culture is subjected to solid state fermentation to obtain a fermentation product, wherein the conditions of the solid fermentation include: a relative humidity of about 85%-95%; a fermentation temperature of about 23 to 35 degrees Celsius; and the fermentation product is hydrolyzed to obtain a a soybean hydrolyzate; the soybean hydrolyzate is sequentially subjected to solid-liquid separation and clarification; and a hydrolyzate containing the peptide product for promoting calcium absorption is collected.

The present invention further provides a peptide product which is used to promote calcium absorption, which is prepared by the above method.

The above and other objects, features and advantages of the present invention will become more apparent from

The method provides a method for producing a peptide product for promoting calcium absorption by using a raw material containing soybean peptide. In one embodiment, the soybean powder is subjected to solid state fermentation decomposition by sputum, followed by hydrolysis reaction, and then carried out. The clarification step gives a clear hydrolyzate. Experiments have shown that the soybean peptide produced by hydrolysis of soy protein has a good effect of inhibiting calcium precipitation and increasing calcium absorption, and can increase the absorption rate of calcium.

The present invention utilizes soybean to produce a soybean peptide, the principle of which is to use a mixture of Aspergillus oryzae, Aspergillus sojae, or a mixture of the above species to treat a soybean protein-containing raw material for solid-state fermentation, wherein the soybean protein is Sources include, but are not limited to, soy flour, defatted soy flour, defatted soy flakes, soy protein isolate, soy meal, bean cake, or combinations thereof. In one embodiment, the inoculum inoculation amount is from about 0.006% to 0.05%, preferably from about 0.008% to 0.02%, more preferably about 0.013%, based on the total weight of the raw materials. In the above solid state fermentation, various buffers can be used, for example: citric acid-sodium citrate, acetic acid-sodium acetate, K ₂ HPO ₄ -KH ₂ PO ₄ , Na ₂ HPO ₄ -NaH ₂ PO ₄ , boric acid-hydrogen peroxide Sodium, trishydroxymethylmethylaminopropanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, trishydroxymethylaminomethane, N-tris-(hydroxymethyl)methylaminoacetic acid, 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid hemi-sodium salt, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 3-(N-morpholine)ethanesulfonic acid Piperazine-N,N'-bis(2-ethanesulfonic acid), sodium citrate citrate, 2-morpholineethanesulfonic acid, etc., regulate the water required for solid-state fermentation, and the required water accounts for the raw materials. The total weight of the mixture with water is from about 45% to about 65%, preferably from about 50% to about 60%, more preferably about 55%. In one embodiment, the solid state fermentation can be carried out at a pH of about 5 to 7; a relative humidity of about 85% to 95%; a fermentation temperature of about 23 to 35 degrees Celsius; and a fermentation time of about 2 to 5 days. .

The above fermentation product is then subjected to a hydrolysis reaction to obtain a soybean hydrolyzate. In one embodiment, the step of performing the hydrolysis reaction comprises: mixing the fermentation product with water at a weight ratio of about 1:1 to 1:3, preferably about 1:2; and the hydrolysis time is about 3-24. The hour is preferably about 6 hours; the hydrolysis temperature is between about 30 and 65 degrees Celsius, preferably about 45 degrees Celsius. Thereafter, the reaction temperature is raised to about 90-95 degrees Celsius to inhibit the activity of the enzyme.

Thereafter, the soybean hydrolyzate is subjected to clarification treatment such as solid-liquid separation. For example, a press can be used to separate the solid and liquid by hydraulic pressure. Then the liquid is heated at 90 ° C, the precipitate produced after 10 minutes, and then the precipitate is removed by a high-speed centrifuge. Finally, the hydrolyzate is filtered using a plate and frame filter press or a microfiltration membrane, and the peptide containing the calcium-absorbing absorption is collected. Hydrolyzate. It should be understood that those skilled in the art can also use any other suitable solid-liquid separation method, as well as other suitable filtration methods, to obtain the soybean peptide product obtained by in vitro test, which has the effect of inhibiting calcium carbonate precipitation. The results of the animal feeding test also showed that feeding the food containing the soybean peptide can effectively increase the calcium absorption rate of the animal.

