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The present study was undertaken to assess the lipid composition and deposition in muscle at three anatomical locations in cultured yellowtail and to investigate the effect of lipid composition and deposition on meat texture. Lipid deposition in muscle was studied by histochemical staining of lipid with Sudan dye. Lipid class composition analysis showed that neutral lipids were the main constituents of lipid in cultured yellowtail and accounted primarily for the variation in muscle lipid content with the anatomical location of meat, as well as with season, whereas the polar lipid content remained almost constant. Furthermore, muscle neutral lipid content was correlated negatively with meat breaking strength; however, no correlation was observed between muscle polar lipid content and meat breaking strength. The histochemical study revealed that, in yellowtail muscle, lipid is preferentially deposited in the myosepta and, with increases in muscle lipid content, additional fat is deposited along sparsely distributed thin connective tissue. It was also observed that the greater the lipid deposition in collageneous connective tissue, the lower the meat breaking strength; presumably, higher lipid deposition in the connective tissue resulted in weakening of the muscle structure.

トカゲエソ肉塩すり身の40°Cでのミオシン重鎖(HC)分解は肉を晒処理しても抑制されなかった。NaClはHC分解を促進し糖類やみりんは影響しなかった。弱アルカリ性でHC分解が最も大きかった。塩すり身の40°C付近での加熱時間が長いほどHC分解が進み,同時にかまぼこのゲル強度が低下した。従って,かまぼこ製造工程においてトカゲエソ肉塩すり身はHC分解によるゲルの劣化を生じやすいことが示された。

In order to examine the effect of meat oxidation on the gel forming ability before grinding the meat with salt, fish meat was washed with CuCl2 solution, and the gel strength as well as total sulfhydryl (SH) groups and sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) patterns were analysed. Washing with CuCl2 solution resulted in a decrease in the total SH content of fish meat and the formation of myosin heavy chain (MHC) dimer through disulfide bonding. The plot of logarithmic gel strength versus protein concentration after heating the washed meat at 80°C in the presence of 3% NaCl to form a gel illustrated that the gel forming ability of meats washed with CuCl2 solution was weaker than the control meat. The gel of meat washed with CuCl2 showed the polymerization of MHC and MHC dimers through disulfide bonding much more than the control meat gel, although a small decrease in the SH group content after heating. Further washing with ethylene diamine tetra-acetic acid (EDTA) solution to remove CuCl2 from the CuCl2-washed meat also resulted in similar behavior for MHC polymerization and SH content as the CuCl2-washed meat, and the gel was still weaker than the control gel. It was found that the oxidation of SH groups during washing with CuCl2 solution accompanied by MHC dimer formation in the meat results in the weakening of its gel forming ability.

A study was conducted to evaluate meat texture (breaking strength), muscle proximate composition and muscle collagen content at three anatomical locations in cultured yellowtail. We report here the contribution of muscle biochemical constituents to the raw meat texture of cultured yellowtail, and the variation in muscle biochemical composition as well as meat texture with the anatomical location of the meat. Meat breaking strength decreased significantly with the anatomical location of the meat from head to tail. Muscle proximate composition also varied with anatomical location; in particular, a large variation was observed in muscle lipid content, which decreased significantly from head to tail. Muscle collagen content was significantly lower in dorsal part meat than in pre-dorsal part and tail part meat. Meat breaking strength was correlated negatively with muscle lipid content and positively with muscle collagen content. It was concluded that muscle lipid and collagen are the two primary muscle constituents having a direct influence on the raw meat texture of cultured yellowtail. It was also demonstrated that the muscle biochemical composition and raw meat texture of cultured yellowtail vary with the anatomical location of the meat and with season. © 2002 Society of Chemical Industry

To clarify the contribution of polymerization of myosin heavy chain (MHC) by disulfide bonding to increased gel strength of cooked gel via preheating, the pastes of walleye pollack surimi (SS and C grades) were preheated at 25°C and 40°C for a variety of hours prior to heating at 80°C for 20 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) patterns of cooked gels were analyzed with and without reducing the samples, which were solubilized in 8 M urea–2% SDS solution. The formation of polymers by disulfide bonding in cooked gels was almost constant in each of the SS and C grade surimi gels despite the period of preheating. Therefore, it was suggested that polymerization by disulfide bonding occurred during cooking at 80°C and not during preheating.

