JP5561642B2 - Yogurt - Google Patents

Description

  The present invention relates to yogurt.

Probiotics refers to preventive medicine that increases the number of good bacteria (lactic acid bacteria, bifidobacteria, etc.) in the gastrointestinal tract, maintains the balance of enteric bacteria, and makes the body less susceptible to disease.
Various dairy products containing lactic acid bacteria are positioned as important foods from the viewpoint of probiotics, and particularly yogurt is widely used as a food that can be easily eaten . However, even if you eat yogurt , it is said that most of the lactic acid bacteria are killed and reach the intestines because they touch the highly acidic stomach acid and bile fluid.
Therefore, recently, a method is known in which lactic acid bacteria are enclosed in acid-resistant capsules and the capsules are mixed into yogurt (Patent Document 1). Also known is a lactic acid beverage or the like devised so that lactic acid bacteria can reach the intestine by selecting so-called vegetable lactic acid bacteria having acid resistance (Patent Document 2).

JP 2000-116320 A JP 2009-82127 A

However, encapsulating lactic acid bacteria in a capsule is awkward and expensive. Moreover, the thing of patent document 2 has the subject that taste will become a thing different from the conventional yogurt.
The present invention has been made to solve the above-mentioned problems, and the object of the present invention is to make lactic acid bacteria survive effectively and reach the intestine without impairing taste and using capsules. There is in providing yogurt that can.

The yogurt according to the present invention is a yogurt containing lactic acid bacteria, and is a porous dried okara that is finely pulverized into a milk raw material consisting of milk and any sugar of glucose, sugar, lactose, melvinose and raffinose. After being added, the lactic acid bacteria are fermented with lactic acid bacteria, contain 0.1 to 20 wt% of the porous dried okara, and the lactic acid bacteria have propagated and settled on the inner walls of the voids from the porous dried okara. Features. It is important here to be dried okara, not raw okara. Originally, okara itself contains only a small amount of carbon sources that are readily available to lactic acid bacteria. Therefore, in order to ferment okara with lactic acid bacteria, it is important to contain low molecular sugars (for example, glucose, sugar, lactose, melvinose, raffinose, etc.) in the okara. In the case of fresh okara, water exists in the porous voids. Therefore, the nutrients of lactic acid bacteria are delivered by natural diffusion, but it takes time. On the other hand, in the case of dried okara, only air exists in the porous void. When dry okara is added to a solution containing nutrients such as sugars, a substance that serves as a nutrient for lactic acid bacteria is instantaneously brought into the porous interior of the fiber. It is suitable for yogurt fermentation that cannot take a long fermentation time. In this way, the porous interior is a comfortable growth environment for lactic acid bacteria.
As a result, the production method of yogurt is very simple and only needs to contain dried okara during lactic acid fermentation, and does not require any special equipment or operation.
As the dried okara, okara obtained by lactic acid fermentation of dried okara can be used. This pre-fermentation means that a protective film made by the lactic acid bacterium itself is selected by selecting the fermented dried okara containing the sugar that is the bait of the lactic acid bacterium for a relatively long time with a relatively low moisture content. The production of biofilms can be promoted, and the protective effect against acid and bile fluid by this protective film can be further expected.

According to the present invention, it is possible to provide a yogurt in which saccharides are brought into the pores of dried okara fibers and fermented, and lactic acid bacteria are propagated and fixed on the inner walls of the voids from the dried okara. Furthermore, it is possible to provide a yogurt that can effectively survive lactic acid bacteria and reach the intestine without impairing the taste and without using a capsule.

Scanning electron micrographs from dried okara are shown. It is a scanning electron micrograph which shows the state which the lactic acid bacteria propagated and settled in the space | gap part from dried okara. The DGGE pattern at the time of using commercially available lactic acid bacteria is shown. The DGGE pattern at the time of using skim milk powder isolation | separation lactic acid bacteria is shown.

Preferred embodiments of the present invention will be described below in detail with reference to the accompanying drawings.
Various methods for producing dried okara are known (for example, JP-A-5-68503, JP-A-2000-4817, etc.).
The production method of dried okara used in the present embodiment is not particularly limited, but in any production method, okara itself is extracted from the stored protein during the production of tofu, mainly from the cell wall. Therefore, the resulting dried okara has a porous structure with a very high porosity connected by cell walls. This hollow (void) part becomes a lactic acid bacteria house (however, an available carbon source is required).
The dried okara is preferably one that has been rapidly dried and finely pulverized under the control of the number of bacteria.
FIG. 1 is a scanning electron micrograph of the dried okara.

