Novel starter cultures to inhibit biogenic amines accumulation during fish sauce fermentation

Bacteria with amine oxidase activity have become a particular interest to reduce biogenic amines concentration in food products such as meat and fish sausages. However, little information is available regarding the application of these bacteria in fish sauce. Hence, our study was aimed to investigate the effect of such starter cultures in reducing biogenic amines accumulation during fish sauce fermentation. Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05 isolated from fish sauce which possess amine oxidase activity were used as starter cultures in this study. Fermentation was held for 120 days at 35 °C. The pH value increased in all samples, while salt concentration remained constant throughout fermentation. Aerobic bacteria count was significantly lower (p < 0.05) in the control than in inoculated samples as a result of starter cultures addition. However, it decreased during fermentation due to the growth inhibition by high salt concentration. Proteolytic bacterial count decreased during fermentation with no significant difference (p > 0.05) among samples. These bacteria hydrolyzed protein in anchovy to produce free amino acid precursors for amines formation by decarboxylase bacteria. The presence of biogenic amines producing bacteria in this study was considered to be indigenous from raw material or contamination during fermentation, since our cultures were negative histamine producers. Amino acid histidine, arginine, lysine and tyrosine concentration decreased at different rates during fermentation as they were converted into their respective amines. In general, biogenic amines concentration namely histamine, putrescine, cadaverine and tyramine increased throughout fermentation. However, their concentrations were markedly higher (p < 0.05) in the control (without starter cultures) as compared to the samples treated with starter cultures. Histamine concentration was reduced by 27.7% and 15.4% by Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively. Both cultures could also reduce other amines during fermentation. After 120 days of fermentation, the overall biogenic amines concentration was 15.9% and 12.5% less in samples inoculated with Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively, as compared to control samples. These findings emphasized that application of starter cultures with amines oxidase activity in fish sauce fermentation was found to be effective in reducing biogenic amines accumulation.     

Biogenic Amines in Dry Sausages as Affected by Starter Culture and Contaminant Amine-Positive Lactobacillus

The effect of an amine-negative starter culture, containing Pediococcus pentosaceus and Staphylococcus carnosus, on the growth and amine formation of an amine-positive contaminant lactic acid bacterium (G 106) was studied in dry sausages. Levels of biogenic amines, precursor amino acids, pH, water activity and microbial counts were measured. Levels of phenylethylamine and tyramine increased in the sausages inoculated with the amine-positive strain. The starter culture did not prevent growth of G 106 or its amine formation capability. However, levels of histamine remained low although G 106 could produce histamine in vitro.     

Type of bacterial starter culture,aging and fermentation effects on some characteristics of inoculated beef sausages

Beef sausage mixes were inoculated with either Pediococcus acidilactici with Staphylococcus xylosus or P. acidilactici with S. carnosus, subdivided and then held for 0, 24, 48 or 72 h at 8–10 °C prior to fermentation. After aging (pre-fermentation holding), the mixes were fermented for 16 h ending at 41 °C. Moisture, protein and fat contents of all sausage mixes did not differ due to holding effects over all starter cultures. The pH of mixes followed the same pattern for all mixes, declining (p < 0.05) from approximately 5.8 to pH 5.2–5.3 at 72 h aging and to 4.4–4.5 after fermentation. Total acidity of the mixes followed an inverse pattern to pH, increasing (p < 0.05) after fermentation although there was no effect due to type of starter culture. Aging had no effect on nonprotein nitrogen (NPN) content as ΔNPN among all cultures. After fermentation, however, sausages held 72 h and inoculated with S. carnosus had higher NPN contents compared to P. acidilactici alone (p < 0.05) and with S. xylosus (p < 0.10). The same effects of starter cultures on changes in total amino acid concentration were observed. Concentrations of individual amino acids showed increases depending on pre-fermentation aging period (0 h versus 72 h) followed by fermentation.     