To confirm the contribution of polymerization and degradation of myosin heavy chain (MHC) during preheating to the gel-forming ability of fish meat paste, walleye pollack surimi paste was preheated at 30∞C and 50∞C prior to heating at 80∞C in the presence of various inhibitors. At 30∞C, ethyleneglycol bis(2-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid (EGTA) and ethylenedi- aminetetraacitic acid (EDTA) inhibited gel formation as well as the polymerization of MHC, whereas dithiothreitol (DTT) and leupeptin promoted gel formation, which was accompanied by the enhance- ment of MHC polymerization and decreased MHC degradation, respectively. At 50∞C, leupeptin inhib- ited MHC degradation and improved gel strength, whereas EGTA, EDTA and DTT had no effect on MHC polymerization and degradation and did not affect gel formation. The results demonstrate that the gel strength of cooked gel (80∞C) is not affected by preheating at 30∞C and 50∞C and does not inhibit polymerization and degradation. Results suggest that the gel strength of cooked gel is depen- dent on the polymerization and degradation of MHC during preheating.

本研究で用いた魚肉水溶性画分中のミオシン重鎖(HC)分解抑制因子(MDI)は市販トリプシンおよびキモトリプシン活性を阻害したことから, セリンプロテアーゼインヒビターと考えられた。一方, 水晒しによって戻りが誘発される魚種の晒肉塩ずり身の40℃付近でのHCの分解は市販セリンプロテアーゼインヒビターで阻害された。従って, MDIは戻りを誘発する晒肉中のセリンプロテアーゼのインヒビターとして作用するものと推察した。単離したMDIは分子量80Kの単量体であった。

マルソウダ加工処理残滓の有効利用のための基礎的知見を得ることを目的として, 高知県沖で漁獲されたマルソウダの脂質および脂肪酸組成の季節変動を調べた。残滓の約42%を占める頭部および眼窩組織の脂質含量は, 一年を通じてそれぞれ5.2a11.6%および7.0a17.9%の間で変動した。両組織の全脂質の脂肪酸では, 22 : 6n-3(DHA)の組成比が通年最も高く, 頭部で22.7%以上, 眼窩組織で24.5%以上を占めていた。以上の結果から, マルソウダ加工処理残滓のうち, 眼窩組織を含む頭部はDHAの供給源として利用できる可能性が確認された。

Proximate, fatty acid, and free amino acid compositions in the muscle of cultured red sea bream fed moist pellet (MP-fish) were compared with those of the fish fed fresh bait (FB-fish). Lipid accumulation in the abdominal cavity of both fish was also examined.The lipid content in the muscle of MP-fish was almost the same as that of FB-fish. On the other hand, the content of lipid in the abdominal cavity of MP-fish was higher than that in FB-fish. In the fatty acid composition of muscle non-polar lipid, the percentages of C18:1 n-9 and C18:2 n-6 in MP-fish were higher than in FB-fish, while the percentage of C22:6 n-3 was lower in the former than in the latter. Fatty acid composition of muscle non-polar lipid was affected by the bait.Free amino acid composition was also affected by the bait. The content of Anserine in MP-fish was higher than that in FB-fish, while the contents of Lys, His, and Arg were much lower.

When carp myosin was heated at 80°C with cysteine, the polymerization of protein through SS bonding (SS polymerization) was promoted. The SS polymerization was promoted even in the presence of both EDTA and cysteine, though it was not promoted in the presence of only EDTA. Cysteine was oxidized into cystine. Cystine also accelerated the SS polymerization of myosin heavy chain during the heating, and it was reduced into cysteine.From these results, it was suggested that acceleration of SS polymerization of myosin heavy chain by adding cysteine is due to the promotion of intermolecular SS bonding by the oxidative effect of cystine which was produced from cysteine during the heating.

To decide the subfragment in carp myosin responsible for the polymerization of myosin heavy chain (HC) through SS bonding during the heating at 80°C, carp myosin was digested with α-chymotrypsin into S-1 and rod, and was heated. From the SDS-PAGE patterns (3% polyacrylamide gel) of the heated samples solubilized in 8M urea-2% SDS, two components were found to be produced. The one was observed on the top of the polyacrylamide gel, and the other was just over HC and was 260 kDa in molecular mass. By treating each component with 10% 2-mercaptoethanol, it was found that the former component was made only from S-1 (80 kDa) and the latter was only from rod (130 kDa).Therefore, it was concluded that in the polymerization of HC through SS bonding during the heating of myosin, SH groups on rod is responsible for the formation of HC dimer and those on S-1 is responsible for the HC polymer formation.

