JPH06253747A - Raw grass additive - Google Patents

Abstract

PURPOSE: To provide a process for producing raw pasture grass by microorganism transformation. CONSTITUTION: This process is to produce raw pasture grass by the microorganism transformation of the raw pasture grass harvest between an aerobic phase and subsequent anaerobic phase characterized by the generation of the anaerobic phase in the presence of lactic acid bacteria forming a Clostridium inhibition factor.

Description

Detailed Description of the Invention

The present invention relates to a process for the production of live grass by first microbial conversion of the live grass crop during an aerobic phase and a subsequent anaerobic phase. The quality of raw grass is subject to wide variation. Fluctuations in climatic conditions are generally considered to be the main factors that cause such variability. Improved quality of hay is often achieved by the addition of bacterial and other hay additives. However, their addition was found to have a low impact on grazing quality under moderate or adverse conditions.

[0002] It is an object of the present invention to provide a pasture additive that provides good quality pasture under various climatic conditions. Many microbial processes occur when live pasture crops are trimmed from the atmosphere. During the first aerobic phase, obligate and facultative aerobic bacteria remain active. The bacteria convert hexoses and oxygen to carbon dioxide and water. As soon as oxygen is depleted, obligately aerobic bacteria lose their activity. As soon as the pH stays above 5,
Enterobacteria and yeast convert hexoses into fatty acids, ethanol and carbon dioxide. However, the lactic acid bacteria present will produce lactic acid which gradually lowers the pH value below 4.

At a pH of 4 and below, the bacterial activity ceases, and the pasture grass will therefore remain well preserved. However, in many cases the pH drop occurs too slowly. Many causes exist under this phenomenon. If the weather prevents premature drying of the pasture crops, plant respiration will last longer. Next, the sugars present will be digested and only a few substrates will survive for full bacterial activity. Therefore, the pH drop will be insufficient. Under conditions of slow pH drop, Clostridia (Cl
ostridia) (ie Clostridium tyrobutyric (Clostridium tyrobut)
yricum)) begins to convert some of the available substrate to butyric acid. Moreover, these bacteria are able to convert the lactic acid produced so far into butyric acid. This step converts two molecules of lactic acid into one of the weaker butyric acid, which leads to an increase in pH. This high pH
And again, proteolytic Clostridia remains active. Their proteolytic clostridia convert proteins to ammonia, which also contributes to the increase in pH.

As a result, the quality of the pasture in terms of nutritional value will be lower (lower dry material, lower protein). Furthermore, the presence of Clostridia in the pasture would cause the presence of Clostridium spores in the raw milk from the pasture-eating dairy cows. This Clostridium contamination will have a negative impact on the quality of dairy products, especially cheese.

It has been found that the nutritional values of pasture grasses produced under unfavorable climatic conditions are improved by inoculation of pasture crops with lactic acid bacteria capable of producing Clostridium inhibitors. Conveniently, high molecular weight Clostridium inhibitors, more particularly Lactobacillus plantarum capable of producing proteinaceous factors may be used.

[0006] In general, such factors are characterized in that their Clostridium inhibitory activity is terminated by trypsin and by heat treatment. This factor is 30
It cannot pass through ultrafiltration membranes with a cut-off value of KD. Examples of Lactobacillus useful in the present invention are Lactobacillus plantarum strains G147 and F1.
23, each of these samples has a deposit number C
As BS 343.92 and CBS 342.92, Cent
raalbureau voor Schimmelcultures at Baarn, Netherl
Deposited with ands.

Suitable live grass crops are grass, clover and alfalha. The crops are stored in such a way that exposure to air is prevented, for example under silos or plastic. Fermentation will occur if the introduction of air is impeded. Pasture additives may be added to increase the rate of fermentation or to direct the type of fermentation. Examples of raw grass additives are sugars, enzymes, acids, salts and inoculums.

The live grass inoculum for use in accordance with the invention is Lactobacillus plantarum with Clostridium inhibitory activity, formulated as a dry or concentrated liquid product. This product is generally suspended in water,
It is then sprayed onto the harvest before and during storage in silos. The cultures contained 10 ⁴ to 10 ⁸ colony forming units (cf
u's) / g harvest, preferably 10 ⁶ cfu's / g harvest.