Effects of starter cultures and nitrite levels on formation of biogenic amines in sucuk

The effects of starter cultures, starter A (Lactobacillus sakei+Staphylococcus carnosus) and starter B (Pediococcus acidilactici+Staphylococcus xylosus+Lactobacillus curvatus), nitrite levels (0, 75, and 150ppm) and ripening period on biogenic amine production were investigated in sucuk (Turkish dry-fermented sausage) production. Changes in biogenic amines, pH, water activity, moisture, non-protein nitrogen, nitrite and counts of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, and moulds-yeasts were determined. Ripening period had a very significant effect (P<0.01) on the all biogenic amines. Sausages produced by fermentation with starter cultures, as compared to natural fermentation (control), had lower amounts of putrescine, cadaverine and tyramine (P<0.05). Starter cultures A and B were found to have almost the same effect on the formation of biogenic amines. Use of nitrite in sucuk production affected the formation of biogenic amines except for spermidine and spermine (P<0.01). Lactic acid bacteria counts in the control group were lower than that of starter culture groups. Lactic acid bacteria, Micrococcus/Staphylococcus, and moulds-yeasts counts increased during ripening period while levels of species of Enterobacteriaceae decreased, which was also found to be under the detectable level (<100cfug(-1)), by day 3 of ripening.     

The use of commercial starter cultures in the production of Turkish semi-dry fermented sausages

 The effects of three different starter cultures (Pediococcus acidilactici, Staphylococcus xylosus plus P. pentosaceus, S. carnosus plus Lactobacillus pentosus) were evaluated during the production of Turkish semi-dry fermented sausages. Sausages were studied during the fermentation phase, after heat processing and after drying for 24 h and 72 h. Chemical and organoleptical results indicated that in the processing of these semi-dry sausages a starter culture of P. acidilactici should be used. The use of this culture significantly reduced the pH, increased the lactic acid content and percentage of total heme pigments converted to the cured pigment and improved the development of the sausages' characteristics, i.e. color, appearance, flavor and general acceptability. Received: 20 February 1998 / Revised version: 7 May 1998     

Characterization of fermented silver carp sausages inoculated with mixed starter culture

To improve the characteristics and functionality, and increase the use of fish muscle, three groups mixed starter cultures (group one: Lactobacillus plantarum-15, Staphylococcus xylosus-12 and Pediococcus pentosaceus-ATCC33316 [S-PXP]; group two: Lactobacillus planatrum-15, Staphylococcus xylosus-12 and Lactobacillus casei subs casei-1.001 [S-PXC]; and group three: Staphylococcus xylosus-12, Lactobacillus casei subsp. casei-1.001 and P. pentosaceus-ATCC33316 [S-XCP]) were inoculated in minced silver carp muscle to produce a fermented fish product. During the 48 h fermentation at 30 °C, silver carp muscle inoculated with mixed starter cultures resulted in a rapid pH decrease, suppression in the increase of thiobarturic acid (TBARS) values, total volatile base nitrogen (TVB-N), trimethylamine (TMA), and the growth of spoilage bacteria and pathogens, and had higher whiteness than the control (without any starter) (P<0.05). The changes in SDS-PAGE indicated extensive hydrolysis of muscle protein occurred during fermentation. This study showed that the mixed starter cultures could substantially improve the flavor, digestibility, and nutritional value of the silver carp muscle.     

The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages

The main objective of this work was to examine the effects of using five types of commercial starter cultures in fermented sausages. During the fermentation stage, changes in proteolytic characteristics were observed in fermented sausages. Proteolytic activity was high in Ls_b + Sc:(Lactobacillus sakei + Staphylococcus carnosus) and Pp + Sx:(Pediococcus pentosaceus + Staphylococcus xylosus) starter-inoculated sausages during processing. Moreover, a slight increase in proteolytic activity was detected during storage in both these sausages. Sarcoplasmic and myofibrillar proteins were also affected by this starter culture addition, during the fermentation, ripening and intense proteolysis were observed in both the fermented sausages. The content of free amino acids was similar at the beginning of the fermentation stage for all the studied batches. However, the high differences in the content of free amino acids at the end of the process could be attributed to the starter culture activity.     