Myosin from carp muscle was heated at 80°C in 0.6M NaCl solutions containing various kinds of buffer (pH 6.8), and the polymerization behavior of proteins through SS bonding (SS polymerization) was analyzed by SDS-polyacrylamide gel electrophoresis. In the phosphate buffer and Tris-HCl buffer, the oxidation of SH groups in myosin and SS polymerization of myosin heavy chain were promoted, which suggested that metal ions from the reagents used might be related to them. In Tris-maleate buffer and Tris-maleic anhydride buffer, the content of SH groups decreased more than in the phosphate buffer and in Tris-HCl buffer, but the decrease was not inhibited by a metal chelating reagent (EDTA) Moreover, in Tris-maleate buffer, SS polymerization was inhibited, and in Tris-maleic anhydride buffer, SS polymerization through covalent bonding other than SS bonding occurred. Therefore, it was concluded that these two buffers are not suitable for the research of SS polymerization.

The polymer of protein through SS bonding on the top of 5% SDS-PAGE gel was found to be made up of only a myosin heavy chain from the analysis of two-step SDS-PAGE, and from the results it was a dimer, trimer, tetramer, and above-decamer with 1.8% SDS-PAGE gel. The polymerization was suppressed in the presence of N-ethylmaleimide, dithiothreitol, or EDTA. The polymerization of myosin light chains as well as myosin heavy chain was promoted in the presence of transition ion (Cu+, Cu2+), while Ca2+ and Mg2+ did not affect the polymerization. From the above results, it was made clear that the polymerization of protein through SS bonding during the heating of myosin is the reaction that the SH groups of myosin heavy chain are oxidized in the presence of catalysts such as copper ion, and contribute to the smooth and swift formation of the polymer larger than decamer.

For the sake of utilizing the washed meat from Type IV (the fish which exhibits a modori at around 40°C in the washed meat, not in the unwashed meat) fish, this meat was mixed with the unwashed meat and the washed meat from the fish Types I and II which have excellent gel-forming ability. Type I is the fish which does not exhibit a modori at all in the unwashed meat and the washed meat, and Type II is the fish which exhibits the modori at 60°C only in the unwashed meat. The mixed meat pastes with the unwashed meat of these two types did not exhibit the modori at 40°C, while that with the washed meats exhibited it. However, the modori at 60°C was occurred in the mixture with the unwashed meat of Type II. The gel-forming ability at 80°C was improved in the mixed meat pastes. Sarcoplasmic protein fraction of the meat of not only Type IV but also Type I inhibited the modori at 40°C as well as the myosin heavy chain (HC) degradation of the washed meat pastes from Type IV. The floating lipid on the wash water did not have the inhibitory effect. Therefore it was suggested that an inhibiting factor against the modori at 40°C of the washed meat may be contained in the sarcoplasmic protein fraction of fish meats.

Two-step heating (2S heating) is one of the processes used for strengthening kamaboko gel. The usefulness of 2S heating on meat pastes from various modori-inducing types of fish (Type I-IV) was examined. 2S heating was carried out by heating at 40°C for various periods, and subsequently at 80°C for 20 min. The gel strength of 2S heated gels was compared with that of gel heated only at 80°C for 20 min to evaluate the effectiveness of 2S heating. 2S heating was not effective for the washed meat gels from Type IV, whose washed meat indicates modori-phenomenon at 40°C. The gel strength of the 2S heated gels decreased with heating time at 40°C, accompanied by degradation of myosin heavy chain. These results imply that the degradation of myosin heavy chain is responsible for gel disintegration and that the degraded product of myosin heavy chain does not contribute to the gel forming ability of the meat. The results also indicate that 2S heating (40→80°C) to strengthen the gel fails when the washed meat of fish Type IV is used.