  It is said that galactose is the most abundant saccharide in the soy cell wall and occupies 35% by weight (“brewed ingredients” edited by Japan Brewing Association, 1999). This galactose has high affinity with the lectin constituting the cell wall of lactic acid bacteria (Oxford Journals. Life Sciences. Glycobiology 10 (11) 1193-1199, 2000). Therefore, lactic acid bacteria can propagate on the cell wall using galactose in the cell wall of soybean (okara) as a scaffold. Specifically, it was actually confirmed that a large number of lactic acid bacteria grew and settled in the cavity from dried okara (scanning electron micrograph in FIG. 2). In this way, lactic acid bacteria settle in the cavity of dried okara, so when eaten as yogurt, the degree of attack by gastric acid and bile fluid decreases, and lactic acid bacteria that survive and reach the intestine increase. .

Moreover, in the polysaccharide constituting the soybean cell wall, the amount of pectin is high next to hemicellulose (glucose, xylose) (about 30 wt%). This pectic substance is insoluble (hard) when acidic, and soluble (swells) when neutral or alkaline. Therefore, when passing through the stomach and duodenum, it is insoluble depending on stomach acid and bile fluid and maintains its shape. It is considered to be.
The addition amount from dried okara is preferably 0.1 to 20 wt%. If it is less than 0.1 wt%, the amount of lactic acid bacteria that live and reach the intestines is small, and if it exceeds 20 wt%, the texture of yogurt is affected. The amount added from dried okara is optimally about 0.1 to 1 wt% from the texture of yogurt.

<Example 1> Lactic acid fermentation from dried okara First, the dried okara itself was lactic acid fermented using lactic acid bacteria for yogurt.
1) Fermentation method Dried okara (bean flour # 300, manufactured by Misuzu Corporation) Add 100 g of 30% 20% molasses containing 2.5% sodium acetate, place this 130 g in a bag, sterilize by autoclaving (121 ° C, 20 minutes) .
As an inoculum for fermentation, 12 mL of water was added to 8 mL of a culture solution of yogurt isolate (Lactobacillus delbuchii subsp. Bulgaricus) isolated from commercially available yogurt, and water was added to 8 mL of an isolated culture solution of L. paracasei isolated from a commercially available supplement. Three types were used: 12 mL added and commercially available lactic acid bacteria species (manufactured by Kyodo Daigyo Co., Ltd.) (3 lactic acid bacteria: Lactobacillus delbuchii subsp. Bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512) culture solution 8 mL.
The bag was sealed and fermented at 35 ° C. for 14 days (water content 39%, sugar content 2.9%).

The above is shown in Table 1.

  Separately, a culture solution obtained by culturing these three bacteria in MRS medium at 35 ° C. for 24 hours was used as a living cell control.

2) Survival rate survey in the virtual digestive tract To examine the survival of lactic acid bacteria in the digestive tract, treatment with gastric pH (pH 2-3, treatment time 0.5-1h) and bile fluid (0.4% bile powder, 0.5h) ( Acid treatment), and the effect of okara was seen by comparing the number of viable bacteria.
A specific acid treatment method is shown below.
Acid treatment method: Suspend 1 g of fermented okara and 1 g of the culture solution of the viable cell control group in 4.2 mL of physiological saline. Thereto is added 5.0 mL of 0.2 M hydrochloric acid-potassium hydrochloride buffer (pH 1.18) to bring the pH to about 2.0. Leave in a 37 ° C water bath for 40 minutes, then add 0.4 mL of 10% bile solution to a concentration of 0.4% and treat in a 37 ° C water bath for 30 minutes. Next, the pH is returned to neutrality around 7.2-7.5 with 6M aqueous sodium hydroxide solution at room temperature, diluted with physiological saline containing 0.5% of a surfactant (Tween 80), and a BCP-added plate agar medium, Anero Anaerobic culture using a pack jar (Mitsubishi Gas Chemical Co., Ltd.) was performed, and the number of viable bacteria was measured.
The acid-untreated group was diluted with 9 mL of physiological saline, and the viable cell count was measured on a BCP-added plate agar medium.

The results are shown in Table 2.
Survival rate = (series of number of viable bacteria after acid and bile treatment) / (series of number of untreated viable bacteria)
As is clear from Table 2, the number of viable bacteria after acid treatment corresponding to the digestive tract was reduced to 1 / 10,000 to 1 / 100,000 in the liquid culture bacteria (control) that did not contain okara compared to untreated. End up. On the other hand, in the case of fermented okara (test), it can be seen that the survival rate of the lactic acid bacteria after the acid treatment is as extremely high as 1/10 to 1/100 compared with the untreated.
In addition, the scanning electron micrograph of FIG. 2 is a photograph which shows the reproduction state of the lactic acid bacteria in the acid untreated from fermentation okara using the commercially available lactic acid bacteria seed | species. In this way, lactic acid bacteria grow and settle on the inner wall of the cavity of porous dry okara, so it is difficult to be attacked by gastric acid and bile fluid, and it is likely to live and reach the intestines more often. It is done.