The effect of different starter cultures and ripening temperatures on formation of biogenic amine in Turkish fermented sausages

Different starter culture added groups (Group I: Lactobacillus sake, Staphylococcus xylosus; Group II: Lactobacillus plantarum, Staphylococcus carnosus; Group III: Lactobacillus curvatus, Staphylococcus carnosus, and Staphylococcus xylosus) and control group sausage samples were produced experimentally at two different temperatures (22 and 26 °C). In raw materials and all groups, by the 0th, 2nd, 5th and 7th days of the ripening period, the analysis of HPLC and biogenic amine (tyramine, histamine, cadaverine, putrescine, phenylethylamine, tryptamine, spermine, and spermidine) amounts, microbiological (Lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, Enterococcus spp., and moulds–yeasts) and chemical (pH, dry matter, and salt) have been done. During the ripening period, there is no statistical discrepancy (P > 0.05) in terms of biogenic amine amounts, microbiological, and chemical values detected from the sausage samples produced at 26 and 22 °C. However, in both levels of temperature, there is statistical discrepancy (P < 0.001) detected in terms of tyramine, putrescine values and the count of Enterococcus spp. between the starter culture added samples and control group samples. By this study, it has been stated that the ripening temperature does not make any statistical discrepancy (P > 0.05) for all values but the use of starter culture prevents the formation of biogenic amine in Turkish fermented sausages.     

The application of staphylococci with flavour-generating potential is affected by acidification in fermented dry sausages

Differences in the production of bacterial metabolites with potential impact on fermented sausage flavour were found in meat simulation medium when comparing different strains of Staphylococcus xylosus and Staphylococcus carnosus as starter cultures. Overall, higher levels of 3-methyl-1-butanol and acetoin were found for S. xylosus, with some intraspecies variability. In addition, sausage fermentation parameters affected staphylococcal growth and metabolism. Strong acidification, as in Northern-European types of fermented dry sausage, inhibited S. xylosus 3PA6 but not S. carnosus 833. During a milder, Southern-European type of acidification, both strains displayed survival over time. During in situ sausage trials, variations in the degree of acidification and the choice of starter microorganisms were of importance, whereas modifications in fat and salt contents had no effects. Staphylococcus sciuri αSg2, Staphylococcus succinus 4PB1, and S. xylosus 3PA6 were unable to survive the fermentation of a Northern-European type of fermented dry sausage, characterized by low or no 3-methyl-1-butanol and acetoin production. Inoculation with S. sciuri αSg2, S. succinus 4PB1, or S. xylosus 3PA6 led to 3-methyl-1-butanol and acetoin production in Southern-European type of fermented dry sausages, which was not observed with S. carnosus 833.     

不同乳酸菌发酵剂对发酵红肠品质的影响

为研究乳酸菌发酵对红肠品质的影响,将发酵技术应用于本无发酵工艺的红肠制品中,筛选出能够提高红肠品质的乳酸菌发酵剂。分别将常应用于发酵肉制品的7 种商业乳酸菌发酵剂（木糖葡萄球菌＋戊糖片球菌（THM-17）、木糖葡萄球菌＋清酒乳杆菌＋类植物乳杆菌（PRO-MIX5）、木糖葡萄球菌＋肉葡萄球菌＋清酒乳杆菌（WBL-45）、木糖葡萄球菌＋戊糖片球菌＋植物乳杆菌（VHI-41）、木糖葡萄球菌＋戊糖片球菌＋植物乳杆菌（SHI-59）、肉葡萄球菌＋木糖葡萄球菌（WBX-43）和戊糖片球菌＋木糖葡萄球菌＋肉葡萄球菌＋乳酸片球菌（VBM-60））及8 种单菌（弯曲乳杆菌、戊糖乳杆菌、清酒乳杆菌-1、戊糖片球菌、木糖葡萄球菌、肉葡萄球菌、清酒乳杆菌-2、植物乳杆菌）以107 CFU/g的接种量接种至腌制后的肉馅中,拌馅灌肠后于35 ℃、80%湿度条件下发酵12 h,取样测定发酵后样品的乳酸菌数和细菌总数,再经干燥、蒸煮、烟熏、烘烤制得成品,测定其感官、pH值、色差、质构、亚硝酸盐、硝酸盐、生物胺及N-亚硝胺含量等指标。结果表明：15 种发酵剂中以木糖葡萄球菌和植物乳杆菌2 种乳酸菌发酵剂应用效果较好,所制得产品pH值分别为5.26和5.04,色泽美观,弹性适中,亚硝酸盐残留量（10.84、10.13 mg/kg）低,可显著抑制N-亚硝胺的形成（N-二甲基亚硝胺含量分别为1.29、2.51 μg/kg）,生物胺总量较低。由此说明,木糖葡萄球菌和植物乳杆菌能够显著提高红肠产品的安全品质。     