The gel-forming characteristics of unwashed and washed meat from three lizardfish species (maeso Saurida undosquamis, wanieso Saurida wanieso, and tokageeso Saurida elongata) were investigated at various heating temperatures from 30 to 80°C for 20 minutes and 2hours. Judging from evaluation of gel strength by tensiometer and a folding test accompanied by degradation of myosin heavy chain (MHC) by SDS-PAGE analysis, the gel degradation of unwashed meat gels occurred at 40, 60, and 70°C in tokageeso, while it occurred at 60 and 70°C in maeso and at 60°C in wanieso. Washing of meat did not improve the gel strength or MHC degradation of meat gel at 40°C in tokageeso or at 60°C in maeso. Moreover, washing of meat showed an enhancing effect on MHC degradation of maeso and wanieso meat gels at 40°C. These findings indicate that the gel-degrading factors (GDFs) which existed in these species were not only sarcoplasmic (Sp) and myofibrillar (Mf) type active at 60°C but also Mf type active at 40°C and Sp type active at 70°C.

Gel-forming ability at 30-90°C of unwashed or washed meat from 19 fish species, which were caught off Kochi Prefecture and are usually used for Age-mono (a fried product of fish meat paste), was investigated to utilize their meat as raw materials for Kamaboko.The fish species used could be classified into the following 4 groups. Type I (3 species): neither unwashed nor washed meat gels disintegrated at any temperature examined. Type II (3 species): unwashed meat gels disintegrated at about 60°C, but washed ones improved. Type III (6 species): both unwashed and washed meat gels disintegrated at about 60°C. Type IV (7 species): unwashed meat gels disintegrated at about 60°C, but washed ones newly disintegrated at about 40°C. The gel forming ability at 80°C of Type I (unwashed or washed meat) and Type II (washed meat) was enough for Kamaboko and that of Type III was also improved to some extent by washing.It was suggested that washing the meat is not necessary for Type I and is useful for Types II and III, but that it is not good for Type IV. It should be emphasized that the fish of Type IV are a novel group in view of the fact that the Modori-phenomenon was newly induced at around 40°C by washing the meat, accompanied by the degradation of myosin heavy chain.

The inhibitory effects of several selected proteinase inhibitors, soybean trypsin inhibitor, leupeptin, antipain, E-64, NEM and EDTA, on myosin heavy chain (MHC) degradation of unwashed and washed meat gels from three lizardfish species, maeso or true lizardfish Saurida undosquamis, wanieso Saurida wanieso, tokageeso or shortfin lizardfish Saurida elongata, were investigated in order to identify the proteolytic activities of gel-degrading factors (GDFs) existing in mat. It was apparent that the proteolytic activities of GDFs responsible for MHC degradation of meat gels from all species at 40°C resulted from two myofibril-associated (Mf) type proteinases, mainly of serine proteinase and slightly of cysteine proteinase. In addition, as indicated by the amino acid patterns obtained from TCA-soluble fraction of meat gels with the presence of various inhibitors, these proposed proteinases were not distinct in these three species. With regard to the MHC degradation at 60°C, the proteolytic activity present in GDFs of maeso, proposed as myofibril-associated gel degrading factors (Mf-GDFs), belonged to cysteine proteinase while that of wanieso and tokageeso were sarcoplasmic-type serine proteinases. The Mf-GDF corresponding to the MHC degradation at 60°C, which occasionally occurs in wanieso meat gel, showed the proteolytic activities of serine proteinase.

Gel forming ability of miscellaneous fish caught in Tosa Bay off Kochi was examined to find good materials or utilize these fish. Nineteen species of fish used were classified into 4 groups according to the effect of meat washing on the modori appearance. Type I: both unwashed meat and washed meat show no modori and an excellent gel forming ability (3 species), Type II: unwashed meat shows modori around 60°C and washed meat do not show modori (3 species), Type III: both unwashed meat and washed meat show modori around 60°C (3 species), and Type IV: unwashed meat shows modori at 60°C and washed meat shows modori around 40°C (10 species), which was a type newly found. Preheating of washed meat of Type IV should not be carried out principally around 40°C. Mixing with the meat of Type I or II improves the gel-forming ability. A 40°C modori-inhibiting factor should exist in sarcoplasmic fraction. In addition modori appearance in washed meat of Type IV was due to the activation of myofibril type protease by removing the modori-inhibiting factor that might inhibit protease activity.