<Example 2>
1. As inoculum prepared <br/> used species yoghurt prepared yogurt using the following lactic acid bacteria.
1) Cooperative dairy industry: 3 lactic acid bacteria (Lactobacillus delbuchii subsp. Bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512)
2) Cafe Factory: Caspian Sea yogurt (Lactococcus lactis subsp. Cremoris)
3) Plain yogurt A: Koiwai (Bifidobacterium, Acidophilus)
4) Plain yogurt B: Morinaga Milk Industry (Bifidobacterium longum BB536)
5) Plain Yogurt C: Meiji Dairy (LG21)
6) Plain yogurt D: Meiji Dairy (Lactobacillus bulgaricus2038, Streptococcus thermophilus1131)

Inoculum 1) and 2)
Commercial milk --- 500g
Inoculum ---- 1g / pack
Sugar ----- 50g
Dried Okara (Bean Flower # 300) --- 5wt%

In the case of yogurt friend varieties Commercial milk --- 500g
Plain yogurt--50g
Sugar ----- 50g
Dried Okara (Bean Flower # 300) --- 5wt%

  Both the test group (including 5 wt% dried okara) and the control group (without okara) were fermented at 35 ° C. for 16 hours. As above, 10 wt% sugar was added to aid fermentation.

Yogurt performance = Any of the bacteria was a commercial inoculum or an inoculum based on a commercially available yogurt, and delicious yogurts were obtained as shown in Table 3, respectively.

<Example 3> Survival survey of fermented yoghurt bacteria using okara Cooperative milk industry bacteria for yogurt production : Using commercially available products (Lactobacillus delbuchii subsp. Bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis LKM512), fermented 8 g of dried okara (bean flour # 300) and 160 mL of milk containing 10% sugar at 46 ° C It was. There were two types of fermentation time: standard 12h and fully matured 40h. A was prepared by fermenting lactic acid with dried okara as A, B as a conventional yogurt without dried okara, and C was prepared by simply mixing dried okara with B for 12 hours fermented yogurt.

2. Survival survey in the virtual digestive tract In order to reproduce the environment from the stomach to the upper small intestine in vitro, 6.1 mL of physiological saline and 2.5 mL of 0.2 M hydrochloric acid-potassium hydrochloride buffer (pH 1.3) are added to 1 g of the sample. In addition, the pH was adjusted to around 2.0 and maintained at 37 ° C. for 40 minutes. Thereafter, 0.4 mL of 10 wt% bile solution was added and treated at 37 ° C. for 30 minutes. After the treatment, the pH was raised to around 7.2-7.5 with 6M aqueous sodium hydroxide solution (60-70 μL) and left for 40 minutes. This sample was diluted in the same manner as described above, and the viable cell count was measured on a BCP-added plate agar medium. In the acid-untreated group, the number of viable bacteria was measured in the same manner as described above.

3. Results The results are shown in Table 4.

4. Fermentation for 12 hours As shown in Table 4, when milk made with dried okara and sugar is fermented with lactic acid bacteria at 46 ° C for 12 hours to make yogurt, it does not look like milk that does not contain conventional okara. It reached the number.
When this is treated with the above-mentioned acid digestive tract, conventional yogurt that does not contain dried okara and dried okara are simply mixed with fermented yogurt from x10 ⁸ to x10 ³ 5 While the number of sprouting bacteria was reduced, the one fermented with dried okara still had a three-digit decrease from × 10 ⁸ to × 10 ⁵ . As a result, yogurt fermented with dried okara retained 100 times the number of lactic acid bacteria as compared with normal yogurt without additives.

5. Fermentation for 40 hours Yogurt usually finishes fermentation in 12-24 hours, but here it is fermented for 40 hours at 46 ° C for yogurt to promote further entry of the lactic acid bacteria into the voids made.
Although it was a lactic acid bacterium that was relatively resistant to temperature, a decrease in the number of viable bacteria was observed against the intention, whether it was an effect of low pH in addition to high temperature.
The result was the same as in the case of 12 hours, and fermented dried okara had a double-digit bacterial count reduction inhibitory effect.