Effect of the use of autochthonous Lactobacillus curvatus, Lactobacillus plantarum and Staphylococcus xylosus strains on microbiological and biochemical properties of the Sardinian fermented sausage

In this study, a starter culture was tentatively made by testing autochthonous Lactobacillus and Staphylococcus strains, isolated from traditional ‘Salsiccia Sarda’ sausage. Three sausage-making trials were carried out using an association of Staphylococcus xylosus + Lactobacillus curvatus (A), Staphylococcus xylosus + Lactobacillus plantarum (B), and Lactobacillus plantarum alone (C). Microbiological profiles, free amino acids, biogenic amines, and free fatty acids were evaluated after 21 days of ripening. Sausages A and B showed the highest number of lactic acid bacteria that produced a rapid acidification of substrates and reduced the number of spoilage microorganisms. Sausages made with L. plantarum alone or together with S. xylosus had the highest content of free amino acids, whereas the one produced with S. xylosus + L. curvatus showed the lowest content of total biogenic amines and best acceptance score. No significant differences among samples were found as to the free fatty acids concentration.     

Effect of autochthonous starter cultures on the volatile flavour compounds of Chinese traditional fermented fish (Suan yu)

Effect of autochthonous starter cultures on the volatile flavour compounds of Chinese traditional fermented fish was studied. Lactobacillus plantarum 120, Staphylococcus xylosus 135 and Saccharomyces cerevisiae 31, isolated from Suan yu, were selected as starter cultures. Volatiles were extracted by headspace solid‐phase microextraction (SPME) and analysed by gas chromatography–mass spectrometry technology (GC‐MS). Esters and alcohols were the main components of volatiles, accounting for over 50 percentage points in all samples. The highest content of esters (3034.54 μg kg^?1) was observed in S1 inoculated with L. plantarum 120, while the highest content of alcohols (2164.53 μg kg^?1) and ketones (379.98 μg kg^?1) was detected in S3 inoculated with S. cerevisiae 31. The content of acids and aldehydes was lower in inoculated samples. Principal component analysis revealed that the volatile composition was primarily influenced by the nature of the starter cultures. L. plantarum 120 and S. xylosus 135 could accelerate fermentation.     

Improvement of the quality and abatement of the biogenic amines of grass carp muscles by fermentation using mixed cultures

BACKGROUND: To improve the quality of processed grass carp (Ctenopharyngodon idellus) products and control the accumulation of hazardous substances therein, minced grass carp slices were salted for 6 h at room temperature and then inoculated with mixed starter cultures of Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen and Monascus anka and fermented for 12 h at 30 °C. The changes in some characteristics and biogenic amine contents of the fermented muscles were investigated. RESULTS: During the 12 h fermentation at 30 °C, muscles inoculated with mixed starter cultures showed a rapid decrease in pH from 6.0 to 5.1 and suppression of the growth of enterobacteria and pseudomonads. The fermented muscles exhibited better colour, appearance, flavour and overall acceptability than the control (P < 0.05). The changes in non‐protein nitrogen and free amino acid contents of the fermented muscles and in their sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles indicated that severe hydrolysis of muscle proteins occurred during fermentation. The accumulation of biogenic amines in the muscles was efficiently reduced by fermentation with mixed starter cultures. CONCLUSION: Fermentation with mixed starter cultures of L. casei, S. lactis, S. cerevisiae Hansen and M. anka significantly improved the characteristics of grass carp muscles and controlled the accumulation of biogenic amines. Copyright © 2009 Society of Chemical Industry     

The influence of environmental parameters on the catabolism of branched-chain amino acids by Staphylococcus xylosus and Staphylococcus carnosus