The method for preparing a peptide product for promoting calcium absorption provided by the invention is easy to obtain and low in cost, and in addition, in the production of the soybean peptide, the liquid fermentation process is relatively easy to control, but the yield thereof Generally, it is lower than solid state fermentation, and the cost of liquid fermentation equipment is also high. Further, in the embodiment provided by the present invention, the step of autoclaving is omitted, and therefore, the peptide product obtained by the present invention has a low manufacturing cost and can be eaten by a patient suffering from lactose intolerance.

[Examples] Example 1. Production of calcium absorption soy peptides

Using defatted soybean meal as raw material, inoculate Aspergillus sojae, wherein the inoculum inoculum is 0.013% of the total weight of the soybean raw material, and the water is adjusted by a buffer of 0.25% by weight of CH ₃ COONa/0.25% HNaSO ₃ and pH=5.8. Up to 55%, cultured in an environment of 25 to 30 ° C and a relative humidity of 95% or more for 2 days.

Then, the hydrolysis reaction is carried out, and the soybean meal is mixed with water, wherein the ratio of soybean meal to water is 1:2, the hydrolysis time is 6 hours, the hydrolysis temperature is 45 ° C, and the temperature is further increased to 90-95 ° C for 10 minutes to inhibit the enzyme. active.

Next, the clarification treatment of the hydrolyzate is carried out, and the soybean hydrolyzate is first placed in a filter bag using a press, and laid flat in a press tank to separate the solid and liquid by oil pressure. The liquid was then heated to a temperature of 90 ° C for 10 minutes, and the precipitate was removed by a telecentric high-speed centrifuge (16,000 rpm, Taichung, manufactured by Quantai Co., Ltd.), and finally filtered using a plate and frame filter press (Seitz Orion, made in Germany) or The hydrolyzate was filtered using a microfiltration membrane (0.3 μm) to obtain a clarified soybean hydrolyzate.

Example 2. Effect of Soy Hydrolysate on Inhibition of Calcium Carbonate Precipitation

According to Jin et al. (J. Agnc. Food Chem. 2000) calcium carbonate crystallization experiment slightly modified, 1 ml sample was mixed with 1 ml concentration of 40 mM calcium chloride and 1.2 ml of distilled water, then added 0.8 ml concentration of 40 mM carbonic acid The reaction was carried out for 1 hour with sodium hydrogen and maintained at pH = 8.5 by titration with 0.1 N sodium hydroxide. The mixed solution at the end of the reaction was centrifuged (centrifugal force: 2000 g), and the supernatant was taken to measure the calcium concentration using the calcium measuring reagent group (product of Taiwan Merck), which is the soluble calcium content.

In the experiment for inhibiting calcium carbonate crystallization, the soy protein hydrolyzate obtained in Example 1 has an inhibitory effect on calcium carbonate crystals, and the effect is evaluated as the minimum sample concentration for completely inhibiting calcium carbonate crystals under alkaline conditions (pH=8.5). If calcium carbonate precipitates, the calcium content of the supernatant decreases; otherwise, the calcium content of the supernatant does not change. Fig. 1 is a graph showing the effect of inhibiting calcium carbonate crystallization in soybean protein hydrolysate compared with casein phosphopeptide, and the results showed that the minimum inhibitory concentration of casein phosphopeptide protein (CPP) which promotes calcium absorption was 1.76 x 10 ^-4 %. The protein of the soy protein hydrolyzate of the present invention has a minimum inhibitory concentration of 2.16 x 10 ^-3 %.

Example 3. Effect of inhibition of soybean hydrolysate

In this example, different protein source materials were selected for comparison, and 1 ml sample was mixed with 1 ml of 40 mM calcium chloride and 1.2 ml of distilled water, and then reacted for 1 hour by adding 0.8 ml of 40 mM sodium hydrogencarbonate. The pH was maintained at 8.5 using 0.1 N sodium hydroxide titration. The reaction mixture was centrifuged (centrifugal force: 2000 g), and the supernatant was used to measure the calcium concentration using the calcium measuring reagent group (product of Taiwan Merck), and under the alkaline condition (pH=8.5), the carbonic acid was completely inhibited. The lowest sample concentration of calcium crystallization is used as an evaluation criterion for soluble calcium content.