6). Effect of cold storage Yogurt is produced, then cooled and distributed, and there is a time lag of several days to two weeks until it reaches the customer's hand (general shelf life is below 10 ° C for two weeks). During this time, lactic acid fermentation is said to continue slowly.
After the end of fermentation, it was investigated whether the effect of adding okara to yogurt when kept cold or to further improve it.

The results are shown in Table 5.

As shown in Table 5, as compared with the control not containing okara, the result of co-fermenting with dried okara survived by acid treatment and the like with the number of lactic acid bacteria increased by two orders of magnitude.
Refrigerated storage did not reduce or increase the effects of acid resistance from okara.

<Example 4> Lactic acid bacteria in human feces When eating oak yogurt, it was investigated whether lactic acid bacteria were observed in the stool.
1. For commercially available lactic acid bacteria species
Yogurt fermented with 8% sugar and 1% dried okara and normal yogurt without dried okara were made with commercially available lactic acid bacteria. First, about 100 g of normal yogurt was eaten continuously for 4 days, 48 hours after the last day of ingestion, stool was collected and stored frozen. Next, the fermented yogurt containing 1% dried okara was similarly taken, and the stool was collected and stored frozen.
For each of these stools, the chromosomal DNA of the contained bacteria was extracted, and the polymerase chain reaction with a GC clamp using a primer specific for lactic acid bacteria and a general bacterial primer within the variable region of the 16S ribosomal DNA gene (PCR) was performed. The amplified fragment was subjected to denaturing gradient gel electrophoresis (DGGE). The results are shown in FIG.

Primer and PCR conditions are as follows.
SG-Lab-0159-aS-20 (GGA AAC AG (A / G)) on the forward side as a primer specific to lactic acid bacteria (genus Lactobacillus, Lueconostoc, Pediococcus, Wessella) within the variable region of 16S ribosomal DNA General bacterial S-*-Univ-0515-aA-24 (ATC GAT TTA CCG CGG CTG CTG GCA) was used on the reverse side of TGC TAA TAC CG (reference: Hans GHJ Heilig et al., Appl. Environ. Microbiol. Jan. 2002, p. 114-123).
For PCR, Taqr-polymerase was used, and DNA fragments were amplified for 35 cycles at an annealing temperature of chromosomal DNA and primers of 63 ° C.

FIG. 3 shows a DGGE pattern when the above-mentioned commercially available lactic acid bacteria are used.
Of the detected DNA bands, bands a, b, and c common to lane 1 of lactic acid bacteria and lane 3 of stool after ingesting yogurt were cut out from the gel, purified, DNA sequence determined, DNA database Species were identified from the base sequence information accumulated in The results are summarized in Table 6.
As a result, in the band “b”, Lactobacillus delbuchii subsp. Bulgaricus, the same lactic acid bacterium used in the production of yogurt, was found in the stool ingesting yogurt with okara.
The DGGE conditions and staining conditions are as follows.
The concentration gradient of the gel denaturant was 35-50%. Electrophoresis was performed at a gel temperature of 60 ° C. and a constant voltage of 220 V for 5 hours. The gel was removed from the glass plate, DNA was stained with a gel star nucleic acid stain (Takara) at room temperature for 30 minutes, and the staining band on an ultraviolet illuminator was excited to detect the DNA.

2. In the case of non-fat dry milk isolated lactic acid bacteria species As in 1, a yoghurt containing 1 g dried okara was prepared using isolated lactic acid bacteria (Lactobacillus paracasei), and 100 g was fed only once. Thereafter, stool samples were collected 24 hours, 6 days, and 10 days later, and bacterial DGGE bands were obtained in the same manner as in 1.

FIG. 4 shows a DGGE pattern when the above skim milk powder isolated lactic acid bacterium is used.
Bacteria were identified from the gene sequence in the same manner as in band 1 in the stool after ingesting yogurt from lane 2 but not in normal lane in lane 1.
As a result, Lactobacillus paracasei used for yogurt was found in human feces after 24 hours, 6 days, and 10 days after feeding.
The discovery of a gene fragment of lactic acid bacteria in human feces does not imply the existence of live lactic acid bacteria. However, foods eaten by humans are usually excreted in 24 hours. The presence of the lactic acid bacteria DNA fragment after 48 hours after feeding indicates that the lactic acid bacteria ingested as yogurt reach the intestines alive and proliferate.

Claims (1)

In yogurt containing lactic acid bacteria, pulverized porous dried okara is added to milk raw material consisting of milk and any sugar of glucose, sugar, lactose, melvinose and raffinose, and then lactic acid bacteria The yogurt is characterized by containing 0.1 to 20 wt% of the above-mentioned porous dried okara and fermenting and fixing lactic acid bacteria on the inner wall of the void portion of the porous dried okara.