Degradation of the amino acids leucine, isoleucine and valine into branched flavour compounds by Staphylococcus xylosus and Staphylococcus carnosus was studied using resting cell cultures added to a defined reaction medium under different environmental conditions relevant to sausage fermentation. The environmental conditions studied were temperature (12–28°C), NaCl concentration (4.0–12.0% (w/w)) acidity (pH 4.8–5.8) and addition of manganese (0–2.5 mg Mn/kg). Flavour compounds were sampled by automatic static headspace collection and separated/quantified using gas chromatography/flame ionization detection (GC/FID). Main volatile catabolic products of leucine, isoleucine and valine were 3-methylbutanoic, 2-methylbutanoic and 2-methylpropanoic acids, respectively. The generation of branched flavour compounds was influenced significantly by most of the investigated environmental parameters, but in particularly by temperature and NaCl.Raising the temperature increased the production of branched-chain acids by both S. xylosus and S. carnosus, whereas increasing NaCl concentration had a clear negative effect, reducing the concentrations of branched-chain acids for both species but in particularly for S. carnosus. The acidity of the medium was also important for both Staphylococcus spp., but with opposite effects. Lowering pH decreased the generation of branched-chain acids by S. carnosus but increased the generation by S. xylosus. In addition, several two-factor interactions between temperature, NaCl concentration and pH were significant. Manganese addition increased the branched-chain acid concentration slightly for S. xylosus but not for S. carnosus.     

Comparison of the effects of a commercial and an autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter culture on the sensory and safety properties of a traditional Iberian dry‐fermented sausage “salchichón”

The effect of the addition of an autochthonous starter culture and commercial starter culture on the physico‐chemical and sensory characteristics of the dry‐fermented sausage ‘salchichon’ was investigated. Sausages were prepared with a mixture of Pediococcus acidilactici MC184 and Staphylococcus vitulus RS34 as the autochthonous starter culture (P184S34), ripened for 86 days, and compared with a commercial starter batch (CS1). Strains of acid lactic bacteria and Staphylococcus were identified in the samples along the ripening process which demonstrated that the inoculation of sausages assures their implantation during the ripening process. However, P184S34 starter culture showed a higher inhibitory effect on enterobacteria and coliform flora than the CS1 batches, guaranteeing a better quality hygienic sausages. Dry‐fermented sausages ripened with P184S34 showed higher amounts of NPN and volatile compounds derived from amino acid catabolism than the CS1 batches. Especially important was the result found in biogenic amines, since the P184S34 reduced their accumulation compared to the commercial starter batch. The inoculation of a decarboxylase‐negative autochthonous starter culture P184S34 reduced the biogenic amine accumulation guaranteeing safety and homogeneity of the products without producing a negative effect on the sensory characteristics of the traditional fermented sausages.     

Effect of fermentation with mixed starter cultures on biogenic amines in bighead carp surimi

The effects of one group mixed starter cultures, combined with Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen, and Monascus anka, and a batch without starter as control on biogenic amines accumulation in the bighead carp surimi during fermentation were investigated. Determination of five different biogenic amines was carried out by reverse‐phase high‐performance liquid chromatography with UV detection. Results showed that mixed starter cultures decreased the pH quickly from initial pH value of 6.4 to 5.2, inhibited the growth of contaminant microorganisms, such as Enterobacteriaceae and Pseudomonas, present in the raw materials, and suppressed the accumulation of histamine, tyramines, spermine and spermidine. Fermentation inoculated with combination of Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen, and Monascus anka with negative‐decarboxylase activity may prevent biogenic amine formation in the fermented ssurimi and improve hygienic quality of fermented surimi.     