The dark muscle content in fish was measured by a rapid direct gravimetric method. This method was modified by using heat treated fish which was more convenient than using fresh fish. The fish were placed in a plastic bag and immersed in a water bath at 70°C for 30min and then brought to room temperature and the muscle was separated. The ratio of dark muscle to whole muscle (dark muscle ratio) in common mackerel measured by this method was lower than that of the usual method with fresh fish, but was the same as obtained by the indirect method using an estimate of transverse section. This method had the advantage of ease and rapidity (about one hour per fish), but some loss occurred since the heat released a fluid portion (drip) from the fish body. However, it was thought that this did not hinder the estimate of the dark muscle ratio, since drip loss was proportional in dark and white muscle. The dark muscle ratio using this method in 16 fish species were shown to be greater than 12% in the so-called red meat fish species, 4-9% in intermediate fish species and less than 3% in white meat fish species.

In order to elucidate the degradation of inosinic acid (IMP) in the dark muscle of fish, the levels of adenine nucleotides and their related compounds were compared in the white and dark muscles of yellowtail Seriola quinqueradiata and in common mackerel Scomber japonicas during iced storage. The struggling of 30 minutes before death affected the degradation rate of IMP in the dark muscle of yellowtail, while it did not affect in the white muscle. During iced storage, the degrada-tion of IMP proceeded very rapidly in the dark muscle of common mackerel. IMP was degraded optimally at pH6 and pH8 in muscle homogenates of common mackerel. The activity in the dark muscle homogenate at pH6 was 5 times higher than that of the white muscle homogenate, whereas the activity at pH8 was nearly the same in both muscle homogenates. The fact that the high activity at pH6 was present in the supernatant of the dark muscle homo-genate indicated that this enzyme is easily soluble in water. In view of these findings, the fact that the pH of red meat fish such as mackerel falls quickly to around 6 after death suggests that the enzyme activity is mainly responsible for the rapid IMP degradation in the dark muscle.

It has been shown that inosinic acid (IMP) hydrolyzing enzyme has high activity at pH 6.0 in the dark muscle of common mackerel Scomber japonicas and is easily soluble in water. This suggests the possibility that the enzyme in the dark muscle may affect the degradation of IMP in the white muscle of common mackerel. It was found that the IMP degradation in the white muscle homogenate was accelerated by mixing the dark muscle homogenate during a 4 days period of storage in ice. A similar phenomenon was also clearly observed when the minced white muscle was mixed with 10% of minced dark muscle during a 7 days period of storage in ice. After 8 days of storage in ice, when the contents of ATP-related compounds in the white muscle of common mackerel were determined in the fillets with and without the dark muscle, it was observed that IMP was slightly higher and inosine was slightly lower in the fillet without the dark muscle than in the fillet with the dark muscle. These results strongly indicate that the enzyme of the dark muscle affected the degradation of IMP in the white muscle of common mackerel.

Extractive nitrogenous constituents of dark muscles were determined in 13 species of fish having deep-seated dark muscle (skipjack, frigate mackerel), fin dark muscle (blow fish, surgeon fish) and superficial dark muscle (nine other species) and compared them with those of white muscles.The concentration of total extractive nitrogen and creatine were high in the dark muscle as compared with the white muscle. Of the ninhydrin positive substances, taurine concentration was much higher in the dark muscle than in the white muscle, and His, Ala, Lys and Glu were also high in the dark muscle. But His was higher in concentration in the white muscle of pelagic fishes, Except in larval fish, taurine content in the dark muscle of the common mackerel did not vary according to body weight or lipid content. Using chromatography for determining the ninhydrin positive substance in the dark muscle, a few unidentified peaks appeared near the taurine peak. Trimethylamine concentration was higher in dark muscle. But the volatile basic nitrogen concentration was less in the dark than in the white muscle. The total amount of nucleotides and related compounds was less in dark muscle than in white muscle. But the ratio of nucleotides to the total extractive nitrogen was larger in the dark than in the white muscle. Therefore, it was thought that no difference in quantity of nitrogen was found between the two kinds of muscles. However, constitutively, there was large difference in the inosin monophosphate concentration in the dark muscle than the white muscle.