Figure 2 is the effect of different protein source materials on the inhibition of calcium carbonate crystallization. Among them, free amino acids such as monosodium glutamate (MSG) and glycine have poor inhibitory effects; followed by proteins such as isolated soy protein (ISP), casein, and chicken of essence (essence of chicken). The commercially available product, which is a chicken protein extract), is superior to the first two types in inhibiting the precipitation of calcium carbonate; and the soy hydrolysate of the present invention is the best. It can be seen that a single free amino acid or a complete protein is not chelated with calcium ions as in the case of protein hydrolysates. After hydrolysis of proteins to different extents, they have a better effect of inhibiting calcium precipitation. Therefore, the protein material of the peptide type, such as chicken essence, and the method for decomposing soy protein hydrolyzate by using the bacteriocin enzyme are all good calcium precipitation precipitants.

Example 4. Effect of calcium-promoting peptide on calcium absorption in animals

The experimental method is similar to that of Lai Ai Ji (The Journal of the Taiwan Nutrition Society, Vol. 21, No. 2, 1996), but with minor modifications, adult female rats (Bltw: SD; purchased from Lesco Biotech Co., Ltd.) are kept in separate countries. In the metabolic cage, the room temperature was maintained at 23±2°C, and the light was irradiated for 12 hours. After the food was removed for one day, the soybean protein isolate was added as the control group or the calcium-promoting peptide was used as the experimental group. The number is 10, the animals are fed ad libitum, the feces are collected daily, the feed is taken after 6 days, the total amount of food is taken, and after waiting for one day to exclude the feed, the calcium content in the excreted feces is collected and analyzed. . Calcium analysis is carried out by nitric acid and then measured by atomic absorption spectrometer.

The total intake of calcium is estimated by the amount of feed eaten by the amount of calcium in the feed. The absorption rate of calcium (total intake - calcium content in feces) / total intake.

Figure 3 shows the calcium absorption rate of mice fed with calcium-promoting peptide and soy protein. Comparing the absorption rate of soy protein or calcium-promoting peptide, it was found that the absorption rate of calcium-enhancing peptide was significantly higher than that of the larger soybean protein, and the mean value was ±95% confidence interval, and there was a significant difference between the two (p<0.05).

Table 1. Soy protein isolate (control group) and calcium-enhancing peptide (experimental group) feed formulation (quantity of each component per gram of feed, in grams).

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Fig. 1 is a graph showing the effect of inhibiting the crystallization of calcium carbonate in comparison with the casein phosphopeptide by the soy protein hydrolyzate.

Figure 2 is the effect of different protein source materials on the inhibition of calcium carbonate crystallization.

Figure 3 shows the calcium absorption rate of the rats fed the calcium-promoting peptide and the soy protein feed, with a mean ±95% confidence interval, with significant differences between the two (p < 0.05).

Claims (4)

A method for preparing a peptide product for promoting calcium absorption, comprising: mixing a raw material containing soy protein with water to form a mixture having a water content of about 45% to 65% by weight; and inoculating the bacterium The mixture obtains a culture, wherein the bacillus flora comprises: Aspergillus oryzae, Aspergillus sojae, or a mixture thereof, and the inoculation amount of the sputum flora is about 0.006%-0.05% by weight of the soybean protein material; The culture is subjected to solid state fermentation to obtain a fermentation product, wherein the conditions of the solid state fermentation include: relative humidity of about 85%-95%; fermentation temperature of about 23 to 35 degrees Celsius; fermentation time of about 2 to 5 days; 5~7; performing the hydrolysis reaction of the above fermentation product to obtain a soybean hydrolyzate, wherein in the hydrolysis reaction, the weight ratio of the fermentation product to water is about 1:1 to 1:3, and the hydrolysis time of the hydrolysis reaction For about 3-24 hours, the hydrolysis reaction has a hydrolysis temperature of about 30-65 degrees Celsius; the soybean hydrolyzate is sequentially subjected to clarification treatment and solid-liquid separation; and the peptide product containing the calcium absorption is collected. Hydrolyzate. The method for preparing a peptide product for promoting calcium absorption according to the first aspect of the patent application, wherein the soybean protein-containing material comprises soybean powder, defatted soybean powder, defatted soybean flakes, soy protein isolate, soybean meal, Bean cake, or a combination of the above. The method for preparing a peptide product for promoting calcium absorption according to claim 1, wherein after the hydrolysis reaction is completed, the reaction temperature is further increased to about 90-95 degrees Celsius to inhibit enzyme activity. A peptide product for promoting calcium absorption, which is prepared by the method described in claim 1 of the patent application.