[0009]

【Example】

Example 1 Growth of Lactobacillus Lactobacillus was isolated by plating appropriate dilutions of starting material on MRS agar plates. R
The composition of MS medium is given in Table 1 (De Man, JCR
ogers, M. and Sharpe, ME (1960) J.Appl.Bacteriology
23 , 130-135).

[0010]

[Table 1]

After incubation at 30 ° C. under anaerobic conditions for 48 hours, isolated colonies were selected. Selected colonies were grown in MRS broth supplemented with glucose (2% w / v) and calcium carbonate (6% w / v) at 30 ° C. under anaerobic conditions for 48 hours.

Characterization of Clostridium Inhibitors The Clostridium inhibitors produced by Lactobacillus strains G147 and F123 were further characterized for thermostability, trypsin sensitivity and molecular size.

Thermostable Cell-free supernatants of each Lactobacillus strain were incubated for a period of time at constant temperature and the residual inhibitory activity was measured.

[0014]

[Table 2]

The results (summarized in Table 2) show that the inhibitors investigated are resistant to Pasteurization. However, they are inactivated by cooking and sterilization. The inhibitor produced by strain F123 appears to be slightly more thermosensitive than the inhibitor produced by strain G147.

Trypsin-sensitive trypsin (1 mg) was added to 1 ml of cell-free supernatant of F123 and G147 strains. Subsequently, at pH 8, the supernatant was incubated at 37 ° C for 1 hour. afterwards,
The pH was lowered to 5.8 and the residual Clostridial inhibitory activity was measured. In all cases tested, the inhibitory activity disappeared completely.

Molecular size F123 and cell-free supernatants of G147 strain were
tricon30 filter system (Amicon)
I left it to ultrafiltration in. 2 of this filter system
The two compartments were separated by a filter with a cut off of 30 KD. Transfer 5 ml of each supernatant to the upper compartment of the filter system and
Centrifugation (1000 g) was allowed for 0 min and the contents of both compartments [permeate (lower compartment); retentate (upper compartment)] were tested for inhibitory activity. The results are summarized in Table 3.

[0018]

[Table 3]

Example 2 Acidification of grass The grass was mowed and pre-dried in the field to a dry mass content of 30%. Next, about 2 cm of grass
Cut into pieces. Cut shredded grass into 0, 10 ⁵ , 10 ⁶
Colony forming units / g forage (cfu / g) were inoculated with strain G147. Inoculation was done by spraying the bacterial suspension on the grass using a plant sprayer during the mixing of the grass with a concrete mixer. For each treatment, eight 11 bottles were each filled with 400 g of grass and the bottles were stored at room temperature.

During the experiment for each treatment, two bottles were incubated at incubation times 0, 24, 48 and 7
It opened in 2 hours. Aqueous extract (30g grass + 270g
Deionized water in Stomacher for 5 minutes)
Were prepared from individual bottles and their pH was measured.

Results : During the experiment, the acidification rate of the pasture inoculated with strain G147 exceeded that of the control and it was dependent on the inoculation level (Fig. 1).

Example 3 Anti-Clostridium activity in live grass As in Example 2, grass cut to about 2 cm pieces was used. Care was taken to use sulphate-free grass to prevent Clostridia inhibition due to nitrate deterioration.

In experiments 3.1, 3.2 and 3.3, grass was inoculated with Lactobacillus cells in an amount of 10 ⁵ and 10 ⁶ cfu / g grass. Deionized water was used as a control. Has a septum (experiments 3.1 and 3.2) or no septum (experiment 3.3) for individual treatments
Three 1 liter bottles were filled with 400 g of grass.

Each Lactobacillus suspension was sprayed onto grass (25 ml suspension / kg grass). Experiment 3.2
And 3.3, apart from Lactobacillus, the soil extract was sprayed onto the grass (25 ml extract / kg grass),
The starting amount of Clostridium spores was increased. The soil extract was prepared by mixing 1 kg of meadow soil and 780 ml of deionized water.

In Experiment 3.4, the grass was treated with 10 ⁶ cf.
Inoculated with Lactobacillus suspension at u / g. Deionized water was used as a control. 2. For individual processing
5 kg of grass was filled into a 10 l experimental silo. Each Lactobacillus strain was applied to grass using a plant sprayer (25 ml / kg) and the grass was mixed using a concrete mixer. To increase the number of Clostridium spores, soil suspension (1 kg of pasture soil + 75
0 ml deionized water) was applied (25 ml / kg grass). The dry mass content of the grass was reduced by adding 100 ml deionized water / kg.