In a previous paper it was observed that the high peak of an unidentified substance appeared just before taurine peak on the chromatogram of amino acid analysis from dark muscle of fishes. After hydrolyzing the dark muscle extracts of common mackerel Scomber japonicus, this peak almost disappeared and a new peak was observed in an earlier position on the chromatogram. The peak was not considered to be that of a peptide because other amino compounds were produced only in trace amounts after the hydrolysis, except glycine which was produced from nucleotide and its relat-ed compounds such as inosinic acid in the muscle extracts. Because crystallization could not be performed successfully before the hydrolysis, the substance was isolated from the hydrolyzed dark muscle extracts by the 2-step column chromatography using AG 50 W×8 (H+ form, 200-400 mesh, 2.6×40cm column) and AG 2×8(OH- form, 20-50 mesh, 2.6×40cm column). The molecular weight of the isolated compound was estimated to be 169 from the results of ultimate analysis and 13C-NMR spectra. An IR spectrum indicated peak characteristics of -COOH, -NH2 and -SO3H at 1750cm-1, 1600cm-1 and 1230cm-1, respectively. From these results, the isolated compound was identified as cysteic acid. It was assumed from the chromatographic behaviour of the substance before and after the hydrolysis that cysteic acid-related compound existed in the dark muscle of fish.

The effect of furylfuramide (FF) on germination and outgrowth of two bacterial spores was studied in a slide culture, in order to define the point at which FF acts to prevent the developmental process, from a morphological point of view. Concentrations of FF higher than minimal inhibitory concentration (MIC) did not interfere with the reduction of refractility. However, the developmental process stopped there, and the germinated spores eventually lysed before elongation occurred, shedding the empty spore coats. At less than MIC, many distorted cells appeared and gradually lysed. The bacteriostatic action of FF was shown as a result of the prolongation of their outgrowth. FF acted to arrest the development of bacterial spores not at germination, but after the stage from “swelling” to “emergence”. A discrepancy between the two organisms existed in that many refractile spores of B. coagulans, isolated from spoiled fish sausage, still remained on the culture after 35 hours at 37°C, but not in the case of B. subtilis.

Nitrofuran derivativses, firstly developed as a chemotherapeutic agent for human diseases, have been utilized as feed additives in the United States and as food preservatives in Japan. AF-2 (2-(2-furyl)-3-(5-nitro-2-furyl) acryl amide) and AF-5 (3-amino-6-(5-nitrofuryl-2-ethenyl)-1, 2, 4-triazine hydrochloride), two new nitrofurans, have been found to have high antibacterial activity in vitro and very low chronic toxicity as compared with nitrofurazone (F) and nitrofuryl acryl amide (Z) which are currently being used as food preservatives in Japan. In this study, preservative effects of these two new nitrofurans were tested in use with Kamaboko (fish cake), fish sausage and fresh fish. They showed strong preservative effects for these foods. AF-2 and AF-5, both showed a preservative effect for Kamaboko at a 5 ppm ratio which was more effective than a 250 ppm ratio of Neofuraskin (trade name of powder sold by Ueno Seiyaku Co., containing 2% of F and 8% of Z). (Table 2. and 3). For fish sausage, AF-2 and AF-5 showed almost the same preservative effect. A 5 ppm ratio of AF-2 or AF-5 was as effective as a 250 ppm ratio of Neofuraskin. But the effect of AF-5 for these two kinds of foods was more varied than that of AF-2 during the experiments. (Fig. 1). This phenomenon might be caused by the instability of this compound in foodstuffs. When the fish were dipped in druged sea water, the preservative effect of AF-2 and AF-5 for fish was almost the same as that of chlorotetracycline, with AF-2 slighly more effective than AF-5. Both were far more effective than Z. (Table 5. and Fig. 2).

The effects of furylfuramide (FF) on the germination and successive outgrowth of two bacterial spores were investigated by measuring the change in optical density of spore suspensions under several exposing systems to FF, in order to substantiate the microscopic observations which suggested that this drug acted to inhibit the outgrowth not at germination, but during the stage from “swelling” to “emergence” in the developmental process of the bacterial spore. The results obtained were summarized as follows: 1. FF did not inhibit the germination in physiological germinant solution even at ten times more than MIC for both microbes, although in PGY medium the time required to reach minimum optical density was prolonged in proportion to the concentration of FF in the case of B. coagulans. 2. When the dormant spores were exposed to FF in phosphate buffer, it had no effectiveness on either germination or outgrowth, but when the microbes were exposed in physiological germinant, the effect of FF was shown as the prolongation of the lag phase of the outgrowth in the medium without FF.