Application of Autochthonous Mixed Starter for Controlled Kedong Sufu Fermentation in Pilot Plant Tests

Traditional sufu is fermented by back‐slopping and back‐slopping has many defects. The objective of this study was to apply autochthonous mixed starter to control Kedong sufu fermentation. Sufu was manufactured using back‐slopping (batch A) and autochthonous mixed starter (batch B) with Kocuria kristinae F7, Micrococcus luteus KDF1, and Staphylococcus carnosus KDFR1676. Considering physicochemical properties of sufu, 150‐day sufu samples from batch A and 90‐day sufu samples from batch B met the standard requirements, respectively. Considering sensory characteristics of sufu, 150‐day sufu samples from batch A and 90‐day sufu samples from batch B showed no significant differences (P > 0.05). The maturation period of sufu was shortened by 60 d. Profiles of free amino acids and peptides partly revealed the mechanism of typical sensory quality and shorter ripening time of sufu manufactured by autochthonous mixed starter. In final products, content of total biogenic amines was reduced by 48%. Autochthonous mixed starter performed better than back‐slopping. Fermentation had a positive influence on the quality, safety, and sensory properties of sufu. The application of autochthonous mixed starter does not change the sensory characteristics of traditional fermented sufu. In addition, it reduces maturation period and improves their homogeneity and safety. It is possible to substitute autochthonous mixed starter for back‐slopping in the manufacture of sufu.     

The effects of a starter culture on the formation of biogenic amines in Turkish soudjoucks

The effects of starter culture containing Lactobacillus sake, Pediococcus pentosaceus, Staphylococcus carnosus plus Staphylococcus xylosus on the formation of biogenic amines during ripening of Turkish soudjoucks were investigated. Determination of eight different biogenic amines was carried out by reverse-phase high performance liquid chromatography (HPLC) using diode array detection. It was found that aerobic plate counts (APC) decreased in the samples with added starter culture. Lactic acid bacteria counts increased in both the controls and samples with added starter during ripening. Total Enterobacteriaceae (ENT) counts decreased on the 30th day for vacuum packed and starter added samples. Yeast counts decreased only in vacuum packed and starter added samples on the 30th day. No biogenic amines were found in ground meat samples. Putrescine (PU) and tyramine (TYR) were determined in all control samples during the storage at 4°C. Starter addition inhibited formation of PU but not TYR.     

微生物发酵对牛肉调味基料的增香作用

以牛骨肉末为原料,在传统肉味香精的制备工艺中增加发酵技术,研究比较不同菌种发酵对牛肉调味基料的赋香效果。将牛骨肉末经过热压浸提、酶解后分成5组:不接种发酵剂的对照(CK)组;分别接种0.02%意大利萨科WBL-45(肉葡萄球菌+木糖葡萄球菌+清酒乳杆菌)、WBX-43(肉葡萄球菌+木糖葡萄球菌)、BOM-13(清酒乳杆菌)和THM-17(木糖葡萄球菌+戊糖片球菌)的实验组,30℃发酵12 h后进行美拉德反应制成牛肉调味基料。采用感官评定结合电子鼻和电子舌对其挥发性气味和滋味成分进行分析。结果表明,经发酵处理得到的牛肉调味基料在气味和滋味上与CK组之间差异显著(p<0.05),而4种菌株之间比较,接种THM-17对牛肉调味基料的赋香作用更为明显。实验说明,选用适宜菌株进行微生物发酵对牛肉调味基料具有明显的赋香效果,从而为工业化生产调味基料提供了新思路和新方法。     

北工商海洋杆菌诱导戊糖片球菌产生细菌素及降黄酒生物胺功效研究

利用功能微生物控制杂菌污染,是黄酒生产中控制生物胺含量的有效途径。从黄酒麦曲中分离筛选出新菌种[北工商海洋杆菌(Pontibacter beigongshangensis)]和新菌株[戊糖片球菌(Pediococcus pentosaceus)M25]。北工商海洋杆菌能够诱导戊糖片球菌产生细菌素,对黄酒中产生物胺的腐生葡萄球菌(Staphylococcus saprophyticus)、阪崎肠杆菌(Cronobacter sakazakii)、乳酪短杆菌(Brevibacterium casei)、芽孢杆菌(Bacillus)和阴沟肠杆菌(Enterobacter cloacae)均具有抑制作用。北工商海洋杆菌和戊糖片球菌自身不产生物胺。添加北工商海洋杆菌和戊糖片球菌,制备强化功能曲,利用强化功能曲酿造黄酒,可使黄酒中的生物胺含量降低29.8%,而对黄酒的风味物质影响不明显。因此,未来北工商海洋杆菌和戊糖片球菌可以作为降生物胺功能菌株应用在黄酒酿造中。