During experiments 3.1 and 3.2, the weight loss and hydrogen concentration of the pasture grass were measured periodically. For each treatment in experiment 3.1, one bottle was removed after 21 days and its contents frozen. For the individual treatment in Experiment 3.2, the contents of two bottles were
Removed after days and frozen.

The number of Clostridium spores and the contents of ethanol, volatile fatty acids, lactic acid and ammonia from these frozen samples were determined. During experiment 3.3, the contents of the two bottles were removed after 6 weeks and the number of Clostridium spores and the contents of ethanol, volatile fatty acids, lactic acid and ammonia were determined from those samples.

During experiment 3.4, the hydrogen concentration of the individual silos was measured periodically, and the pasture samples were measured for pH, ethanol, volatile fatty acids, lactic acid and ammonia, and lactic acid bacteria, yeast. , For removal of enterobacteria and Clostridium spores.

Results of Experiment 3.1 : The results of the experiment after 21 days of grass incubation are summarized in Table 4. Here, RCM means the number of Clostridium spores according to the RCM plate method (log cfu /
g); HAC means acetic acid content (mmol / kg); H
B means butyric acid content (mmol / kg); and HL means lactic acid content (mmol / kg). Little difference between control and inoculated pasture was shown, all pasture was well preserved (low pH) and contained only low amounts of Clostridium spores (<10 ² cfu / g).

[0030]

[Table 4]

Results of experiment 3.2 : After 6 days of incubation, a slight increase in water production and weight loss was found in the control pasture compared to the inoculated pasture. The results summarized in Table 5 (data after 26 days of incubation) lead to the support of the hypothesis that this increase was due to the activity of Clostridium. The abbreviations in Table 5 have the same meanings as in Table 4; MPN is the number of Clostridium spores according to the MostProbable Number (MPN) tube method (log cf
u / g).

[0032]

[Table 5]

Results of experiment 3.3 : The observations made in experiment 3.2 were confirmed in this experiment after 42 days of incubation: the control grass was higher than the inoculated grass. It showed pH and butyric acid levels, higher weight loss and higher numbers of Clostridium spores (Table 6: WL means weight loss (g / kg).

[0034]

[Table 6]

Results of Experiment 3.4 : It is shown in Figure 2 that the pH of the two inoculated grasses dropped to about 3.8 within 5 days (pH 4.1 was found in the control). Was done). Subsequent onset of pH rise occurred later in the inoculated grass (41 days) and then in the control grass (21).
Day).

[Brief description of drawings]

FIG. 1 is a graph showing incubation time versus acidification rate of pasture grass inoculated with various strains.

FIG. 2 This is a plot of live grass inoculated with various strains.
It is a graph which shows the incubation time with respect to pH.

FIG. 3 is a graph showing incubation time versus butyric acid levels in pasture inoculated with various strains.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hans Kay. Founder, Weasp, Sae, The Netherlands. Yeh van Houtenlarn 36

Claims (11)

[Claims] 1. A method of producing live grass by microbial conversion of a live grass crop during an aerobic phase and a subsequent anaerobic phase, the method comprising:
The anaerobic phase is Clostridium.
um) occurring in the presence of lactic acid bacteria that produce an inhibitory factor. 2. The method according to claim 1, wherein the anaerobic phase occurs in the presence of lactic acid bacteria that produce a proteinaceous Clostridium inhibitor. 3. The Lactobacillus plantarum (Lact) wherein the anaerobic phase produces a Clostridium inhibitor.
2. The method according to claim 1, which occurs in the presence of the bacterium Obacillus plantarum. 4. The method of claim 3, wherein the anaerobic phase occurs in the presence of Lactobacillus plantarum which produces a protein Clostridium inhibitor. 5. Use of a lactic acid bacterium which produces a Clostridium inhibitor for the production of grass. 6. Use of a lactic acid bacterium which produces a proteinaceous Clostridium inhibitor for the production of grass. 7. The use of Lactobacillus plantarum which produces a Clostridium inhibitor in the production of hay. 8. The use of Lactobacillus plantarum which produces a proteinaceous Clostridium inhibitor for the production of hay. 9. Lactobacillus plantarum capable of producing a Clostridial inhibitor. 10. Lactobacillus plantarum capable of producing a proteinaceous Clostridium inhibitor. 11. A live grass inoculum containing the Lactobacillus according to any one of claims 9 to 10.